Autumn, winter and spring dynamics of aphid Sitobion avenae and parasitoid Aphidius rhopalosiphi interactions

The potential of parasitoids for aphid control during summer has been well documented. Few results are available on the impact of parasitoid populations on aphid hosts during autumn and winter and on the dynamics of their interactions during this period. The population development of Sitobion avenae, in Belgium, is analysed, from October to April, in the presence and absence of the parasitoid Aphidius rhopalosiphi. In the presence of parasitoids in winter, aphid populations decreased markedly and remained low at the beginning of spring. Induction of winter diapause in A. rhopalosiphi was observed during November at a mean temperature of 6.3°C and a decreasing photoperiod from 9.5–8.5 h of day light. A large range of A. rhopalosiphi mummy colourations, between dark and light, was noticed. This range of colouration did not allow a clear-cut distinction between diapausing and non-diapausing individuals of A. rhopalosiphi. The influence of seasonal weather and particularly temperature conditions on parasitoid mortality, strategy for overwintering and aphid population dynamics are discussed.     

Environmental and physiological factors for diapause induction and termination in the aphid parasitoid,Aphidius ervi (Hymenoptera: Aphidiidae)

In the aphid parasitoid Aphidius ervi Haliday, the incidence of diapause around the critical photoperiod was dependent on temperature. At a photoperiod of 12 h light:12 h dark, a significantly higher proportion of diapausing individuals was found at 12 degrees C than at 15 degrees C. The second larval stage of the parasitoid was most sensitive to photoperiod and localised-illumination experiments showed that the parasitoid responded independently of the aphid host. The sex ratio of the population which emerged after rearing in short-days and/or after diapause was male biased. Topical application of the juvenile hormone-mimic, pyriproxyfen, before or during diapause delayed emergence of Aphidius ervi but did not induce diapause or influence its termination. The ecdysteroid mimic RH 5849 induced diapause in a few treated individuals but did not affect diapause termination.     

Physiology of diapause and cold hardiness in overwintering pupae of the apple leaf miner Phyllonorycter ringoniella in Japan

Abstract The apple leaf miner Phyllonorycter ringoniella (Matsumura) (Lepidoptera: Gracillariidae) overwinters as a diapausing pupa. The diapause rate reaches 100% in early October. Diapause intensity decreases gradually from early October and diapause terminates in early February. The fresh body weight of diapausing pupae is 1.6 times that of non-diapausing pupae. The main cryoprotectant in P. ringoniella pupae is trehalose. Three stages are distinguishable as indicated by the correlations between diapause intensity, levels of cold hardiness and the trehalose content: diapause induction occurred in October, diapause development from November to December, and post-diapause quiescence from January to April. During diapause induction, the pupae accumulate low levels of trehalose and do not survive exposure to −15 °C. During diapause development, the pupae gradually accumulate more trehalose and show some ability to survive exposure to −15 °C, but not to −20 °C. During post-diapause quiescence, the pupae accumulate relatively more trehalose and cold hardiness fully develops, but decreases quickly in April. The trehalose content in pupae sampled in December is unaffected by acclimation temperatures in the range 0–30 °C, but decreases in pupae sampled in March after acclimation at temperatures from 5 to 15 °C. These results suggest that overwintering pupae of P. ringoniella have the ability to accumulate trehalose and develop a high level of cold hardiness during diapause development.     

Transgenerational phenotypic plasticity of diapause induction and related fitness cost in a commercial strain of the parasitoid Aphidius ervi Haliday

Diapause is an adaptation that insects have evolved to synchronize their life cycle with that of seasonal climatic changes and resources availability. However, cues for its induction are not always clear and, in some cases, a maternal effect may be involved. At the population level, just a part of the individuals may exhibit diapause with important consequences in terms of winter survival. Moreover, clear indicators of diapause state are difficult to identify. Diapause induction was thus investigated in the aphid parasitoid species Aphidius ervi Haliday (Hymenoptera: Braconidae) developing in the aphid Sitobion avenae (Hemiptera: Aphididae) at four crossed photothermal regimes (16 °C and 8 °C, 16:8 h L:D and 8:16 h L:D), and during 2 successive generations. We analyzed the reliability of changes in mummy color to assess for the diapausing state compared to dissections, and we measured parasitoid morphological and physiological traits. We observed that the proportion of dark brown mummies increased after one generation under low photothermal regime compared to other regimes. No diapause was recorded at 16 °C, 16:8 h L:D, while we observed 16.2% and 67.5% diapause incidence at 8 °C, 8:16 h L:D, at 1st and 2nd generation, respectively. Diapause induction is thus increased by short day-length conditions and low temperatures as well as by maternal effects. All parasitoid life-history traits (weight, size, fat content, water content, egg-load, and longevity) were affected by the photothermal regime and/or the generation. These results raise new questions on the environmental thresholds needed to induce diapause and on survival and adaptation potential of commercially available parasitoid strains in different environments.     

Host behaviour modification by the endoparasitoid Aphidius nigripes: a strategy to reduce hyperparasitism

Abstract. 1. One possible component of successful parasitism by insect parasitoids is the reduction of predation and hyperparasitism through the modification of host behaviour.
2. Just prior to death, potato aphids, Macrosiphum euphorbiae (Thomas), containing diapausing larvae of the parasitoid Aphidius nigripes Ashmead have been shown to leave the host plant to mummify in concealed sites, while those parasitized by non-diapausing individuals generally leave the aphid colony to mummify on the upper leaf surfaces.
3. The present study examines the within plant distribution of aphids and mummies containing non-diapausing A.nigripes , and compares parasitoid survival among microhabitats under field conditions.
4. On potato, Solunum tuberosum L., a significant proportion of aphids were found on lower leaf surfaces, whereas mummies were usually on the upper surfaces, particularly in the apical stratum of the plant canopy.
5. In both field surveys and experimental manipulations, parasitoid survival was higher on the upper surface of apical leaves than in other microhabitats, due to lower levels of hyperparasitism and, to a lesser extent, lower predation. This suggests that the pressure exerted by natural enemies has influenced the evolution of host behaviour modification.     

Effects of acclimation and diapause on the thermal tolerance of the two-spotted spider mite Tetranychus urticae

The two-spotted spider mite, Tetranychus urticae, is a worldwide pest species that overwinters as diapausing females. Cold hardening is presumed to start during diapause development to ensure the successful overwintering of this species. To address this hypothesis, we compared cold tolerance between non-diapausing and diapausing females. We measured supercooling point (SCP) and survival to acute cold stress by exposing the mites at a range of sub-zero temperatures (from −4 to −28 °C for 2 h). The mean SCPs of non-diapausing and diapausing females were −19.6±0.5 and −24.7±0.3 °C respectively, and freezing killed the mites. Diapausing females were significantly more cold tolerant than non-diapausing ones, with LT₅₀ of −19.7 and −13.3 °C, respectively. Further, we also examined the effects of cold acclimation (10 d at 0 or 5 °C) in non-diapausing and diapausing females. Our findings indicated that diapause decreased SCP significantly, while cold acclimation had no effect on the SCP except for non-diapausing females that were acclimated at 5 °C. Acclimation at 5 °C enhanced survival to acute cold stress in diapausing and non-diapausing females, with LT₅₀ of −22.0 and −17.1 °C, respectively. Altogether, our results indicate that T. urticae is a chill tolerant species, and that diapause and cold acclimation elevate cold hardiness in this species.     

Consequences of acclimation on survival and reproductive capacities of cold-stored mummies of Aphidius rhopalosiphi (Hymenoptera: Aphidiinae)

The cold storage of parasitoid mummies is a crucial point during mass production of parasitoids for aphid control in wheat, Triticum spp. In this study, the effect of acclimation to cold before storage of mummies containing Aphidius rhopalosiphi DeStefani-Peres (Hymenoptera: Braconidae: Aphidiinae) was evaluated on survival, sterility, and fecundity of parasitoids. Groups of 1-d-old and 3-d-old mummies were stored at -5 degrees C for 10 d without acclimation or after one of five different acclimation treatments. One-day-old and 3-d-old mummies contain prepupa and postmetamorphosis but not yet sclerotinized adult parasitoids, respectively. The offspring and sex ratio of stored parasitoids were compared with a control that was left at rearing temperatures (20 degrees C). Without acclimation, two-thirds of the parasitoids died during storage, for both ages of mummies tested. For stored 1-d-old mummies, subsequent survival increased under progressive exposure to low temperatures and reached 67.3% after 480 min of acclimation. In the same way, percentage of male sterility decreased with acclimation duration, whereas female fecundity increased. For stored 3-d-old mummies, subsequent survival improved with the duration of the acclimation treatment, but not male sterility and female fecundity.     

Deciphering the Metabolic Changes Associated with Diapause Syndrome and Cold Acclimation in the Two-Spotted Spider Mite Tetranychus urticae

Diapause is a common feature in several arthropod species that are subject to unfavorable growing seasons. The range of environmental cues that trigger the onset and termination of diapause, in addition to associated hormonal, biochemical, and molecular changes, have been studied extensively in recent years; however, such information is only available for a few insect species. Diapause and cold hardening usually occur together in overwintering arthropods, and can be characterized by recording changes to the wealth of molecules present in the tissue, hemolymph, or whole body of organisms. Recent technological advances, such as high throughput screening and quantification of metabolites via chromatographic analyses, are able to identify such molecules. In the present work, we examined the survival ability of diapausing and non-diapausing females of the two-spotted spider mite, Tetranychus urticae, in the presence (0 or 5°C) or absence of cold acclimation. Furthermore, we examined the metabolic fingerprints of these specimens via gas chromatography-mass spectrophotometry (GC-MS). Partial Least Square Discriminant Analysis (PLS-DA) of metabolites revealed that major metabolic variations were related to diapause, indicating in a clear cut-off between diapausing and non-diapausing females, regardless of acclimation state. Signs of metabolic depression were evident in diapausing females, with most amino acids and TCA cycle intermediates being significantly reduced. Out of the 40 accurately quantified metabolites, seven metabolites remained elevated or were accumulated in diapausing mites, i.e. cadaverine, gluconolactone, glucose, inositol, maltose, mannitol and sorbitol. The capacity to accumulate winter polyols during cold-acclimation was restricted to diapausing females. We conclude that the induction of increased cold hardiness in this species is associated with the diapause syndrome, rather than being a direct effect of low temperature. Our results provide novel information about biochemical events related to the cold hardening process in the two-spotted spider mite.     

Are aphid parasitoids from mild winter climates losing their winter diapause?

Temperature is both a selective pressure and a modulator of the diapause expression in insects from temperate regions. Thus, with climate warming, an alteration of the response to seasonal changes is expected, either through genetic adaptations to novel climatic conditions or phenotypic plasticity. Since the 1980s in western France, the winter guild of aphid parasitoids (Hymenoptera: Braconidae) in cereal fields has been made up of two species: Aphidius rhopalosiphi and Aphidius matricariae. The recent activity of two other species, Aphidius avenae and Aphidius ervi, during the winter months suggests that a modification of aphid parasitoid overwintering strategies has taken place within the guild. In this study, we first performed a field survey in the winter of 2014/15 to assess levels of parasitoid diapause incidence in agrosystems. Then, we compared the capacity of the four parasitoid species to enter winter diapause under nine different photoperiods and temperature conditions in the laboratory. As predicted, historically winter-active species (A. rhopalosiphi and A. matricariae) never entered diapause, whereas the species more recently active during winter (A. avenae and A. ervi) did enter diapause but at a low proportion (maximum of 13.4 and 11.2%, respectively). These results suggest rapid shifts over the last three decades in the overwintering strategies of aphid parasitoids in Western France, probably due to climate warming. This implies that diapause can be replaced by active adult overwintering, with potential consequences for species interactions, insect community composition, ecosystem functioning, and natural pest control.     

A large-scale rearing method for Peristenus digoneutis (Hymenoptera: Braconidae), a biological control agent of Lygus lineolaris (Hemiptera: Miridae)

Releases of Peristenus digoneutis against Lygus spp. in North America have been conducted for many years; however, no published procedures for mass production of the biological control agent were available. A laboratory rearing method was developed using Lygus lineolaris as the host to enhance establishment efforts and provide large numbers of wasps for inundative releases into high value fruit crops. Experiments were conducted to determine optimum host:parasitoid density and rearing temperature. The effects of nymph:wasp ratios and temperature on parasitism and wasp survival showed a 20:1 ratio at 20°C provided high parasitism (256 parasitized nymphs/wasp over lifetime) and excellent wasp survival of 27 days. Experiments on diapause-inducing conditions for P. digoneutis demonstrated that fluctuating temperatures of 23°C (day) and <16°C (night) and corresponding photo phases of 16 h light, for rearing parasitized nymphs, produced 100% diapausing parasitoids whereas non-diapausing parasitoids were only produced at more than 16 h light. Furthermore, parasitized Lygus nymphs need to be transferred to short day conditions no later than 10 days after parasitism to produce diapausing parasitoids. Critical life stages for exposure to conditions inducing diapause, the egg, first and second instar parasitoid larva, occurred from 0 to 10 days at 24°C constant temperature. Increased time in cold storage reduced the number of days to first emergence of parasitoids from diapausing cocoons when transferred to warm temperatures. The optimum storage time for diapausing P. digoneutis is between 25 and 44 weeks, depending upon the length of time that cocoons remain at warm conditions prior to chilling.     

Cold‐induced expression of diapause in Praon volucre: fitness cost and morpho‐physiological characterization

Cold exposure (2°C for 7 days) in constant darkness at mummy stage induces diapause expression in 9% of the Praon volucre Haliday population. Diapausing parasitoids show a significant delay in emergence time compared with nondiapausing counterparts. A diapause‐mediated polyphenism is observed in mummy colour, with diapausing mummies being clearly darker than nondiapausing ones. The diapause status of dark mummies is confirmed by a significant reduction in metabolic rate. Diapausing parasitoids also display specific morphological characteristics: they are heavier (fresh and dry mass) and accumulate larger fat reserves than nondiapausing counterparts. The diapause status is associated with a fitness cost in terms of adult longevity. There is no evidence of diapause‐related change in supercooling ability.     

Factors affecting carbohydrate and free amino acid content in overwintering larvae of Enosima leucotaeniella

Amounts of several metabolites were measured in overwintering larvae of Enosima leucotaeniella acclimated to temperatures between -5 and 15 degrees C for 30days. In the diapausing stage, cold hardiness, as shown by the survival rate, began rising below 15 degrees C. Glycogen content decreased as the temperature decreased from 10 to 0 degrees C. Trehalose content rose as the temperature decreased from 15 to 5 degrees C, but remained unchanged as the temperature decreased from 5 and 0 degrees C. Twenty-eight free amino acids were detected in the haemolymph; levels of proline, glutamine and glutamic acid increased at high temperatures, but alanine increased at low temperatures, especially as temperature decreased from 5 to 0 degrees C. Lipid content was unchanged by the different acclimation temperatures. The effects of temperature, diapause and aerobic conditions on the levels of carbohydrates and amino acids in overwintering larvae were analyzed. Alanine levels rose at low temperature only when the larvae were in the diapausing stage. The level of trehalose rose at low temperature in both the diapausing and post-diapausing stages, although it was higher at aerobic conditions in the post-diapausing stage. These results suggest that efficient trehalose synthesis occurs under the combination of low temperature and aerobic conditions of the post-diapausing stage, so that cold hardiness in overwintering E. leucotaeniella larvae may rise to a high level in winter.     

Over-wintering stage polymorphism of a bruchine beetle: geographical variation in optimal diapause strategy

Abstract.  1. Most temperate insects undergo diapause at a specific developmental stage to overcome severe winters. The bruchine beetle Bruchidius dorsalis in a warmer region in Japan, however, has diverse over-wintering stages – non-diapausing larvae, diapausing larvae, and diapausing adults, whereas in a cooler region, it over-winters only as the final instar larva or adult in diapause.
2. The geographical pattern of over-wintering stages in 12 populations was investigated over a wide range along the mainland of Japan. It revealed that a distinct geographical pattern of over-wintering stages exists in relation to temperature conditions. In regions with warmer climates, the proportion of non-diapausing larvae increased and B. dorsalis had a more complex over-wintering stage structure.
3. Life cycles were also compared between two areas of Japan by field experiments. In the cooler area, the first generation over-wintered in the diapausing larval or adult stage. Conversely, in the warmer area, diapause was induced later and some of the first-generation adults produced second-generation offspring before over-wintering.
4. Based on the geographical cline of climates and the differences in cold hardiness among stages, we can demonstrate that the over-wintering stage variation among and within populations results from an adaptive timing of diapause induction in each region, because the late larval or adult diapauses protect pupae or eggs – which unlike other stages are not cold hardy – from being produced late in the season.     

Cold hardiness in summer and winter diapause and post-diapause pupae of the cabbage armyworm, Mamestra brassicae L. under temperature acclimation

Cold hardiness and biochemical changes were investigated in winter and summer pupae of the cabbage armyworm Mamestra brassicae at the diapause and post-diapause stages under temperature acclimation. Diapause pupae were successively acclimated to 25, 20 and then 10 degrees C (warm-acclimated group). Pupae at the diapause and post-diapause stages were successively acclimated to 5, 0, -5 and then -10 degrees C (cold-acclimated groups). Supercooling point values in winter and summer pupae remained constant regardless of the diapause stages and acclimated temperatures. Warm-acclimated pupae at the diapause stage did not survive the subzero temperature exposure, whereas, cold-acclimated pupae achieved cold hardiness to various degrees. Winter pupae were more cold hardy than summer pupae, and pupae at the post-diapause stage were more cold hardy than those at the diapause stage. Trehalose contents in winter pupae rose under cold acclimation. Summer pupae accumulated far lower trehalose contents than winter pupae, with the maximal level occurring in winter pupae at the post-diapause stage. Glycogen content remained at a high level in diapause pupae after warm acclimation, whereas it decreased after cold acclimation. Alanine, the main free amino acid in haemolymph after cold acclimation, increased at lower temperatures in both diapause and post-diapause pupae, but the increase was greater in the diapause pupae. These results suggest that cold hardiness is more fully developed in winter pupae than in summer pupae, and cold acclimation provides higher cold hardiness in winter pupae at the post-diapause stage than at the diapause stage.     

Egg development and diapause: ecophysiological and genetic basis of phenological polymorphism and adaptation to varied hosts in the green oak tortrix,Tortrix viridana L. (Lepidoptera: Tortricidae)

Experiments concerned 8 tortrix populations associated with varied oak species. They showed that the egg development included a phase of diapause. Completion of embryogenesis at 20 degrees C was used as a criterion for whether diapause was completed. Under semi-natural conditions diapause terminated in late autumn or early winter, then eggs developed continuously, without postdiapause winter quiescence, even in severe cold. The eggs from the populations associated with holm or cork oak completed diapause then hatched later than those from the populations associated with sessile or pubescent oak. Reciprocal crossbreedings confirmed that this phenological polymorphism was genetically determined. Under constant temperatures the physiological state of diapausing eggs, assessed by measuring their cold requirements to complete diapause by exposure to 8 degrees C, varied gradually with increasing age. This diapause development was strongly temperature-dependent. Cold requirements of diapausing eggs were much higher in a late-hatching than in an early-hatching population. This explains the phenological polymorphism of the tortrix: the more eggs need cold, the later they complete diapause in autumn, and the later they hatch in spring. Egg cold requirements varied widely within populations too, which resulted in large variations in the date of diapause end among individuals. The date of egg hatch was influenced by the temperatures occurring during diapause and postdiapause, but apparently not by photoperiod.     

The roles of ERK and P38 MAPK signaling cascades on embryonic diapause initiation and termination of the silkworm, Bombyx mori

To clarify the molecular mechanisms of Bombyx diapause, we focused on mitogen-activated protein kinases (MAPK), which are the major components of signal transduction cascades that regulate cell proliferation, differentiation, and stress responses. In the present study, cloning of Bombyx extracellular signal-related kinase (ERK), MAPK-ERK kinase (MEK), and p38 MAPK cDNA, revealed that their amino acid sequences have close similarity with those of other species. We analyzed the roles of the kinases in diapause initiation and termination by immuno-blotting with anti-phospho-kinase antibodies. Phospho-MEK levels remained consistently high in non-diapausing eggs, then declined after the diapausing stage in diapausing eggs, and began to increase 45 d after transfer to 5 degrees C upon diapause termination. The phospho-ERK and phospho-MEK profiles were similar, suggesting that ERK phosphorylation is regulated by MEK. The phospho-p38 MAPK levels declined 36 h after oviposition in diapausing eggs, and increased at 15-30 d at 5 degrees C in yolk cells, suggesting that p38 MAPK has a role in diapause initiation and termination. Phospho-ERK levels were maintained with diapause-interrupting treatment and declined with diapause-sustaining treatment. ERK phosphorylation is considered to have a role in diapause termination and in the resumption of development.     

Establishment potential of the predatory mirid <Emphasis Type="Italic">Dicyphus hesperus</Emphasis> in northern Europe

The predatory mirid Dicyphus hesperus Knight (Hemiptera: Miridae) is native to North America. The species has been used for the control of glasshouse whitefly on aubergine in the Netherlands, and is currently being evaluated for continued and wider release in Europe. Field and laboratory studies were conducted on a population collected from southern California, USA, to assess the cold tolerance and potential for outdoor establishment under prevailing northern European climates. The supercooling points (whole animal freezing temperatures) of nymphal and adult insects were around −20°C. The lethal temperatures (LTemp₅₀) of non-diapausing nymphs and adults and diapausing adults were close to their respective freezing temperatures at −17.6, −17.6 and −19.2°C. At 5°C, the LTime₅₀ was 54, 101.7 and 117.5 days for fed nymphs, non-diapausing and diapausing adults respectively. When first instar nymphs were placed in the field in winter, starved samples died out after 70 days, but 5% of the fed nymphs survived until the end of winter (140 days) and developed to adult on return to the laboratory. After a similar 5-month field exposure, 50% of fed diapausing adults and 15% of fed non-diapausing adults were still alive at the end of winter, whereas starved diapausing adults died after 140 days. On return to the laboratory after 5 months in the field, both diapausing and non-diapausing adults mated and laid eggs, forming viable populations. Overall, the field and laboratory experiments indicate that this population of D. hesperus is able to enter diapause and that winter temperatures are not a barrier to establishment in northern Europe.     

Characteristics of summer diapause in the onion maggot,Delia antiqua (Diptera: Anthomyiidae)

Characteristics of summer diapause in the onion maggot, Delia antiqua, were clarified by laboratory experiments. Temperature was the primary factor for the induction of summer diapause in this species. The critical temperature for diapause induction was approximately 24 degrees C, regardless of the photoperiod. At 23 degrees C, the development of the diapausing pupae was arrested the day after pupariation, when about 7% of the total pupal development had occurred in terms of total effective temperature (degree-days). The most sensitive period for temperature with regard to diapause induction was estimated to be between pupariation and "pupation" (i.e., evagination of the head in cyclorrhaphous flies). Completion of diapause occurred at a wide range of temperatures (4-25 degrees C): The optimal temperature was approximately 16 degrees C, at which temperature only five days were required for diapause completion. The characteristics of summer diapause in D. antiqua are discussed in comparison with those of summer dormancy in a congener D. radicum and those of winter diapause in D. antiqua.     

Long-term reduction of cold hardiness following ingestion of ice-nucleating bacteria in the Colorado potato beetle,Leptinotarsa decemlineata

We investigated the effect of ingestion of ice-nucleating bacteria on the supercooling capacity and cold hardiness of the Colorado potato beetle (Leptinotarsa decemlineata Say), a freeze-intolerant species that overwinters as adults in shallow, terrestrial burrows. Ingestion of ice-nucleating bacteria (Enterobacter agglomerans, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas syringae), fed on slices of potato tuber, caused an abrupt decrease in supercooling capacity. No change occurred in the supercooling capacity of beetles fed Escherichia coli, as this species lacks ice-nucleating activity. Ingestion rates showed that tubers treated with different species were equally palatable. During diapause induction beetles evacuated food from their guts, but nevertheless retained sufficient ice-nucleating bacteria to diminish supercooling. Beetles fed P. fluorescens and P. putida exhibited reduced supercooling even after an 8-wk exposure to simulated winter conditions. Furthermore, P. fluorescens was isolated 10-wk post-ingestion from diapausing beetles. Our data suggest that ingested bacteria may be retained by insects during entry into diapause and that the cold hardiness of candidate crop pests, such as L. decemlineata, may be reduced by feeding them ice-nucleating bacteria prior to winter diapause.     

Diapause detection and monitoring in the Mediterranean corn stalk borer

Abstract. Sesamia nonagrioides Lefebvre diapauses as mature larvae. Once diapause is induced, larvae continue to grow and moult with no apparent changes, so distinction between non-diapausing and diapausing larvae is difficult. In the present work two physiological markers of diapause induction are obtained and their efficacy in detecting diapause onset and in monitoring diapause development in field populations is evaluated. The first marker is based on the differential capacity of two photoregimes, LD 0:24 h and LD 16:8 h, to reverse diapause induction. When larvae of the first and second stadia were subjected to the diapause-inducing LD 12:12 h light cycle and then transferred to LD 0:24 h, larvae pupated after a development duration significantly higher than that of the larvae transferred to LD 168 h. This criterion was used to monitor diapause development in overwintering larvae until field-collected larvae submitted to LD 0:24 h pupated synchronously with those submitted to LD 16:8 h. This occurred between late January and early March. The second marker refers to development of imaginal wing discs. A relative index of the tracheal mass area divided by the total disc area diminishes steadily during development in non-diapausing sixth-instar larvae until it becomes zero just before pupation, whereas it remains constant in diapausing larvae. Though wing disc development was blocked in diapausing sixth-instar larvae, the disc continued to grow and, consequently, the total area of the disc was not a suitable marker of diapause induction. When the constancy of the relative area index was used to monitor diapause development and termination in overwintering larvae, the results agreed with those obtained using the first criterion and with previous field observations.