Resetting of the circannual rhythm of the varied carpet beetle Anthrenus verbasci by low‐temperature pulses

The varied carpet beetle Anthrenus verbasci L. has a circannual pupation rhythm and pupates in the spring in the wild. The change in photoperiod acts as a predominant zeitgeber for this rhythm. However, it is unclear whether the change in ambient temperature acts as a zeitgeber. The present study examines the effects of low‐temperature pulses on this circannual rhythm by exposing larvae kept under constant short‐day conditions (LD 12 : 12 h) at 20 °C to a lower temperature of 15, 10 or 5 °C for 8 or 12 weeks at various phases. Larval development and pupation are suppressed during exposure to low temperature, with this pupation being induced in sufficiently grown larvae within 2 months of a return to 20 °C. These results are attributed to the exogenous suppression and stimulation of pupation, rather than being related to the circannual rhythm (i.e. masking of the circannual rhythm by temperature). Furthermore, long‐term observations demonstrate the existence of phase‐dependent phase shifts of circannual rhythm as a result of low‐temperature pulses. Circannual phase response curves to low temperature are constructed on the basis of the phase shifts obtained. A low‐temperature pulse as a winter signal can reset the circannual rhythm of A. verbasci. It is probable that both temperature and photoperiod play a role in the entrainment of this circannual rhythm to a natural year.     

Endogenous timing mechanism controlling the circannual pupation rhythm of the varied carpet beetle Anthrenus verbasci

This paper describes the detailed characteristics of the circannual pupation rhythm in Anthrenus verbasci determined by laboratory experiments under various photoperiods and temperatures. The frequency distribution of larval duration showed a periodic pattern over 2-3 years and the period was 37-40 weeks under a constant short-day photoperiod (light:dark 12:12) at 20 degrees C. This rhythm showed temperature compensation to some extent under a short-day photoperiod between 17.5 degrees C and 27.5 degrees C. Under alternations of a long-day (light:dark 16:8) and a short-day photoperiod, pupation occurred 21-24.5 weeks after transfer from a long-day to a short-day photoperiod. Therefore, we concluded that the timing of pupation in A. verbasci is controlled by a circannual rhythm and its zeitgeber is a change in photoperiod. Furthermore, when larvae were transferred from a long-day to a short-day photoperiod at various ages, the larval duration after the photoperiodic transfer depended on the time of the transfer. This difference can be explained by phase-dependent phase shifts in the circannual rhythm.     

Evaluation of the Frequency Demultiplication Hypothesis of Circannual Pupation Rhythm in the Varied Carpet Beetle Anthrenus verbasci (Coleoptera: Dermestidae)

The present study was reported to examine whether the circannual pupation rhythm in Anthrenus verbasci is derived from a circadian rhythm through a process of frequency demultiplication. This frequency demultiplication hypothesis requires that the period of the circannual rhythm is proportional to the period of the entrained circadian rhythm. When larvae were reared under constant photoperiods of T = 20h, 22h, 24h and 26h at 20°C, the larval duration differed among the photoperiodic conditions, although there was no positive correlation between the period of T cycle and larval duration. The differences among photoperiods may have been caused by the duration of the photophase. Therefore, the results did not support the frequency demultiplication hypothesis.     

Evaluation of the Frequency Demultiplication Hypothesis of Circannual Pupation Rhythm in the Varied Carpet Beetle Anthrenus verbasci (Coleoptera: Dermestidae)

The present study was reported to examine whether the circannual pupation rhythm in Anthrenus verbasci is derived from a circadian rhythm through a process of frequency demultiplication. This frequency demultiplication hypothesis requires that the period of the circannual rhythm is proportional to the period of the entrained circadian rhythm. When larvae were reared under constant photoperiods of T = 20h, 22h, 24h and 26h at 20°C, the larval duration differed among the photoperiodic conditions, although there was no positive correlation between the period of T cycle and larval duration. The differences among photoperiods may have been caused by the duration of the photophase. Therefore, the results did not support the frequency demultiplication hypothesis.     

A circadian system is involved in photoperiodic entrainment of the circannual rhythm of Anthrenus verbasci

In the circannual pupation rhythm of the varied carpet beetle, Anthrenus verbasci, entrainment to annual cycles is achieved by phase resetting of the circannual oscillator in response to photoperiodic changes. In order to examine whether a circadian system is involved in expression of the periodic pattern and phase resetting of the circannual rhythm as photoperiodic responses, we exposed larvae to light-dark cycles with a short photophase followed by a variable scotophase (the Nanda-Hamner protocol). When the cycle length (T) was a multiple of 24 h, i.e., 24, 48, or 72 h, short-day effects were clearer than when T was far from a multiple of 24 h, i.e., 36 or 60 h. Exposure to light-dark cycles of T = 36 h had effects similar to exposure to long-day cycles of T = 24 h. The magnitude of phase shifts depended on the duration and the phase of exposure to the cycles of T = 36 or 60 h. It was therefore concluded that a circadian system is involved in photoperiodic time measurement for phase resetting of the circannual oscillator of A. verbasci.     

A phase response curve for circannual rhythm in the varied carpet beetle <Emphasis Type="Italic">Anthrenus verbasci</Emphasis>

We know that entrainment, a stable phase relationship with an environmental cycle, must be established for a biological clock to function properly. Phase response curves (PRCs), which are plots of phase shifts that result as a function of the phase of a stimulus, have been created to examine the mode of entrainment. In circadian rhythms, single-light pulse PRCs have been obtained by giving a light pulse to various phases of a free-running rhythm under continuous darkness. This successfully explains the entrainment to light-dark cycles. Some organisms show circannual rhythms. In some of these, changes in photoperiod entrain the circannual rhythms. However, no single-pulse PRCs have been created. Here we show the PRC to a long-day pulse superimposed for 4 weeks over constant short days in the circannual pupation rhythm in the varied carpet beetle Anthrenus verbasci. Because the shape of that PRC closely resembles that of the Type 0 PRC with large phase shifts in circadian rhythms, we suggest that an oscillator having a common feature in the phase response with the circadian clock, produces a circannual rhythm.     

Responsiveness to photoperiodic changes in the circannual rhythm of the varied carpet beetle, <Emphasis Type="Italic">Anthrenus verbasci</Emphasis>

The circannual pupation rhythm of Anthrenus verbasci is entrained to an environmental cycle by changes in photoperiod. Exposure of larvae reared under short-day conditions to 4 weeks of long days can induce phase-dependent phase shifts. In the present study, we examined the range of photoperiodic changes effective for phase shifts at 20°C. For larvae under light/dark (LD) 12:12 conditions, 4-week exposure to LD 14:10 caused a clear phase delay, as great an extent as that brought about by exposure to LD 15:9 and LD 16:8. In contrast, the delay brought about by exposure to LD 13:9 was slight. For larvae under LD 10:14, exposure to LD 14:10 and LD 16:8 for 4 weeks induced a phase delay, but exposure to LD 12:12 did not. These results indicate that a clear phase delay is induced when the photoperiodic change exceeds a critical value in the photophase between 13 and 14 h, regardless of the amplitude of the change. Although phase advances were smaller than phase delays, they depended on the amplitude of photoperiodic changes rather than the absolute photophase duration in contrast to the case of the phase delay.     

Phase resetting and phase singularity of an insect circannual oscillator

In circadian rhythms, the shape of the phase response curves (PRCs) depends on the strength of the resetting stimulus. Weak stimuli produce Type 1 PRCs with small phase shifts and a continuous transition between phase delays and advances, whereas strong stimuli produce Type 0 PRCs with large phase shifts and a distinct break point at the transition between delays and advances. A stimulus of an intermediate strength applied close to the break point in a Type 0 PRC sometimes produces arrhythmicity. A PRC for the circannual rhythm was obtained in pupation of the varied carpet beetle, Anthrenus verbasci, by superimposing a 4-week long-day pulse (a series of long days for 4 weeks) over constant short days. The shape of this PRC closely resembles that of the Type 0 PRC. The present study shows that the PRC to 2-week long-day pulses was Type 1, and that a 4-week long-day pulse administered close to the PRC’s break point induced arrhythmicity in pupation. It is, therefore, suggested that circadian and circannual oscillators share the same mode in phase resetting to the stimuli.     

Plasticity in the timing of a major life‐history transition and resulting changes in the age structure of populations of the salamander Hynobius retardatus

Variation in age and size at life‐history transitions is a reflection of the diversifying influence of biotic or abiotic environmental change. Examples abound, but it is not well understood how such environmental changes influence the age structure of a population. I experimentally investigated the effects of water temperature and food type on age and body size at metamorphosis in larvae of the salamander Hynobius retardatus. In individuals grown at a cold temperature (15 °C) or given Chironomidae as prey, the time to metamorphosis was significantly prolonged, and body size at metamorphosis was significantly enlarged, compared with individuals grown at a warmer temperature (20 °C) or fed larvae. I also examined whether larval density (a possible indicator of cannibalism in natural habitats) generated variation in the age structure of natural populations in Hokkaido, Japan, where the climate is subarctic. Natural ponds in Hokkaido may contain larvae that have overwintered for 1 or 2 years, as well as larvae of the current year, and I found that the number of age classes was related to larval density. Although cool water temperatures prolong the larval period and induce later metamorphosis, in natural ponds diet‐based enhancement of development translated into a shorter larval duration and earlier metamorphosis. Geographic variation in the frequency of cannibalism resulted in population differences in metamorphic timing in H. retardatus larvae. It is important to understand how environmental effects are ultimately transduced through individual organisms into population‐level phenomena, with the population response arising as the summation of individual responses. Without a thorough comprehension of the mechanisms through which population and individual responses to environmental conditions are mediated, we cannot interpret the relationship between population‐level and individual‐level phenomena. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 102 , 100–114.     

Nutrient balance in medfly, Ceratitis capitata, larval diets affects the ability of the developing insect to incorporate lipid and protein reserves

The Mediterranean fruit fly [Ceratitis capitata Wiedemann (Diptera: Tephritidae)], or medfly, is mass produced in many facilities throughout the world to supply sterile flies for sterile insect technique programs. Production of sterile males requires large amounts of larval and adult diets. Larval diets comprise the largest economic burdens in the mass production of sterile flies, and are one of the main areas where production costs could be reduced without affecting quality and efficacy. The present study investigated the effect of manipulating diet constituents on larval development and performance. Medfly larvae were reared on diets differing in the proportions of brewer's yeast and sucrose. We studied the effect of such diets on the ability of pupating larvae to accumulate protein and lipids, and on other developmental indicators. Except for diets with a very low proportion of brewer's yeast (e.g., 4%), pupation and adult emergence rates were in general high and satisfactory. The ability of pupating larvae to accumulate lipid reserves and proteins was significantly affected by the sucrose and yeast in the diet, and by the proportion of protein to carbohydrates (P/C). In contrast to previous nutritional studies conducted with other insects, low P/C in medfly larval diets (with excess dietary carbohydrates) resulted in pupating medfly larvae having a relatively reduced load of lipids; medfly larvae protein contents in these diets were, as expected, relatively low. Similarly, high P/C ratios in the diet produced larvae with high protein and lipid contents. Differences with other insects may be due to differential post‐ingestion regulation where a high dietary carbohydrate diet reduces the lipogenic activity of the larvae, and induces a shift from lipid to glucose oxidation. Larvae reared on low P/C diets spent more time foraging in the diet than larvae maintained on a high P/C diet, suggesting a compensatory mechanism to complement nutrient intake. The results suggest that the content of brewer's yeast, the most expensive diet component, could be fine‐tuned without apparently affecting fly quality.     

Effect of some phytopathogenic fungi and their metabolites on growth of Heliothis virescens (F.) and its host plants

Eleven fungal isolates and their secondary metabolites incorporated into artificial diet were tested for oral toxicity to the tobacco budworm (TBW) by examining larval weight, efficiency of conversion of ingested food to body tissue (ECI), pupal weight, days to pupation, and mortality. Two isolates of Altemaria alternata, two isolates of Fusarium moniliforme, three isolates of F. oxysporum and an isolated of F. solani reduced larval weight 90–99% after 7 days and inhibited pupation. ECI was reduced 34–96% in control groups. One isolate of A. alternata reduced pupal weight by 67% and increased the time to pupation three‐fold. One isolate of Cladosporium cladosporioides reduced larval weights by 56% and pupal weights by 7%. In a preference test of these isolates incorporated at a 1:4 ratio into artificial diet, 48% of the larvae were found on diet cubes containing autoclaved rice, 19% on standard diet, 10% on C. cladosporioides, 6–9% on F. solani, 8% on A. alternata and 3% on F. moniliforme. The fusarial toxins, T‐2 and diacetoxyscirpenol (DAS), were the most active compounds against TBW larvae among the 10 microbial toxins tested. T‐2 toxin reduced larval weight by 87%, reduced ECI by 62%, reduced pupal weight by 33% and delayed pupation by 1 week DAS caused similar but less severe effects than T‐2 toxin. AAL‐toxin inhibited larval growth and reduced pupal weights by 20% and 13%, respectively. A. alternata, F. moniliforme and F. solani were also phytotoxic to alfalfa (Medicago sativa,), crimson clover (Trifolium incarnatum) and wild geranium (Geranium dissectum,), which are early season plant hosts of TBW.     

How do food quality and larval crowding affect performance of the autumnal moth,Epirrita autumnata?

Outbreak densities of autumnal moth, Epirrita autumnata (Borkhausen) (Lepidoptera: Geometridae), lead to high larval crowding. Phenotypic responses of E. autumnata to larval crowding and to food quality were studied by measuring growth and consumption as well as pupal weight and fecundity. Crowding may trigger increased consumption and faster development to avoid impending food shortage on good quality food. This is suggested by the result that on a good‐quality diet, the growth of crowded larvae was better than that of solitary larvae, though they did not consume more food than solitary larvae. Crowded larvae also completed the last instar earlier than solitary larvae. The fecundity of crowded autumnal moths was not lower than the fecundity of solitarily grown autumnal moths. This may provide conditions for extra rapid population build‐up of E. autumnata. During the population increase phase the crowding effect may facilitate larval performance; however, at peak density the crowding starts to have negative effects on the performance of larvae. On a poor‐quality diet, the performance of crowded and solitary larvae did not differ. The growth of larvae was better on a good‐quality diet than on a poor‐quality diet, due to higher efficiency in food utilization. Larvae feeding on low‐quality diet did not prolong their development time, but pupated at smaller size; this resulted in lower fecundity. A decrease in food quality can be seen as a cue of oncoming food shortage and resource depletion; it may be advantageous to pupate at a smaller size and ensure survival till reproduction, rather than risk prolonging development to achieve larger size and higher fecundity.     

Food availability controls the onset of metamorphosis in the dung beetle Onthophagus taurus (Coleoptera: Scarabaeidae)

In nature, larvae of the dung beetle Onthophagus taurus (Schreber 1759) are confronted with significant variation in the availability of food without the option of locating new resources. Here we explore how variation in feeding conditions during the final larval instar affects larval growth and the timing of pupation. We found that larvae respond to food deprivation with a reduction in the length of the instar and premature pupation, leading to the early eclosion of a small adult. To achieve pupation, larvae required access to food for at least the first 5 days of the final instar (= 30% of mean third‐instar duration in control individuals), and had to exceed a weight of 0.08 g (= 58% of mean peak weight in control individuals). Larvae that were allowed to feed longer exhibited higher pupation success, but increased larval weight at the time of food deprivation did not result in increased pupation success except for larvae weighing > 0.14 g. Larvae responded to food deprivation by initiating and undergoing the same sequence of developmental events, requiring the same amount of time, as ad libitum‐fed larvae once those had reached their natural peak weight. Our results reveal a striking degree of flexibility in the dynamics and timing of larval development in O. taurus. They also suggest that premature exhaustion of a larva's food supply can serve as a cue for the initiation of metamorphosis. Premature metamorphosis in response to food deprivation has been documented in amphibians, but this is, to the best of our knowledge, the first time such a behaviour has been documented for a holometabolous insect. We discuss our findings in the context of the natural history and behavioural ecology of onthophagine beetles.     

Xanthophylls are key constituents of semi‐synthetic diets for continuous breeding of Pieris brassicae without food plant

Semi‐synthetic or fully artificial diets are well established in culturing laboratory insects on a large scale. In Pieris brassicae L. (Lepidoptera: Pieridae), a published semi‐synthetic diet supplemented with sinigrin, a chemical label of cruciferous plants, is well accepted by the larvae but the adults fail to produce offspring; continuous breeding, therefore, requires the addition of cabbage powder to the diet. We set out to identify essential substances to eliminate the need for cabbage in our long‐term mass culture. The studies revealed that the dietary difference, which becomes most evident at the adult stage only, is because of a severe lack of carotenoids in the cabbage‐free version. This was uncovered in larvae, fed cabbage‐supplemented diet, that respond to blue light of sufficient intensity by high cuticle melanization on pupation; larvae fed the carotenoid‐deficient sinigrin‐supplemented diet fail to respond in this way. Males were found to be more sensitive than females to blue light in this respect. The essential substance mediating this photo response was found to be lutein and/or zeaxanthin, two abundant xanthophylls of green leaves. β‐Carotene was only marginally active. These xanthophylls are known to serve as precursors to the synthesis of 3‐hydroxyretinal, the exclusive visual chromophore of lepidopteran insects; retinal, derived from β‐carotene, is not (substantially) used by these insects. In conclusion, the absence of flying and mating activity in the adults from the sinigrin diet may indicate visual deficiency owing to a lack of a specific structural class of carotenoids in the larval food. Our results enabled us to formulate a semi‐synthetic diet that allows continuous mass rearing of P. brassicae without cabbage at any stage including oviposition. In addition to the key constituents sinigrin and xanthophylls, this superior diet includes tryptophan, as methyl ester, and α‐tocopherol.     

Contrary food requirements of the larvae of two Curtonotus (Coleoptera: Carabidae: Amara) species

The larvae of carabids that are granivorous as adults can be granivorous, omnivorous or carnivorous. The differences in larval food preferences of Amara aulica and Amara convexiuscula, two closely related species of the subgenus Curtonotus, were studied. Survival and duration of development of non‐diapausing first and second instar larvae were compared. The third instar larvae were not studied because they go into diapause prior to pupation. The larvae were fed diets consisting of insect larvae (Tenebrio molitor), seeds of Artemisia vulgaris, Tripleurospermum inodorum, Urtica dioica or Cirsium arvense and a mixed diet consisting of insect larvae and seed. Larvae of A. aulica required seeds for successful development, whereas those of A. convexiuscula developed on both an insect diet and seeds of Artemisia. The difference in larval food requirements facilitates the coexistence of these closely related species, which frequently share the same habitat.     

Food‐availability dependent premature metamorphosis in the bean blister beetle Epicauta gorhami (Coleoptera: Meloidae), a hypermetamorphic insect that feeds on grasshopper eggs in the larval stage

Larvae of the bean blister beetle, Epicauta gorhami, feed on only grasshopper eggs and undergo hypermetamorphosis with pseudopupal diapause in the fifth instar. Whether E. gorhami larvae enter pseudopupal diapause or pupate directly from the fourth instar is controlled by temperature and photoperiod. In nature, larvae are confronted with a significant variation in the availability of food, suggesting the possibility that feeding conditions may also affect the diapause incidence. Here, we addressed this issue by changing the feeding conditions in the fourth instar under conditions of 16 h light : 8 h dark (LD 16 : 8) at 25°C. Food deprivation reduced the length of instar and increased the tendency to pupate, leading to the early eclosion of a small adult. Even non‐feeding fourth‐instar larvae pupated. Regardless of the timing of food deprivation, the post‐feeding larval period was constant and equivalent to that of ad libitum‐fed larvae, suggesting that premature exhaustion of the food supply triggers the initiation of pupation. In agreement with these results, when larvae were fed on intact grasshopper egg pods of various sizes from four species, those that fed on smaller egg pods had a decreased tendency to pseudopupate (i.e., to enter diapause). Food‐deprived larvae showed a clearer photoperiodic response and had a shorter critical day‐length. Thus, in E. gorhami, feeding conditions do not affect pupation success, but do affect the tendency to pupate or pseudopupate. This is the first report of the occurrence of premature pupation in carnivorous insects. We discuss our findings in the context of the natural history and behavioral ecology of E. gorhami.     

Bacterial microbiota assemblage in Aedes albopictus mosquitoes and its impacts on larval development

Interactions between bacterial microbiota and mosquitoes play an important role in mosquitoes’ capacity to transmit pathogens. However, microbiota assemblages within mosquitoes and the impact of microbiota in environments on mosquito development and survival remain unclear. This study examined microbiota assemblages and the effects of aquatic environment microbiota on the larval development of the Aedes albopictus mosquito, an important dengue virus vector. Life table studies have found that reducing bacterial load in natural aquatic habitats through water filtering and treatment with antibiotics significantly reduced the larva‐to‐adult emergence rate. This finding was consistent in two types of larval habitats examined—discarded tires and flowerpots, suggesting that bacteria play a crucial role in larval development. Pyrosequencing of the bacterial 16S rRNA gene was used to determine the diversity of bacterial communities in larval habitats and the resulting numbers of mosquitoes under both laboratory and field conditions. The microbiota profiling identified common shared bacteria among samples from different years; further studies are needed to determine whether these bacteria represent a core microbiota. The highest microbiota diversity was found in aquatic habitats, followed by mosquito larvae, and the lowest in adult mosquitoes. Mosquito larvae ingested their bacterial microbiota and nutrients from aquatic habitats of high microbiota diversity. Taken together, the results support the observation that Ae. albopictus larvae are able to utilize diverse bacteria from aquatic habitats and that live bacteria from aquatic habitats play an important role in larval mosquito development and survival. These findings provide new insights into bacteria's role in mosquito larval ecology.     

Effect of larval density on development and diapause of Sesamia nonagrioides (Lef.) (Lep., Noctuidae) under laboratory conditions

The effect of larval density on development and diapause of Sesamia nonagrioides (Lef.) was examined. Larvae were grown on an artificial diet at densities of five, 10, 20, 30 and 40 larvae per box, under two environmental (diapause-including and diapause-averting) conditions. High larval densities resulted in increased mortality under both experimental conditions tested. Although crowded larvae showed a higher pupation rate than those reared under low densities, small differences occurred between mean larval developmental time at different densities under developmental condition. In spite of this, crowding proved effective on the intensity of diapause of this species and the duration of diapause was shorter for crowded larvae. Pupal weights of both sexes were significantly reduced at high densities while there were significant differences between pupal weights formed by larvae reared under diapause conditions and those formed by larvae reared under nondiapause conditions at all density treatments. The pupal period was significantly longer under diapause conditions than that under development conditions but density was not found to play a role on the time of the emergence of the adults produced by larvae reared under developmental or diapause conditions.     

Effects of temperature on the development and circannual control of pupation in the carpenter moth, <Emphasis Type="Italic">Cossus insularis</Emphasis> (Lepidoptera: Cossidae), reared on an artificial diet

The effects of temperature on larval development and the timing of pupation in the carpenter moth, Cossus insularis (Staudinger) (Lepidoptera: Cossidae) were examined by artificial rearing under different temperatures and the same photoperiod (15L:9D). Although C. insularis pupated and emerged at 20, 25, and 30 °C, the pupation rate was lower at 20 °C than at 25 and 30 °C. These results suggest that the optimum temperature range for preadult development is 25–30 °C. The duration of larval development was about 260 days for the first pupation group at 25 and 30 °C, and at least 600 days at 20 °C. Therefore, the C. insularis generation time was 2 years or more, as the total effective temperature for development from hatching to the pupal stage was unlikely to be reached within 1 year in Tokushima Prefecture. The second group pupated at 25 °C, about 200 days after the first group. This periodicity of pupation was likely due to the free-running period of the circannual rhythm. Furthermore, although only the first group pupated at 30 °C, the peak was almost synchronous with the first group at 25 °C. These results indicate that the timing of the first pupation group in C. insularis is temperature compensated. Therefore we propose that the presence of an endogenous rhythm during the development of C. insularis is evidence for a circannual rhythm related to the timing of pupation.