Brain-independent development in the moth Sesamia nonagrioides

The caterpillars of Sesamia nonagrioides developing under long-day (LD) photoperiod pupate in the 5th or 6th instar whereas under short day (SD) conditions they enter diapause and undergo several extra larval molts. The diapause is terminated within 1-3 instars upon transfer of SD larvae to the LD conditions. Brain removal from the 6th instar larvae promotes pupation followed by imaginal development; however, one third of the SD larvae and 12% of the LD larvae debrained at the start of the instar first undergo 1-2 larval molts. The incidence of larval molts is enhanced by the brain implants. Exclusively pupal molts occur in the LD larvae debrained late in the 6th instar. Decapitation elicits pupation in both LD and SD larvae, except for some of the 4th and 5th and rarely 6th instar that are induced to a fast larval molt. The pupation of decapitated larvae is reverted to a larval molt by application of a juvenile hormone (JH) agonist. No molts occur in abdomens isolated from the head and thorax prior to the wandering stage. Abdomens isolated later undergo a larval (SD insects) or a pupal (LD insects) molt. Taken together the data reveal that in S. nonagrioides (1) several larval molts followed by a pupal and imaginal molt can occur without brain; (2) an unknown head factor outside the brain is needed for the pupal-adult molt; (3) brain exerts both stimulatory and inhibitory effect on the corpora allata (CA); (4) larval molts induced in CA absence suggest considerable JH persistence.     

Control of larval-pupal-adult molt in the moth Sesamia nonagrioides by juvenile hormone and ecdysteroids

Sesamia nonagrioides (Lepidoptera: Noctuidae) larvae reared under long day (LD; 16L:8D) conditions pupate after 5 or 6 larval instars, whereas under short day (SD; 12L:12D) conditions they undergo up to 12 additional molts before pupating. This extended period of repeated molting is maintained by high levels of juvenile hormone (JH). Previous work demonstrated that both LD and SD larvae decapitated in the 6th instar pupate but further development is halted. By contrast, about one-third of SD larvae from which only the brain has been removed, undergo first a larval molt, then pupate and subsequently developed to the adult stage. Debrained LD larvae molt to larvae exceptionally but regularly pupate and produce adults. Implanted brains may induce several larval molts in debrained recipient larvae irrespectively of the photoperiodic conditions. The results of present work demonstrate that the prothoracic glands (PGs) and the corpora allata (CA) of debrained larvae continue to produce ecdysteroids and JHs, respectively. PGs are active also in the decapitated larvae that lack JH, consistent with the paradigm that CA, which are absent in the decapitated larvae, are the only source of this hormone. Completion of the pupal-adult transformation in both LD and SD debrained insects demonstrates that brain is not crucial for the development of S. nonagrioides but is required for diapause maintenance. Application of JH to headless pupae induces molting, presumably by activating their PGs. It is likely that JH plays this role also in the induction of pupal-adult transformation in debrained insects. Application of the ecdysteroid agonist RH 2485 (methoxyfenozide) to headless pupae also elicits molting: newly secreted cuticle is in some cases thin and indifferent, in other cases it bears distinct pupal or adult features.     

Azadirachtin potentiates the action of ecdysteroid agonist RH-2485 in Spodoptera littoralis

Edysteroid agonist RH-2485 induces an immediate and fatal molt in Spodoptera littoralis when added to the diet of the 2nd and 4th instar larvae at 1 ppm, and to that of the 6th instar larvae at 0.001 ppm concentration. Ten times lower doses fed to the larvae continuously allow an apparently normal larval development that is terminated by a supernumerary larval molt. The other effects of RH-2485 include death during metamorphosis and impaired fertility of emerged adults. The number of progeny is reduced even with low RH-2485 doses that do not interfere with moltings; e.g., insects fed 0.0001 ppm since the 2nd, 4th, and 6th instar produce 72%, 62%, and 22%, respectively, less progeny than the controls. Feeding larvae with 10-1000 ppm Suneem oil (containing about 0.1-10 ppm azadirachtin) causes, in a stage- and dose-dependent manner, a cessation or reduction of feeding, delay of molts, death of larvae and pupae, and sterility of emerged adults; with 10 ppm Suneem oil, the number of progeny is reduced by 20-32%. Presence of Suneem oil in the diet does not influence the potential of RH-2485 to induce a prompt molt, but it increases ten times the potency to elicit a supernumerary larval molt. Certain combinations of RH-2485 with Suneem oil provoke up to 3 extra larval molts. Lethal developmental derangements and sterility are more frequent, and the response of larvae of different age is more uniform, when Suneem oil and RH-2485 are combined than when each of these agents is administered alone.     

Supply-Side Constraints Are Insufficient to Explain the Ontogenetic Scaling of Metabolic Rate in the Tobacco Hornworm,Manduca sexta

Explanations for the hypoallometric scaling of metabolic rate through ontogeny generally fall into two categories: supply-side constraints on delivery of oxygen, or decreased mass-specific intrinsic demand for oxygen. In many animals, supply and demand increase together as the body grows, thus making it impossible to tease apart the relative contributions of changing supply and demand to the observed scaling of metabolic rate. In larval insects, the large components of the tracheal system are set in size at each molt, but then remain constant in size until the next molt. Larvae of Manduca sexta increase up to ten-fold in mass between molts, leading to increased oxygen need without a concomitant increase in supply. At the molt, the tracheal system is shed and replaced with a new, larger one. Due to this discontinuous growth of the tracheal system, insect larvae present an ideal system in which to examine the relative contributions of supply and demand of oxygen to the ontogenetic scaling of metabolic rate. We observed that the metabolic rate at the beginning of successive instars scales hypoallometrically. This decrease in specific intrinsic demand could be due to a decrease in the proportion of highly metabolically active tissues (the midgut) or to a decrease in mitochondrial activity in individual cells. We found that decreased intrinsic demand, mediated by a decrease in the proportion of highly metabolically active tissues in the fifth instar, along with a decrease in the specific mitochondrial activity, contribute to the hypoallometric scaling of metabolic rate.     

Developmental variation in the forest tent caterpillar: life history consequences of a threshold size for pupation

In insects, size and age at adult emergence depend on larval growth that occurs in discrete steps or instars. Understanding the mechanisms controlling stepwise larval growth and the onset of metamorphosis is essential to the study of insect life history. We examined the patterns of growth of forest tent caterpillars Malacosoma disstria to quantify variation in the number of instars that larvae undergo before pupation, to identify the mechanisms underlying variation in larval development, and to evaluate the life history consequences of this variation. All caterpillars were reared under the same conditions; at each molt, the date, the head capsule width and the mass of the freshly molted insect were recorded. Logistic regression analysis showed that a threshold size (measured either as mass or head capsule width) must be reached at the beginning of a stadium for pupation to occur at the next molt. This threshold size was higher for females than for males, and as a result, females attained a higher pupal mass than males. To achieve this larger size, females often required more instars than males, despite a higher growth ratio (size increase within an instar). Within each sex, slow growing individuals exhibited more larval instars and longer larval development time, but attained the same pupal mass as faster growing individuals. The combination of a threshold size for pupation, discrete growth steps and variation in the number of these steps can thus complicate relationships between growth rate, pupal mass and larval development time. In our study, growth ratio and number of instars were correlated with development time but not with pupal mass, and no relationship was observed between development time and pupal mass. These findings imply that, in species with variable instar number, one cannot extrapolate overall larval growth from growth during a single instar. Given the constraints of discrete larval growth, variation in instar number provides greater flexibility for insects to compensate for poor growing conditions. In this case, inferior larval growth conditions don't necessarily lead to smaller adult size.     

Juvenile hormone-dependent vitellogenin synthesis in Locusta migratoria fat body: Inducibility related to sex and stage

Juvenile hormone (JH)-dependent vitellogenin (Vg) synthesis in the fat body of Locusta migratoria is normally limited to sexually mature adult females. As a step toward examining the basis of this limitation, we have tested female and male locusts in a series of stages after the third larval molt for inducibility of Vg synthesis by the synthetic JH analog, methoprene. We find that in the fourth and fifth larval instars fat body of both sexes can be induced to produce Vg, but in the adult stage females respond strongly while no more than trace amounts can be induced in males. Quantitative assays show relative responsiveness in the order: adult female > fifth instar female > fifth instar male ? adult male. During the fifth instar of both sexes, maximal vitellogenic response was obtained in midinstar. After the larval-adult ecdysis, female fat body was unresponsive during the first 4 days, then responsiveness increased and by Day 8 after ecdysis fat bodies were fully as competent to produce Vg as at Day 14, the usual maximum of the first vitellogenic cycle due to endogenous JH. Larval and adult female fat bodies implanted into male larvae are competent for Vg synthesis after metamorphosis, so that the differences between adult male and female cannot be imposed by the male milieu intérieur during the larval-adult molt. In male and female precocious adults, produced by treatment of fourth instars with precocene, fat body responded to methoprene as in normal adults. We conclude that factors intrinsic to the fat body cells, determined early in development, are responsible for differential gene programing in males and females, which is partially expressed by the fifth instar but fully manifest only after a molt in the absence of JH.     

Imaginal discs regulate developmental timing in Drosophila melanogaster

The regulation of body size in animals involves mechanisms that terminate growth. In holometabolous insects growth ends at the onset of metamorphosis and is contingent on their reaching a critical size in the final larval instar. Despite the importance of critical size in regulating final body size, the developmental mechanisms regulating critical size are poorly understood. Here we demonstrate that the developing adult organs, called imaginal discs, are a regulator of critical size in larval Drosophila. We show that damage to, or slow growth of, the imaginal discs is sufficient to retard metamorphosis both by increasing critical size and extending the period between attainment of critical size and metamorphosis. Nevertheless, larvae with damaged and slow growing discs metamorphose at the same size as wild-type larvae. In contrast, complete removal of all imaginal tissue has no effect on critical size. These data indicate that both attainment of critical size and the timely onset of metamorphosis are regulated by the imaginal discs in Drosophila, and suggest that the termination of growth is coordinated among growing tissues to ensure that all organs attain a characteristic final size.     

Regulation of body size during larval development in the German cockroach, Blattella germanica

It may be advantageous for insects to attain a certain standard size when they become adult. Recent studies have demonstrated that in some species of insects, a threshold size must be reached in the last instar in order that the adult will attain the standard size. It has been revealed also, however, that there may be another type of regulation of body size during larval development in the German cockroach. Head width and other characteristics were measured in every larval instar up to the adult. Body size is distributed clearly into a small group and a large group in the 5th instar, and even in the 4th instar. This suggests that a larva has already determined the number of following moults by the late 3rd instar. Adult size is almost the same in 5- and 6-instar types. Among individuals which require the same number of instars to reach the adult stage, the coefficient of variation in body size increases until the 3rd instar then decreases to the adult stage. Smaller individuals in a particular instar after the 3rd tend to grow more than larger ones during that instar. These results suggest that a larva regulates its body size after the 3rd instar in order to attain the ‘norm’ for adult size.     

Relationship between an increase of juvenile hormone titer in early instars and the induction of diapause in fully grown larvae of Sesamia nonagrioides

The larvae of Sesamia nonagrioides (Lepidoptera: Noctuidae) grown at 25 degrees C and long photoperiod (16:8h light:dark) pupate in the 5th or 6th (mostly) larval instar, while the larvae reared under a short photoperiod (12:12h) enter diapause during which they consume some food and undergo up to 12 (usually 3-4) stationary larval molts. Diapause programming includes an increase of juvenile hormone (JH) titer in the hemolymph from about 20 to 50 nM in the 4th and 5th instar larvae (titer in earlier instars was not measured). JH I, II, and III are present in approximate ratio 1-2:10:1. The JH titer drops to zero before pupation but remains around 20 nM during diapause. Perfect extra larval molts associated with a body weight increase can be induced in the non-diapausing larvae with a JH analogue (JHA). The weight rise is due to accumulation of reserves and not to a general body growth. The timing of extra molts is similar to the molting pattern of the diapausing larvae only when JHA is present since early larval instars. In the diapausing larvae, JHA application affects neither molting periodicity nor the body weight. It is concluded that (1) Increased JH titer in early larval instars is a part of diapause programming; (2) The extension of larval stage in the diapausing larvae, but not the timing pattern of extra molts, is due to continuously high JH titer; (3) The diapause program includes low food intake, maintenance of a certain body weight, and periodic larval molts.     

A Novel Technique for Identifying the Instar of Field-Collected Insect Larvae

Many field studies of insects have focused on the adult stage alone, likely because immature stages are unknown in most insect species. Molecular species identification (e.g., DNA barcoding) has helped ascertain the immature stages of many insects, but larval developmental stages (instars) cannot be identified. The identification of the growth stages of collected individuals is indispensable from both ecological and taxonomic perspectives. Using a larval–adult body size relationship across species, I present a novel technique for identifying the instar of field-collected insect larvae that are identified by molecular species identification technique. This method is based on the assumption that classification functions derived from discriminant analyses, performed with larval instar as a response variable and adult and larval body sizes as explanatory variables, can be used to determine the instar of a given larval specimen that was not included in the original data set, even at the species level. This size relationship has been demonstrated in larval instars for many insects (Dyar’s rule), but no attempt has been made to include the adult stage. Analysis of a test data set derived from the beetle family Carabidae (Coleoptera) showed that classification functions obtained from data sets derived from related species had a correct classification rate of 81–100%. Given that no reliable method has been established to identify the instar of field-collected insect larvae, these values may have sufficient accuracy as an analytical method for field-collected samples. The chief advantage of this technique is that the instar can be identified even when only one specimen is available per species if classification functions are determined for groups to which the focal species belongs. Similar classification functions should be created for other insect groups. By using those functions together with molecular species identification, future studies could include larval stages as well as adults.     

Distinguishing between anticipatory and responsive plasticity in a seasonally polyphenic butterfly

Seasonal generations of short-lived organisms often differ in their morphological, behavioural and life history traits, including body size. These differences may be either due to immediate effects of seasonally variable environment on organisms (responsive plasticity) or rely on presumably adaptive responses of organisms to cues signalizing forthcoming seasonal changes (anticipatory plasticity). When directly developing individuals of insects are larger than their overwintering conspecifics, the between-generation differences are typically ascribed to responsive plasticity in larval growth. We tested this hypothesis using the papilionid butterly Iphiclides podalirius as a model species. In laboratory experiments, we demonstrated that seasonal differences in food quality could not explain the observed size difference. Similarly, the size differences are not likely to be explained by the immediate effects of ambient temperature and photoperiod on larval growth. The qualitative pattern of natural size differences between the directly developing and diapausing butterflies could be reproduced in the laboratory as a response to photoperiod, indicating anticipatory character of the response. Directly developing and diapausing individuals followed an identical growth trajectory until the end of the last larval instar, with size differences appearing just a few days before pupation. Taken together, various lines of evidence suggest that between-generation size differences in I. podalirius are not caused by immediate effects of environmental factors on larval growth. Instead, these differences rather represent anticipatory plasticity and are thus likely to have an adaptive explanation. It remains currently unclear, whether the seasonal differences in adult size per se are adaptive, or if they constitute co-product of processes related to the diapause. Our study shows that it may be feasible to distinguish between different types of plasticity on the basis of empirical data even if fitness cannot be directly measured, and contributes to the emerging view about the predominantly adaptive nature of seasonal polyphenisms in insects.     

Body size regulation in copepod crustaceans

Body size affects survival probabilities, reproductive output and individual fitness in many organisms. In freshwater zooplankton, traits ranging from demographic rates to community composition depend on body size, and predation is most often identified as the selection pressure determining body size. We examined the extent to which stage-specific growth trajectories and body sizes of copepod crustaceans are constrained, independent of selection. We used exuviae shed at each molt to quantify the relationship between size at molting and growth during the subsequent instar for two common, herbivorous calanoid copepods, Boeckella triarticulata and Diaptomus leptopus. Individuals of both species were raised under diets of different food quality or quantity, and at different temperatures. Size at molting varied little among individuals of both species, as a consequence of a persistent negative relationship between size at molting and subsequent (absolute) growth increment. Individuals that were small when they molted grew more during the subsequent instar than individuals that were large. This relationship was statistically significant for nearly all instars of both species raised in different food or temperature conditions, and not affected by food quality, food quantity or temperature. Our results indicate that body size is constrained or regulated over much of the copepod life cycle, independent of the effects of environmental conditions (food, temperature, predation). Received: 26 December 1998 / Accepted: 13 September 1999     

Isolation and developmental expression of two nuclear receptors, MHR4 and betaFTZ-F1, in the tobacco hornworm, Manduca sexta

The cDNAs for two members of the nuclear receptor superfamily were isolated from the tobacco hornworm, Manduca sexta. The deduced amino acid sequence of MHR4 shows 93-95% identity in the DNA-binding domain and the first portion of the hinge (D) region with the germ cell nuclear factor (GCNF)-related factors (GRFs) of the silkworm, Bombyx mori, and the mealworm, Tenebrio molitor, and with a genomic sequence from the fruit fly, Drosophila melanogaster. Northern blot hybridization showed that a 7.5 kb MHR4 mRNA appeared in Manduca abdominal epidermis just as the ecdysteroid titer began to decline during the larval molt, disappeared about 12 h later, then transiently reappeared shortly before larval ecdysis. During the pupal and adult molts, a similar pattern of expression was seen (the very end of the adult molt was not studied). At peak times of expression in the epidermis, MHR4 mRNA was also present in fat body and the central nervous system (CNS). The deduced amino acid sequence of Manduca FTZ-F1 is 100% and 96% identical to that of B. mori and Drosophila betaFTZ-F1, respectively, in the DNA-binding domain and the adjacent hinge region including the FTZ-F1 box. Northern blot analysis showed that the >9.5 kb betaFTZ-F1 mRNA appeared in Manduca epidermis during the decline of the ecdysteroid titer in the larval, pupal and adult molts as the first peak of MHR4 mRNA declined, then it disappeared in the larval and pupal molts before the second peak of MHR4 appeared. betaFTZ-F1 mRNA was also found in fat body and the CNS at the time of peak expression in the epidermis during the larval and pupal molts. Both MHR4 and betaFTZ-F1 mRNAs were found in the testis during the onset of spermatogenesis in the prepupal period.     

Biostable insect kinin analogs reduce blood meal and disrupt ecdysis in the blood-gorging Chagas’ disease vector,Rhodnius prolixus

Rhodnius prolixus is a blood-gorging hemipteran that takes blood meals that are approximately 10 times its body weight. This blood meal is crucial for growth and development and is needed to ensure a successful molt into the next instar. Kinins are a multifunctional family of neuropeptides which have been shown to play a role in the control of feeding in a variety of insects. In this study, two biostable Aib-containing kinin analogs were tested to see if they interfere with blood-feeding and subsequent development into the next instar. One of the analogs, 1729 (Ac-R[Aib]FF[Aib]WGa), had no effect on the size of the blood meal or on the subsequent molting of the insect into the next instar. This analog also did not interfere with either short-term or long-term diuresis. The second analog, 1728 ([Aib]FF[Aib]WGa), appeared to be an antifeedant. Insects feeding on blood containing this analog (15 μM) only consumed 60% of the blood meal taken by insects fed on blood without analog. Insects feeding on blood containing 1728 had a slower rate of rapid diuresis (diuresis in the first 3–5 h after feeding) leading to less urine being excreted by 5 days post feeding. The consequence of these effects was that insects fed on 1728 did not molt. This data indicates that the biostable Aib-containing analog 1728 disrupts normal growth and development in the blood-feeding insect, R. prolixus.     

Canalization of development and ecdysteroid timing during the last instar in lubber grasshoppers

Development in many phytophagous, holometabolous insects is flexible at the beginning but inflexible at the end of the last larval instar. A prominent feature of the inflexible period is a peak in hemolymph levels of ecdysteroids. We tested whether this pattern holds true for the final molt of a phytophagous, hemimetabolous insect, Romalea microptera (the Eastern lubber grasshopper). We fed one group of grasshoppers a high quantity diet (H) throughout the 5th (final) instar and a second group a low quantity diet (L) throughout the instar. Three other diet treatments involved starting the instar on the high diet and then abruptly switching to the low diet at 3, 8, or 13 days (H3L, H8L, and H13L respectively) and continuing the low diet until adult molt. Diet treatment did not affect the maximum hemolymph level of ecdysteroids (E(max)); this peak typically reached ~4000 ng/ml. Ecdysteroid levels were elevated for ~4 days in all groups. In contrast, diet significantly affected age at adult molt and age at E(max) such that H = H13L = H8L < H3L = L. We identified estimates of thresholds for weight gain (20% initial weight) and hemolymph ecdysteroids (100 ng/ml), after which diet did not affect the time to the adult molt. The weight gain threshold was less precise than the ecdysteroid threshold. These results suggest that R. microptera has an extended period of inflexible (canalized) development during the final instar that includes a peak of ecdysteroids. We hypothesize this pattern holds for many phytophagous, hemimetabolous insects.     

Effects of ecdysteroid agonist RH-2485 reveal interactions between ecdysteroids and juvenile hormones in the development of Sesamia nonagrioides

Larvae of Sesamia nonagrioides developing under long day (LD) conditions pupate in the 5th or 6th instar, whereas under the short day (SD) conditions, they undergo several supernumerary larval molts and are regarded as diapausing. The development in early larval instars occurs in the LD larvae at a moderate and in the SD larvae at a high juvenile hormone (JH) titer; ecdysteroid titer cycles similarly under both conditions. The transformation to pupa is initiated by a burst of ecdysteroids at undetectable JH levels, whereas extra larval molts in the diapausing larvae are associated with moderate JH titer and irregular rises of ecdysteroids. Application of 0.2 ppm RH-2485 to the diet of the 6th instar larvae promotes hormonal changes supporting metamorphosis in the LD larvae and slightly accelerates larval molts in the diapausing SD larvae. The 0.5- and 1-ppm doses revert these patterns of endocrine regulations to a mode typical for early larval instars. Particularly dramatic is a JH titer increase provoked within 24 h in the LD larvae. After the treatment, both the LD and SD larvae undergo a series of larval molts, suggesting that hormonal programming of the larval development has been stabilized. A few insects receiving 1 ppm RH-2485, and a high proportion of those fed with 5 ppm RH-2485, deposit two cuticles within a single apolysis and die.     

The Ontogeny of Sexual Size Dimorphism of a Moth: When Do Males and Females Grow Apart?

Sexual dimorphism in body size (sexual size dimorphism) is common in many species. The sources of selection that generate the independent evolution of adult male and female size have been investigated extensively by evolutionary biologists, but how and when females and males grow apart during ontogeny is poorly understood. Here we use the hawkmoth, Manduca sexta, to examine when sexual size dimorphism arises by measuring body mass every day during development. We further investigated whether environmental variables influence the ontogeny of sexual size dimorphism by raising moths on three different diet qualities (poor, medium and high). We found that size dimorphism arose during early larval development on the highest quality food treatment but it arose late in larval development when raised on the medium quality food. This female-biased dimorphism (females larger) increased substantially from the pupal-to-adult stage in both treatments, a pattern that appears to be common in Lepidopterans. Although dimorphism appeared in a few stages when individuals were raised on the poorest quality diet, it did not persist such that male and female adults were the same size. This demonstrates that the environmental conditions that insects are raised in can affect the growth trajectories of males and females differently and thus when dimorphism arises or disappears during development. We conclude that the development of sexual size dimorphism in M. sexta occurs during larval development and continues to accumulate during the pupal/adult stages, and that environmental variables such as diet quality can influence patterns of dimorphism in adults.