Environmental conditions terminate reproductive diapause and influence pheromone perception in the long‐lived moth Caloptilia fraxinella

Reproductive diapause enables long‐lived insects to time mating with environmental conditions suitable for offspring development. Plasticity in the perception of pheromones used in sexual communication may enable mate‐finding at the appropriate time of year. The moth Caloptilia fraxinella (Ely) (Lepidoptera: Gracillariidae) undergoes a 9‐month reproductive diapause, during which the male response to pheromone is plastic and is highest during the period of reproductive activity. The mechanisms controlling this pheromone response plasticity are not well‐understood, and the aim of the present study is to determine the main factors involved. In the present study, the impact of temperature, photoperiod, juvenile hormone analogue (JHA) and adult nutrition on diapause termination are tested using electroantennogram (EAG) and behavioural response to pheromone in male C. fraxinella. Eclosion in a state of reproductive diapause occurs in most males; diapause is maintained under short‐day or cool conditions indoors, or under natural conditions outdoors. Exposure to long‐day, warm conditions over a period of 4 weeks causes a small number of males to become behaviourally responsive to pheromone; a larger number of males become behaviourally responsive over a period of 3 months of post‐eclosion. Treatment with a JHA impacts male EAG and the behavioural response to pheromone during the period of reproductive diapause. A carbohydrate food source is not required by reproductively active adult male C. fraxinella to respond to pheromone and express mate location behaviours. The main factors involved in controlling male pheromone response plasticity and the implications of these factors for the C. fraxinella population in its expanded range are discussed.     

Effects of nutrition and methoprene treatment upon reproductive diapause in Caloptilia fraxinella (Lepidoptera: Gracillariidae)

Abstract Female Caloptilia fraxinella exhibit a prolonged reproductive diapause immediately post adult emergence in mid‐summer until the next spring when mating, egg development and oviposition on fresh Fraxinus spp. leaflets occur. Factors that effect the termination of reproductive diapause are investigated in this species. Caloptilia fraxinella diapausing adults held in overwintering conditions (2 °C, LD 0 : 24 h) for 24 weeks terminate diapause after placement for 2 weeks in simulated summer conditions (24 °C, LD 16 : 8 h) only if they are provided with 10% sugar water. Exogenous application of the Juvenile Hormone (JH) analogue methoprene to moths in both early‐ (summer) and mid‐ (autumn) reproductive diapause demonstrates that JH affects diapause termination but a carbohydrate nutrition source also mediates mating and vitellogenesis. Mating between moth pairs early in diapause occurs only after treatment with methoprene and provision with sugar water. However, there is no impact of mating on the propensity of females to produce vitellogenic oöcytes. Moths collected in the autumn in mid‐diapause respond in a dose‐dependent fashion to methoprene treatment and the response is greater than that of moths early in diapause collected in the summer. Treatment with methoprene and access to sugar water results in vitellogenic oöcytes in 18.75% of females from mid‐diapause moth pairs treated with 0.01 μg methoprene per insect and in all females from pairs treated at the two highest doses of methoprene (0.1 and 1 μg per insect). Mating occurs only between moths in mid‐diapause treated with the two highest doses of methoprene and these doses induce 91% and 100% mating, respectively. Both control and methoprene‐treated males in mid‐diapause held under summer conditions mate successfully and pass a spermatophore to their methoprene‐treated female partner. These data demonstrate that female C. fraxinella undergo a prolonged reproductive diapause in which termination is dependent on JH and further mediated by a carbohydrate nutrition source. The production of vitellogenic oöcytes is independent of mating. These data also provide evidence that response of moths in diapause to exogenous applications of methoprene differs throughout the diapause period and between male and female C. fraxinella.     

Size and protein content of accessory glands in adult male Caloptilia fraxinella in different physiological states

Reproductive diapause is a strategy employed by some insects to coordinate reproduction with the appropriate environmental conditions for mate location and offspring development. Male Caloptilia fraxinella Ely (Lepidoptera: Gracillariidae) eclose in July in reproductive diapause, and remain unmated until May of the next year, when they emerge from overwintering sites in a reproductively active state. The present study assesses characteristics of male sex accessory glands (SAG) throughout the adult life stage when males are reproductively active and in early and late reproductive diapause. Male SAG are longer and positively correlate with moth body size when males are reproductively active in the spring compared with males in reproductive diapause in the summer and autumn. Male SAG also contain significantly more protein during the period of reproductive activity than during reproductive diapause. Access to a carbohydrate nutrition source does not impact male SAG length or protein concentration when males are reproductively active in the spring. Treatment with a Juvenile Hormone analogue in the autumn, but not the summer, tends to increase the total protein concentration compared with that of untreated control moths.     

Nutrition,sex and season contribute to variation in fat and glycerol levels in the long‐lived moth Caloptilia fraxinella

The ash leaf cone roller Caloptilia fraxinella Ely (Lepidoptera: Gracillariidae) is an invasive leaf‐mining moth pest of horticultural ash Fraxinus spp. in the Canadian Prairie Provinces. Caloptilia fraxinella overwinter as adults in reproductive diapause and mating occurs after overwintering in the spring. The effect of a carbohydrate food source on fat and glycerol reserves throughout the long adult life stage of this moth is investigated. Insects collected as pupae are given access to either water or sugar water upon adult eclosion. Moths held under the different feeding regimes are sampled before (summer and autumn) and after overwintering in the spring. Analysis of either glycerol or lipid content is conducted for male and female moths from each collection period. Both moth weight and glycerol concentration are affected by moth sex, food regime and season of collection. Although female moths are heavier than males, a higher glycerol concentration occurs in males. Moths fed sugar are heavier and have a higher glycerol concentration than water‐fed moths late in reproductive diapause and after overwintering. Moths collected in the spring after overwintering are lighter and have a lower glycerol content than moths collected before winter. There is a significant influence of feeding regime and season on moth body lipid content, with sugar‐fed moths having more fat than water‐fed moths; however, this difference is smaller in the summer than the autumn or spring. An initial understanding of the overwintering biology and diapause of this pest is provided in the present study.     

In vivo and in vitro effects of 20-hydroxyecdysone and methoprene on diapause maintenance and reproductive development in Musca autumnalis

Abstract. Face flies overwinter as adults in reproductive diapause. Administration of 20-hyroxyecdysone and/or methoprene induced reproductive development in diapausing flies which were maintained in a diapause-inducing environment. Hormone effects were additive and female flies were more sensitive than males. Release of vitellogenin from cultured fat body was stimulated by 20-hydroxyecdysone or methoprene. Transfer of flies from diapause to diapause-breaking environments induced some to break diapause, but this decreased with the time flies had been in a diapause-inducing environment. In contrast, topical application of methoprene to diapausing flies induced reproductive development irrespective of their ages even when they were kept in the diapause-inducing environment for 80 day degrees above a 12°C base temperature (14.5 days). Therefore diapause induction must depend on hormone levels less than some threshold level. The putative threshold varied according to diapause propensities of different genetic lines. Lines showing high frequencies of diapause required greater amounts of methoprene for reproductive development in diapause conditions than did lines showing low frequencies of diapause.     

Reproductive biology of the arctic collembolan Hypogastrura tullbergi

Terrestrial organisms of the Arctic are faced with strong climatic fluctuations. Predictable seasonality with cold/long winters and short/cool summers are combined with unpredictable between and within year variation. This indicates that various selection pressures act on the reproductive strategies of the populations. The arctic collembolan Hypogastrura tullbergi reproduces in a short period following snow melt. Hatching occurs in late summer, the animals grow to adult size within their second summer and reproduce for the first time in the beginning of their third summer. We performed several experiments to determine the reproductive investment and proximate mechanism that regulate timing and duration of reproduction. We found that H. tullbergi entered a reproductive diapause when reared at constant temperature, a diapause that was terminated by a cold exposure (winter). Surprisingly, cold exposure of small juveniles may also prevent development of a reproductive diapause in adults. Thus, the life-cycle normally spanning 2 yr can potentially be reduced to one year if the overwintering juveniles reach maturity before the end of the reproductive period in the field. After termination of the diapause, the animals reproduced up to 3 times during a period of 6 weeks at 15°C. This reproductive period was considerably longer (measured in degree days) than the one observed in the field. Our results suggested that temperature quiescence, i.e. the inability to reproduce under a certain temperature threshold, may adjust the termination of the reproductive period with current temperature before a new diapause occurs in late summer. The cost of reproduction was low and suggests that it can be adaptive to spread reproduction over more than one year. The results are discussed in relation to the arctic climate and strategies favoured by unpredictable and predictable (seasonal) variations in the environment. The present study forms part of a larger investigation on population dynamics and life history strategies of H. tullbergi from the Arctic.     

Attractiveness and toxicity of an attracticide formulation on adult males of ash leaf cone roller, Caloptilia fraxinella

A biodegradable attracticide formulation containing the pyrethroid insecticide permethrin and the sex pheromone of the ash leaf cone roller, Caloptilia fraxinella (Ely) (Lepidoptera: Gracillariidae), was tested for attractiveness and toxicity to males of this invasive pest of horticultural ash, Fraxinus spp. Marsh. (Oleaceae). Trap capture in attracticide‐baited traps was equal to capture in traps baited with rubber septa lures releasing the known attractive pheromone. Pheromone response by male C. fraxinella was not affected by the presence of permethrin as equal numbers of males were captured in traps baited with the attracticide formulation with and without permethrin. Attraction to the attracticide containing both pheromone and insecticide active ingredients did not vary with dose as 10, 50, and 100 mg droplets of the formulation attracted similar numbers of male moths in a field experiment. Equal numbers of male moths were captured in traps baited with droplets that had aged for 0–5 weeks in a laboratory fume hood before deployment in the field. The permethrin constituent of the attracticide formulation was toxic to male moths treated through tarsal contact and resulted in 73 and 100% mortality at 24 and 72 h post‐treatment, respectively. Leg autotomy occurred in over 80% of males 24 h post‐treatment to formulations containing permethrin. Mortality of moths was greater 24 h after treatment with a fresh attracticide source as compared to a 5‐week‐old source. However, mortality as a result of treatment with fresh and aged attracticide droplets was equal at 48, 72, and 96 h post‐treatment. This formulation has the potential to become a useful tool in an integrated pest‐management system for C. fraxinella on horticultural ash.     

Cyanoprotein: Developmental stage,sex and diapause-dependent expression,and synthesis regulation by juvenile hormone in the bean bug,Riptortus clavatus

Cyanoprotein (CP) synthesis was studied in nymphal and nondiapause adult, diapause adult, and juvenile hormone (JH) treated adult bean bugs, Riptortus clavatus. Hemolymph collected from bugs injected with [³⁵S]-methionine was analyzed by native polyacrylamide gel electrophoresis (PAGE) and fluorography, and CP synthesis was also determined quantitatively by rocket immunoelectrophoresis of hemolymph and counting. In the nymphal stages synthesis of CP-1, 2, 3, and 4 (with CP-4 synthesis predominant) reached a maximum at mid-instar and was not detected at each ecdysis, so that the synthetic activity of CPs changed cyclically in each instar. During the nymphal stages CP synthesis showed the same pattern and level in both females and males, and both diapause and nondiapause oriented bugs. In the adult stage, however, CP synthesis differed in the two sexes and nondiapause or diapause conditions. In nondiapause males CP synthesis was not detected in the adult, but in nondiapause females CP (only CP-1) was synthesized through the reproductive stages. CP-1 accumulated in the egg yolk together with vitellogenin. In diapause adults (both females and males) CP-1 to 4 were synthesized at a very low rate for over 2 months and accumulated in the hemolymph. JH or JH analog (JHA) methoprene and also long day condition, which terminate diapause, switched the main CP synthesis CP-4 to CP-1 in females. CP synthesis in diapause males stopped after JH(A) treatment. The activities of CP synthesis, thus, changed in developmental stages, sexes, and diapause. This is an excellent system for study of specific gene expression and switching controlled by insect hormones and sex. © 1992 Wiley-Liss, Inc.     

Correlation of oxygen consumption, cytochrome c oxidase, and cytochrome c oxidase subunit I gene expression in the termination of larval diapause in the bamboo borer, Omphisa fuscidentalis

The moth Omphisa fuscidentalis (Lepidoptera, Pyralidae) is a univoltine insect with a larval diapause period lasting up to 9 months. We studied changes in O(2) consumption in conjunction with cytochrome c oxidase activity and cytochrome c oxidase subunit I (cox1) gene expression. O(2) consumption changed within a day, showing a supradian rhythm with a ca.12-h cycle at 25 degrees C. During the first two-thirds of the diapause period, from October to March, O(2) consumption was constant until January and then increased by March. Topical application of methoprene, a juvenile hormone analog (JHA), to diapausing larvae terminated the diapause and was associated with an increase in O(2) consumption rate at diapause termination. In JHA-treated larvae, cytochrome c oxidase activity in fat bodies was high at the beginning of the prepupal period and highest at pupation. cox1 expression in fat bodies displayed a transient peak 8 days after JHA application and peaked in the prepupal period. Taken together, our results show that the break of diapause by JHA is associated with the activation of cox1, bringing about an increase in cytochrome c oxidase activity, followed by an increase in O(2) consumption rate.     

Response variability of juvenile hormone and juvenile hormone analogs in workers of the Formosan subterranean termite (Coptotermes formosanus Shiraki)

Juvenile hormone (JH) or juvenile hormone analog (JHA) can induce soldier formation in termites. However, different studies have yielded inconsistent results on the effects of JHA on soldier production in Coptotermes formosanus Shiraki. Using filter paper as the testing substrate, the effects of JH III, pyriproxyfen and methoprene on the intact filter paper left, worker mortality and presoldier formation were tested on six colonies. Our results showed that pyriproxyfen and methoprene were more repellent than JH III. No significant difference in toxicity was observed among methoprene, pyriproxyfen and JH III. JH III and pyriproxyfen did not cause workers to differentiate into presoldiers, but methoprene can induce formation of presoldiers. Colony response variability to methoprene was observed. The confirmation of the effects of JH/JHA on C. formosanus establishes the foundation for molecular studies of soldier differentiation in this species.     

Sensitivities to juvenile hormone and ecdysteroid in the diapause larvae of Omphisa fuscidentalis based on the hemolymph trehalose dynamics index

The final instar larva of the bamboo borer, Omphisa fuscidentalis, is in diapause for 9 months from September to the following June. Trehalose and ecdysteroid concentrations in hemolymph were measured through the larval diapause period and in the pupal stage. The ecdysteroid concentration remained low until November, followed by a gradual increase to about 30 ng/ml in May. The trehalose concentration remained at levels ranging between 40-50 mM until May, and decreased to an almost undetectable level after pupation. Since a juvenile hormone analogue (JHA), methoprene, is capable of terminating diapause by stimulating larval prothoracic glands, we examined its effects on ecdysteroid and trehalose concentrations in larvae in December and February. The hemolymph ecdysteroid increased more quickly in February than in December, indicating that the sensitivity of the prothoracic glands to JHA increased towards the end of diapause termination. Similarly, hemolymph trehalose in February decreased within a few days after JHA application, while in December the decrease occurred in the third week. Exogenous 20-hydroxyecdysone (20E) caused a decrease in trehalose concentration in a dose-dependent manner. The effective dose of 20E, however, did not change from January until April, implying that the sensitivity of tissue(s) to 20E may not change until the end of diapause. Taken together, our results suggest that the sensitivities of tissues to JH and 20E do not increase simultaneously with the progress of diapause development and that termination of larval diapause is not associated simply with the restoration of hormone deficiencies.     

Presence of three diapauses in a subtropical cockroach: control mechanisms and adaptive significance

Abstract.  Diapause is common among insects and is regarded as an adaptive response to periodic occurrence of adverse conditions. It occurs at a particular developmental stage, typically only once in a lifetime. However, little is known about the details of the control mechanism of life cycles with multiple diapauses in insects. In this study, a complex 2-year life cycle with three types of diapause is reported in a subtropical cockroach, Symploce japonica : a winter diapause in mid-nymphal instars, a summer diapause in later nymphal instars, and a reproductive diapause is reported in the adult stage. Nymphal development was extremely slow either at short days (winter diapause) or long days (summer diapause). Nymphs in summer diapause matured rapidly when transferred from long days to short days, indicating that seasonal changes in day-length are the pivotal factor in the control of this life cycle. It is proposed that the main significance of winter diapause in this subtropical species is to enable the nymphs to survive the mild winter successfully with reduced energy demand, and that of summer diapause is to delay adult emergence until late in the autumn for successful induction of the following adult diapause. Adults do not emerge until shortly before winter, yet the presence of diapause in the adult stage does not simply appear to be a response to cope with the winter conditions but, instead, ensures that reproduction will occur early the next year, before summer, because reproduction is greatly hampered at high temperature.     

Juvenile hormone-mediated termination of larval diapause in the bamboo borer, Omphisa fuscidentalis

Larvae of the bamboo borer, Omphisa fuscidentalis are in diapause for more than nine months (Singtripop, T., Wanichaneewa, S., Tsuzuki, S., Sakurai, S. 1999. Larval growth and diapause in a tropical moth, Omphisa fuscidentalis Hampson. Zool. Sci. 16, 725-733). To examine the endocrine mechanisms underlying this larval diapause, we assayed the responsiveness of the diapausing larvae to 20-hydroxyecdysone (20E) and a juvenile hormone analogue (JHA: S-methoprene). 20E injection caused the larvae to halt movement, followed by deposition of a pupal cuticle. Topical application of JHA induced pupation in a dose-dependent manner. JHA also induced pupation of the larvae whose brains were removed before JHA application. In those larvae, the prothoracic glands became active and competent to respond to brain extracts within seven days after JHA treatment, and the hemolymph ecdysteroid concentration began to increase 12 days after JHA application. These results indicate that JHA stimulates the prothoracic glands of diapausing Omphisa larvae, terminating larval diapause, in contrast with previous findings that JH inhibits the brain-prothoracic gland axis and thus maintains the larval diapause. Current results therefore suggest a novel regulatory mechanism for larval diapause in this species.     

Effects of juvenile hormone analogs and 20-hydroxyecdysone on diapause termination in eggs of Locusta migratoria and Oxya yezoensis

To understand the hormonal control of embryonic diapause, juvenile hormone analogs (JHAs), methoprene and hydroprene, and 20-hydroxyecdysone (20E) were applied onto diapause eggs of Locusta migratoria and Oxya yezoensis. These insects enter diapause at the mid-stage of embryogenesis prior to blastokinesis. Topical application of JHAs significantly facilitated diapause termination in both species but JHA-treated embryos underwent abnormal morphogenesis, pigmentation and sclerotization without dorsal closure. The Locusta eggs immersed in the 20E solution for 24h terminated diapause in a dose-dependent manner. We also investigated phosphorylation of extracellular signal-regulated kinase (ERK), a member of mitogen-activated protein kinase (MAPK), during diapause-terminating process of Locusta migratoria and found that ERK was activated either by cold exposure or JHA treatment. The possible involvement of the hormones and ERK in embryonic diapause and the possibility of ecdysteroids synthesis by prothoracic glands of diapause embryo were proposed.     

Overwintering success in adults of the Japanese common grass yellow Eurema mandarina

The evolution of reproductive diapause is controversial in males as compared to females that must overwinter to leave offspring, because late‐autumn males can obtain offspring by pre‐overwintering copulation. The Japanese common grass yellow Eurema mandarina is suitable to examine the evolution of male reproductive diapause, because direct comparisons are possible between males that do and do not exhibit reproductive diapause. Approximately one‐half of males are insensitive to diapause‐inducing conditions, and emerge as non‐diapause summer‐form. Most autumn‐form females mate with summer‐form males in late autumn. Females that have overwintered re‐mate with autumn‐form males before the onset of oviposition. Because last‐male‐precedence is general in sperm competition in Lepidoptera, it is unclear why half of males emerge as summer‐form in late autumn. A potential adaptive benefit for emerging as summer‐form is increased sperm overwintering success, if autumn‐form females have a higher overwintering success than autumn‐form males. In the present study, overwintering success was estimated for both sexes of autumn‐form adults by rearing under seminatural conditions and a mark–release–recapture technique. Both approaches estimated an overwintering success of approximately 5% for both sexes. The absence of difference in overwintering success between the sexes suggests that pre‐overwintering copulation does not increase sperm overwintering success. However, a considerably low overwintering success may explain, at least partly, the presence of summer‐form males in late autumn. The degree of overwintering success might be more important than the sexual differences of overwintering success in the evolution of male reproductive diapause.     

The influence of juvenile hormone analogue on soldier differentiation in the higher termite,Macrotermes michaelseni*

ABSTRACT. The influence of topically-applied or vapour-administered juvenile hormone analogue (ZR-515, methoprene (JHA)), on soldier differentiation in a higher termite species, Macrotermes michaelseni (Sjostedt) (Isoptera; Macro-termitinae) was examined. It was found that even male third larvae, which under natural conditions develop exclusively into major workers, could be induced by JHA to differentiate into presoldiers. Intermediate forms between workers and soldiers were produced from some of the treated individuals. Administered in vapour form, JHA induced a higher proportion of presoldiers and a lower proportion of intermediate forms than it did when administered topically. The rate of presoldier formation was found to be JHA-dose-dependent. Biometric analysis showed that male presoldiers have the largest head capsule size, but the smallest mandibular index. The possible mechanism of soldier induction is discussed.     

Cyanoprotein: Quantitative changes and synthesis in diapause and juvenile hormone analog treated bean bug,Riptortus clavatus

Quantitative changes of cyanoproteins (CPs) in diapause and juvenile hormone (JH) analog treated bean bug, Riptortus clavatus, were analyzed by rocket immunoelectrophoresis (RIE). In diapause-oriented nymphal females and males, CP-A (CP-1, 2 and 3) and CP-B (CP-4) increased and reached a maximum level just before nymphal-adult ecdysis, which was the same in non-diapause female and male nymphs. Both CP-A and B disappeared immediately after adult emergence. After this initial decline CP-4 appeared again in the hemolymph, followed after a few days by CP-1, 2 and 3. CP-A and B then increased slowly but constantly in both diapause female and male adults. Both diapause females and males at day 30 after adult emergence had large amounts of CP-A (CP-1, 2 and 3) and CP-B (CP-4). Treatment of diapause females (day 30) with methoprene induced only CP-1 synthesis and increased CP-A content about twice in both the whole body and the hemolymph, but did not effect on CP-B content. Methoprene treated females developed ovaries which accumulated yolk containing CPegg and vitellin (Vn). In diapause males treated with methoprene CP-A and B were not induced and decreased gradully in concentration, eventually disappearing completely, similar to post-diapause males (30 days after transferred to long day condition) in which CPs were not detected. These results show that methoprene treatment of diapause females and males induced the same dynamical situations of CP-A and B seen in non-diapause adults, i.e. only CP-A was induced in females and CPs disappeared in males. This suggests that CP synthesis is regulated by juvenile hormone.     

METHOPRENE INFLUENCES REPRODUCTION AND FLIGHT CAPACITY IN ADULTS OF THE RICE LEAF ROLLER,Cnaphalocrocis Medinalis (GUENỂE) (LEPIDOPTERA: PYRALIDAE)

Juvenile hormone (JH) influences many aspects of insect biology, including oogenesis‐flight syndrome tradeoffs between migration and reproduction. Drawing on studies of many migratory insects, we posed the hypothesis that JH influences migratory capacity and oogenesis in the rice leaf roller, Cnaphalocrocis medinalis. We treated adults moths (days 1, 2 and 3 postemergence) with the JH analog (JHA), methoprene, and then recorded the influences of JHA treatments on reproduction. JHA treatment on day 1 postemergence, but not on the other days, shortened the preoviposition period, although JHA did not influence total fecundity, oviposition period, or longevity. We infer day 1 postemergence is the JH‐sensitive stage to influence reproduction. Therefore, we treated moths on day 1 postemergence with JHA and recorded flight capacity, flight muscle mass, and triacylglycerol (TAG) accumulation. JHA treatments did not influence flight speed, but led to reductions in flight durations and flight distances. At day 3 posttreatment (PT), JHA‐treated females flew shorter times and less distance than the controls; JHA‐treated males, however, only flew shorter times than the controls. JHA treatments led to reductions in flight muscle mass in females at days 2–3 PT and reductions in TAG content in females at day 3 PT, but, these parameters were not influenced by JHA in males. These findings strongly support our hypothesis, from which we infer that JH is a major driver in C. medinalis oogenesis‐flight syndrome tradeoffs. Our data also reveal a JH‐sensitive stage in adulthood during which JH influences the oocyte‐flight syndrome in C. medinalis.