Pupal Diapause Research Articles

Physiological and biological patterns of a highland and a coastal population of the European cherry fruit fly during diapause

Adult emergence of univoltine temperate insect species and its synchronization with specific host phenological stages is mainly regulated by obligatory pupal diapause. Although a few studies have investigated the factors affecting diapause intensity, little attention has been paid to the physiological alterations and metabolic regulation that take place during diapause. Here, we describe differences in diapause between a highland and a coastal Greek population of the European cherry fruit fly Rhagoletis cerasi, a major pest of sweet and sour cherries in many European countries. Pupae of both populations were exposed to the environmental conditions prevailing in the two areas and diapause termination was observed under laboratory conditions. The regulation of energetic metabolites during the long pupae stage was examined under both field and laboratory conditions. Differences in diapause intensity revealed that the two populations have adapted to the local geographical and climatic conditions and have different requirements for low temperatures to terminate diapause. The coastal population undergoes a shorter diapause and adults emerge more rapidly, especially in the highland area. The highland population failed to terminate diapause (<40% adult emergence) in the coastal area. Both populations draw on their major energetic reserves (lipids and protein) similarly during diapause. Nevertheless, regulation of carbohydrate and glycogen reserves seems to vary between the populations: major peaks of these stored nutrients occur on different dates in the two populations, suggesting a differential regulation. Differences in diapause intensity imply a genetic differentiation between the two populations. The importance of our findings in understanding the physiological patterns during obligatory diapause of a univoltine insect species, as well as the practical implications for the development of specific phenological models for the European cherry fruit fly are discussed.

Sexual difference in the photoperiodic induction of pupal diapause in the flesh fly Sarcophaga similis

The flesh fly Sarcophaga similis enters pupal diapause in response to short days, but averts diapause under long days. This species shows a sexual difference in the photoperiodic induction of diapause, with females having shorter critical daylength than males. Here, we proposed two hypotheses to explain this sexual difference. First, we proposed a sexual difference in the qualitative evaluation of photoperiods. This hypothesis assumes under the external coincidence model that although the photoinducible phase of both sexes locates at late scotophase, in males, it locates at a slightly earlier phase. However, the results of night interruption experiments clearly ruled out this hypothesis. Because we verified that S. similis evaluated photoperiods quantitatively, we next proposed a sexual difference in the quantitative evaluation of photoperiods. This hypothesis incorporates concepts of a hypothetical substance accumulation that shows a diapause-inducing effect and an internal threshold that serves as a reference to determine the diapause/nondiapause developmental program. In long-day exposure experiments and night interruption experiments, females consistently showed a lower incidence of diapause than males. Thus, the present study data satisfactorily meet the second hypothesis, that is a sexual difference in the quantitative evaluation of photoperiods exists in S. similis.

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.

Proteomic and Phosphoproteomic Analysis at Diapause Initiation in the Cotton Bollworm, Helicoverpa armigera

Diapause is a period of developmental arrest that allows a species to adapt to unfavorable conditions. Many insect species reduce metabolic activity and then enter diapause at a certain stage in their life cycles. The cotton bollworm, Helicoverpa armigera, will be destined for pupal diapause when larvae are reared under short daylengths and low temperature. The brain is an important organ for diapause decision, and some signaling molecules from the brain of diapause-destined individuals are released into the hemolymph to regulate diapause. In this study, we performed 2-D gel-based comparative proteomic and phosphoproteomic analyses to search for differentially expressed proteins between nondiapause- and diapause-destined pupal brains. A total of 79 proteins and 23 phosphoproteins showed significant differences between these two groups, and 41 proteins and 10 phosphoproteins were identified by MALDI-TOF/TOF MS. Further, gene expression patterns in diapause- and nondiapause-destined pupal brains were confirmed by RT-PCR or Western blot analysis. These differentially expressed proteins act in the metabolic change, stress response, and signal transduction pathways at early pupal stage for diapause initiation. Thus, these identified proteins may depress metabolism in diapause-destined pupae to lead the insect to enter developmental arrest.

Pupal diapause of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) mediated by larval host plants: pupal weight is important

Facultative diapause, a strategy that allows insects to initiate additional generations when conditions are favorable or to enter diapause when they are not, has a profound effect on the ecology and evolution of species. Most previous studies have concentrated on the role of photoperiod and temperature in inducing facultative diapause in insects. In contrast, here we studied pupal diapause mediated by larval host plants in the cotton bollworm Helicoverpa armigera, and confirmed that pupal weight is a critical factor. Two groups of third instar H. armigera larvae, kept at 25°C with L:D=8:16 and 20°C with photoperiod of L:D=8:16, respectively, were fed on six host plants and on artificial diet (as a control) to determine how larval host plants affect diapause incidence and related traits (such as pupal weight and developmental duration). The data showed larval host plants affected diapause incidence significantly and the effects could be masked by low temperature. Further analysis showed that pupal size, not the length of the sensitive stage, affected the decision to enter diapause. In a further experiment, third-instar to final-stage larvae deprived of artificial diet for 2 days demonstrated a direct relationship between pupal weight and diapause incidence. These results suggest that larval host plants, by affecting pupal size, may influence diapause occurrence in H. armigera. This has important adaptive significance for both over-wintering survival and the possibility for completing an additional generation.

Mechanisms of suspended animation are revealed by transcript profiling of diapause in the flesh fly

Diapause is a widespread adaptation to seasonality across invertebrate taxa. It is critical for persistence in seasonal environments, synchronizing life histories with favorable, resource-rich conditions and mitigating exposure to harsh environments. Despite some promising recent progress, however, we still know very little about the molecular modifications underlying diapause. We used transcriptional profiling to identify key groups of genes and pathways differentially regulated during pupal diapause, dynamically regulated across diapause development, and differentially regulated after diapause was pharmacologically terminated in the flesh fly Sarcophaga crassipalpis. We describe major shifts in stress axes, endocrine signaling, and metabolism that accompany diapause, several of which appear to be common features of dormancy in other taxa. To assess whether invertebrates with different diapause strategies have converged toward similar transcriptional profiles, we use archived expression data to compare the pupal diapause of S. crassipalpis with the adult reproductive diapause of Drosophila melanogaster and the larval dauer of Caenorhabditis elegans. Although dormant invertebrates converge on a few similar physiological phenotypes including metabolic depression and stress resistance, we find little transcriptional similarity among dormancies across species, suggesting that there may be many transcriptional strategies for producing physiologically similar dormancy responses.

The diapause response of Rhagoletis pomonella to varying environmental conditions and its significance for geographic and host plant‐related adaptation

AbstractThe recent shift of Rhagoletis pomonella Walsh (Diptera: Tephritidae) from its ancestral host hawthorn to apple is a model for incipient sympatric speciation in action. Previous studies have shown that changes in the over‐wintering pupal diapause are critical for differentially adapting R. pomonella flies to a difference in the fruiting times of apples vs. hawthorns, generating ecologically based reproductive isolation. Here, we exposed pupae of the hawthorn race to various combinations of pre‐ and over‐wintering rearing conditions and analyzed their effects on eclosion time and genetics. We report certain unexpected results in regards to a combination of brief pre‐winter and over‐wintering periods indicative of gene*environment interactions requiring a reassessment of our current understanding of R. pomonella diapause. We present a hypothesis that involves physiological factors related to stored energy reserves in pupae that influences the depth and duration of Rhagoletis diapause. This ‘pupal energy reserve’ hypothesis can account for our findings and help clarify the role host plant‐related life history adaptation plays in phytophage biodiversity.

Immature Insects: Ecological Roles of Mobility

Immature insects' adaptations for finding the highest quality food and optimal environmental conditions are critical to maximizing a species' success. Optimal food and environmental conditions during immature development increase the insects' survival and shorten their development time; these conditions also result in earlier reproduction and more rapid growth of the insect population. Immature-stage mobility is important in finding suitable food and environment, building shelters, thermoregulating behavior, and evading, escaping, or defending against natural enemies. The immature stages can move distances ranging from a few centimeters to a few hundred meters; whereas adults that can fly can move distances of a few, hundreds, or even thousands of kilometers. Mobility gives immature insects greater access to available food. Neonate larvae often must move large distances to find their first meal or they perish. Air currents, stream flow, or phoresy can carry them farther than walking. Feeding habits of subsequent instars may change as they grow, and larvae may commute between feeding and resting (shelter) sites. An immature insect may feed at many locations during its development, and mature larvae wander in search of a pupation site. Larvae of >361 insect species in 59 families and 5 orders leave their feeding sites to find a pupation site. Among species with nonfeeding, short-lived adults, the immature stages may accumulate food reserves needed for egg production in the adult stage. Larvae select a safe site before entering larval or pupal diapause for overwintering or summer aestivation, and some larvae may remain mobile during diapause. Active pupae or nymphs may increase the chance of successful adult eclosion. In this article, we review the many important ecological roles of immature mobility.

Biology and larval feeding impact of Hypena opulenta (Christoph) (Lepidoptera: Noctuidae): A potential biological control agent for Vincetoxicum nigrum and V. rossicum

A classical biological control program has been initiated against the invasive European swallow-worts Vincetoxicum nigrum and Vincetoxicum rossicum in North America. The noctuid moth Hypena opulenta, discovered feeding on V. rossicum in forests of southeastern Ukraine, is currently under evaluation as a potential biological control agent. In this study the life cycle of H. opulenta and factors affecting diapause induction were evaluated. Additionally, larval impact of H. opulenta on Vincetoxicum spp. performance was tested to determine whether future screening is worthwhile. Adults of H. opulenta begin oviposition 2 days after emergence and produce approximately 600 eggs. Larvae develop through five larval instars and overwinter as pupae. Pupal diapause is facultative, resulting in at least two generations per year. Diapause induction is affected by photoperiod and seasonal changes in plant quality as evidenced by increasing diapause induction when larvae are grown under a short day photoperiod and on senescing plants in the fall. Feeding by two larvae per plant caused reductions in aboveground biomass to V. rossicum resulting in decreased reproductive output (flower, seedpod, and seed production). Only flower production of V. nigrum was negatively affected by larval feeding. The results of this study indicate that H. opulenta is a promising agent against forested populations of V. rossicum and warrants completion of host specificity testing and examination of population dynamics of H. opulenta. This practice of conducting impact assessments of herbivores that are apparent specialist feeders prior to conducting the full battery of host specificity tests should improve agent selection by reducing the costs associated with screening ineffective agents.

Molecular characterization of heat shock protein 90, 70 and 70 cognate cDNAs and their expression patterns during thermal stress and pupal diapause in the corn earworm

Three heat shock protein transcripts, hsp90, hsp70, hsc70, isolated from the corn earworm, Helicoverpa zea, were evaluated for their responsiveness to diapause and thermal stress. These Hsps showed high homology to their counterparts in other species. A phylogenetic analysis of the Hsp90 sequence was consistent with the known classification of insects. Northern blot hybridization indicated the presence of hsp90 transcripts in all tissues, but expression in the brain-subesophageal complex was especially pronounced. The genomic organization of hsp90 examined by Southern blot suggested the presence of a single copy of hsp90 in the H. zea genome. The expression patterns after heat shock indicated that hsp70 and hsp90 were heat-inducible, although hsp70 was more strongly induced than hsp90, and hsc70 was indeed a constitutively expressed member of the hsp70 family. Expression of hsp70 and hsc70 were not altered by the diapause program, but hsp90 was down-regulated at this time. Low temperatures (0-4 degrees C) and recovery from low temperature elicited hsp70 and hsp90 responses, but not an hsc70 response. Thus, unlike several other species, H. zea does not up-regulate hsp70 during pupal diapause, but the down-regulation of hsp90 is consistent with the pattern observed in several other species during diapause. Our results also indicate that hsp90 and hsp70 are responsive to low temperature in both diapausing and nondiapausing pupae.

Genetic analysis of diapause capability and association between larval and pupal photoperiodic responses in the flesh fly Sarcophaga similis

Abstract A variant of the flesh fly Sarcophaga similis, which does not enter pupal diapause even under diapause‐inducing conditions, is established by artificial selection. Interline crosses of the wild‐type and the variant revealed that the diapause capability is inherited in an incomplete dominant manner. Neither sex linkage nor a maternal factor is involved in the mode of inheritance. Genetic and genetic–environmental interactions are involved in the induction of diapause. In the wild‐type, the post‐feeding larval period is prolonged in response to short days. However, in the variant, the duration of the larval period under short‐day conditions is identical to that under long‐day conditions. Long‐day responses under short‐day conditions at both the larval and pupal stages in the variant indicate that the responses are established by common factors in both stages. The factors causing the long‐day responses under short‐day conditions at different developmental stages in the variant remain to be identified. However, it is plausible to hypothesize that common factors regulate ecdysteroid release at both the larval and pupal stages, and that malfunction of the system regulating these factors triggers such ecdysteroid release, irrespective of the photoperiod. Hence, the photoperiodic responses disappear in the variant in both stages. The adult stage of the variant has a functional circadian clock, suggesting that the nondiapause phenotype is not necessarily involved in malfunction of the circadian clock.

Biphasic metabolic rate trajectory of pupal diapause termination and post-diapause development in a tephritid fly

Metabolic depression is a highly conserved feature of insect diapause, and an increase in metabolism is a reliable indicator of diapause termination and the initiation of post-diapause development. The trajectory of metabolic rate following diapause termination can guide the identification of important physiological and developmental landmarks during this developmental transition, yet quantitative descriptions of these trajectories are relatively rare. Here we track changes in metabolic rate from diapause through diapause termination and pharate adult development in Rhagoletis pomonella (Diptera: Tephritidae), a univoltine tephritid fly that diapauses in the pupal stage. Using respirometric monitoring we show that diapause termination and subsequent pharate adult development is characterized by a biphasic increase in metabolic rate. Respiration rate initially increases logistically, reaching a plateau that is followed by a final, exponential increase terminating in adult eclosion. We develop a non-linear model describing this pattern with easily interpretable landmarks, and we map visible landmarks of morphogenesis onto the trajectory. The bulk of visible morphogenesis in pharate adult development occurs relatively late in the trajectory during the final, exponential phase, that matches the U-shaped trajectory of non-diapause individuals. These results mirror a qualitative description of the same trajectories of diapause and non-diapause flesh flies ( Sarcophaga). Overall, our results suggest that (1) the course of diapause breakage and post-diapause development may be evolutionarily conserved in higher flies with pupal diapause, and (2) the biphasic trajectory is distinct from a more continuous trajectory observed during direct development.

Length variation in a specific region of the period gene correlates with differences in pupal diapause incidence in the flesh fly, Sarcophaga bullata

We report differences in the length of a specific region of the circadian clock gene period ( per) that correlate with different capacities for pupal diapause in the flesh fly, Sarcophaga bullata. The conspicuous difference is located in a region we refer to as the putative C-terminal photoperiodic (CP) region. The length of the CP region correlates inversely with the incidence of diapause. A deletion of 33 amino acids in this region correlates with a significant increase in the incidence of diapause, from 78.1% to 93.0%, and an insertion of 9 amino acids in the same area correlates with a drop in the diapause incidence to 4.0%. This correlation suggests a possible functional role for this region of per in photoperiodism.

Clock genes period and timeless are rhythmically expressed in brains of newly hatched, photosensitive larvae of the fly, Sarcophaga crassipalpis

While roles of the clock genes period ( per) and timeless ( tim) are relatively well understood in relation to circadian clocks, their potential roles in insect photoperiodism remain enigmatic. In this study, the expression of per and tim genes under two contrasting photoperiods is described in the central nervous system of photoperiodically sensitive, newly hatched first instar larvae of the flesh fly, Sarcophaga crassipalpis. Using qPCR, diel oscillations were observed in the mRNA levels of both genes under long-day (15 h light:9 h dark, promotes direct development) and short-day conditions (11 h light:13 h dark, induces pupal diapause). Peak per and tim mRNA oscillations were closely associated with the light/dark transition. The conspicuous difference between the two photoperiodic conditions was that the sharp increase in per and tim mRNA abundance occurred during the light phase under long days but during the dark phase under short days. The diel oscillations were, at least in part, driven by an endogenous component, as demonstrated by transferring larvae to continuous darkness. The cells displaying Tim- and Per-like immunoreactivities (Tim- and Per-LIRs) were localized using anti- Drosophila-Per and anti- Chymomyza-Tim antibodies. Per-LIR and Tim-LIR co-localized in three groups of cells in each brain hemisphere. Two other groups, one in the brain hemispheres and the other in the fused ventral nerve ganglion, expressed only the Per-LIR.

Alteration of the pupal diapause program and regulation of larval duration by photoperiod in the flesh fly Sarcophaga similis Meade (Diptera: Sarcophagidae)

Using a water treatment technique that extends the larval period of flesh flies, we investigated the effect of photoperiodic exposure on the alteration of the pupal diapause program and regulation of larval duration in the flesh fly Sarcophaga similis Meade (Diptera: Sarcophagidae). Exposure to only 4 cycles of long days successfully altered the developmental program from diapause to nondiapause, and short days altered the nondiapause developmental program to a diapause program. Larvae recognized all photoperiods of 14L10D, 15L9D, and 16L8D as long days, but their diapause-averting effects were different, i.e., 16L8D showed the strongest effect, 14L10D showed the weakest, and 15L9D showed an intermediate effect. These results indicate that larvae can discriminate long days quantitatively. S. similis also showed a clear photoperiodic response in the larval stage, i.e., larvae reared under short-day conditions pupariated later than those reared under long-day conditions. When diapause-destined larvae were exposed to 3 cycles of long days, some larvae altered their developmental program from diapause to nondiapause and pupariated significantly earlier than individuals destined to diapause.

Rapid elevation of Inos and decreases in abundance of other proteins at pupal diapause termination in the flesh fly Sarcophaga crassipalpis

We analyzed changes in brain proteins 24 h after pupal diapause termination in Sarcophaga crassipalpis by a combination of 2-dimensional gel electrophoresis and mass spectrometry. The proteome analysis revealed significant changes in 20 proteins, 11 of which represented ≥ 2.5-fold changes. Three proteins were present only in the brains of diapausing pupae. Among the most abundant proteins that showed a change, 1 was more abundant, 7 were less abundant, and 2 were absent following diapause termination. The protein that increased in abundance following diapause termination showed highest identity to myo-inositol-1-phosphate synthase (Inos). Proteins that decreased at diapause termination included those showing highest identities to fatty acid binding protein, CG2331-PA, twinstar, catalase, and a histone. Proteins absent at diapause termination included ribosomal protein L17A and one unnamed protein. An increase of Inos protein level was confirmed using Western blot analysis. Attempts to terminate diapause by injection of several Inos-related metabolites failed, thus suggesting that the elevation of Inos at diapause termination is downstream of the physiological regulation that initiates development.

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.

Identification of a novel gene encoding the trehalose phosphate synthase in the cotton bollworm, Helicoverpa armigera

Trehalose and trehalose metabolism are crucial for insect development. We measured the content of polyhydric compounds in the hemolymph of diapause- and nondiapause-destined individuals of Helicoverpa armigera. We found that the trehalose content is much higher in diapause-destined individuals than that in nondiapause individuals. The activity of trehalose-6-phosphate synthase (TPS) during H. armigera larval-pupal development is significantly higher in diapause-type individuals and is closely correlated with the changes in the trehalose content. The cDNA encoding TPS, which converts uridine-5'-diphosphoglucose and glucose-6-phosphate to trehalose-6-phosphate, was cloned from the fat body of H. armigera using rapid amplification of cDNA ends (RACE). The molecular characterization of the cDNA revealed that the mRNA encodes a precursor polypeptide of 826-amino-acid residues, containing Har-TPS at residues 6-507 and a putative trehalose-6-phosphate phosphatase, which converts trehalose-6-phosphate into free trehalose, at residues 512-783. The Har-TPS precursor polypeptide shows 73% identity with that of Drosophila melanogaster. The presence of a 2.8 kb transcript in the fat body and ovary was detected with a northern blot. The Har-TPS mRNA was detected at high levels in the late stage of sixth larval instar and the early middle stage of diapause-destined pupae, which are most likely to respond the changes in TPS activity and trehalose in the hemolymph. The Har-TPS protein was successfully overexpressed in the Bombyx mori baculovirus expression system, and the catalytic activity of Har-TPS was found to be approximately 5-fold higher in B. mori blood infected by the recombined-baculovirus than the control. When diapause is broken, the trehalose content drops significantly and glucose increases rapidly. These results suggest that trehalose is involved in regulating H. armigera pupal diapause.

PBAN/pyrokinin peptides in the central nervous system of the fire ant, Solenopsis invicta

The pyrokinin/pheromone-biosynthesis-activating neuropeptide (PBAN) family of peptides found in insects is characterized by a 5-amino-acid C-terminal sequence, FXPRLamide. The pentapeptide is the active core required for diverse physiological functions, including the stimulation of pheromone biosynthesis in female moths, muscle contraction, induction of embryonic diapause, melanization, acceleration of puparium formation, and termination of pupal diapause. We have used immunocytochemical techniques to demonstrate the presence of pyrokinin/PBAN-like peptides in the central nervous system of the fire ant, Solenopsis invicta. Polyclonal antisera against the C-terminal end of PBAN have revealed the location of the peptide-producing cell bodies and axons in the central nervous system. Immunoreactive material is detectable in at least three groups of neurons in the subesophageal ganglion and corpora cardiaca of all adult sexual forms. The ventral nerve cord of adults consists of two segmented thoracic ganglia and four segmented abdominal ganglia. Two immunoreactive pairs of neurons are present in the thoracic ganglia, and three neuron pairs in each of the first three abdominal ganglia. The terminal abdominal ganglion has no immunoreactive neurons. PBAN immunoreactive material found in abdominal neurons appears to be projected to perisympathetic organs connected to the abdominal ganglia. These results indicate that the fire ant nervous system contains pyrokinin/PBAN-like peptides, and that these peptides are released into the hemolymph. In support of our immunocytochemical results, significant pheromonotropic activity is found in fire ant brain-subesophageal ganglion extracts from all adult fire ant forms (queens, female and male alates, and workers) when extracts are injected into decapitated females of Helicoverpa zea. This is the first demonstration of the presence of pyrokinin/PBAN-like peptides and pheromonotropic activity in an ant species.

Neuropeptide-like precursor 4 is uniquely expressed during pupal diapause in the flesh fly

Suppression subtractive hybridization comparing brains from diapausing and nondiapausing pupae of the flesh fly, Sarcophaga crassipalpis, suggested that the gene encoding neuropeptide-like precursor 4 (Nplp4) was uniquely expressed during diapause. We have sequenced the full-length cDNA encoding Nplp4 and used Northern blots to further evaluate linkage to diapause. The open reading frame of this cDNA encodes a 61-amino acid residue precursor protein containing a predicted 18 residue signal peptide, one 22-amino acid and one 2-amino acid propeptides, and a 19-amino acid neuropeptide. The amino acid sequence of the precursor protein shows 64% identity to Drosophila melanogaster Nplp4; homologues of this precursor protein are not known from species other than these two flies. Nplp4 mRNA levels were quite low in nondiapausing (long day) pupae, but in contrast the gene was highly upregulated in diapausing (short day) pupae. Expression increased at the onset of diapause, remained high throughout diapause, and then decreased 2 days after diapause was terminated. Although the function of this precursor protein and the neuropeptide it yields remain unknown, this close association with diapause suggests a potential role for Nplp4 in initiating and maintaining diapause in the flesh fly.