Timing and ecdysteroid regulation of the molt in last instar greenhouse whiteflies (Trialeurodes vaporariorum)

A system of markers has been devised to track the development of 3rd and 4th instar/pharate adult greenhouse whiteflies. Instars were identified based on measurements of body width and body length. Depending upon the host plant, the product of the two measurements was exceptionally useful in distinguishing between instars. Body depth was used to divide the 3rd instar into eight stages and body depth and color and appearance of the developing adult eye were used to divide the 4th instar/pharate adult into nine stages. Under conditions of L:D 16:8 and a temperature of 26±2°C, the body depth of 3rd instars reared on greenbean increased from 0.025 (stage 1) to 0.2 mm (stage 8) and the instar duration was approximately 3 days. The body depth of 4th instars increased from approximately 0.1±0.02 (Stage 1) to 0.3±0.03 mm (Stage 5) and then remained constant or decreased slightly during adult development. Ecdysteroid titers peaked at approximately 120 fg/μg protein during Stages 3 through 6 of the 4th instar. Based on an external examination of developing 4th instars and the fluctuations in ecdysteroid titer, it appears that adult development is initiated in Stage 4 or 5 4th instars. Results from histological studies support this view. In Stage 4 nymphs, a subtle change was observed in the corneagenous cells of the eye. However, most Stage 4 4th instars possessed wing development characteristic of earlier, immature stages. In all Stage 5 insects, wing development had been initiated and the corneagenous cells had become quite distinct. In Stage 6 whiteflies, the wing buds were deeply folded and by Stage 7, spines were observed on the new cuticle, indicating that the adult cuticle was well-formed by this stage. Our study is the first to investigate the timing and regulation of the molt, to monitor ecdysteroid titers in precisely staged 4th instar whiteflies and to examine the internal anatomical changes associated with metamorphosis in these tiny homopteran insects.     

Ecomorphology and habitat utilization of Cottus species

We investigated relationships between morphological attributes and mesohabitat use in populations of Cottus carolinae. Field studies revealed significant differences between Ozark and prairie stream mesohabitats where populations of C. carolinae occurred. Features of the mesohabitat that were characteristic of each stream type correlated with intra- and inter-specific differences in morphological attributes of Cottus. The morphology of Cottus occurring in the deeper, slower, and siltier prairie stream exhibited larger head width and depth, body width and depth, and caudal peduncle width while those occurring in a shallower, faster flowing Ozark stream had longer pelvic fins, larger eyes, and deeper caudal peduncles. Each population had a morphology tailored to optimize its performance in a specific habitat. Results of laboratory based performance studies using a variable flow chamber revealed that critical current velocities (CCV) of live and preserved specimens were weakly associated with distinct combinations of morphological traits. Specimens with higher CCV measurements typically had longer pelvic fins, larger eyes, and deeper caudal fins (Ozark morphology). Those with lower CCVs had larger head width and depth, body width and depth, and caudal peduncle width (prairie morphology) in studies using cobble as the substrate. Differences in flow regimes between prairie and Ozark streams may generate specific niches for specific morphotypes, and current velocity may exert selective pressure on morphology.     

Genetic and thermal effects on the latitudinal variation in the timing of fin development of a fish Oryzias latipes

Heterochrony, an evolutionary change in developmental processes, is one of the major proximate causes of morphological diversity of organisms. It has been reported in the medaka Oryzias latipes that higher-latitude larvae have a genetic tendency to complete fin ray formation at larger body sizes, which results in relatively shorter anal and dorsal fins in adults. However, this latitudinal, heterochronic variation in fin length in the wild may be partially explained by latitudinal differences in thermal environments, if temperatures affect the timing of fin ray formation. Common-environment experiments revealed that the body size at which fin pterygiophore (a basal skeleton of fin rays) formation was completed was larger in higher-latitude larvae than in lower-latitude larvae at all temperatures examined, supporting the proposal that fin ray formation of the former is genetically delayed. However, phenotypic plasticity in response to temperature was also evident; lower temperatures caused delayed fin ray formation until a larger body size had been achieved in both high- and low-latitude larvae. These observations suggest that habitat temperatures also contribute to the latitudinal difference in the timing of fin development, magnifying phenotypic variation in fin length across latitudes. We discuss reasons for this positive covariance between genetic and environmental effects on the latitudinal, heterochronic variation, from the viewpoint of local adaptation and evolution of phenotypic plasticity.     

Ecdysones and imaginal disc development during the last larval instar of Pieris brassicae

Ecdysone haemolymph levels and in vivo development of imaginal wing discs have been studied during the last larval instar of Pieris brassicae.During this period, β-ecdysone variations show two successive peaks, the first one related to the induction of wandering stage, and the second (main) one to pupal cuticle synthesis. The observed situation is very similar to that of Manduca sexta. Imaginal wing disc growth is composed of several genetically programmed steps that need the presence of ecdysone, but do not appear very closely linked to circulating hormone levels. It seems that ecdysone haemolymph peaks should be considered as periods where ecdysone levels are above a threshold value.     

Correlations between epidermal DNA synthesis and haemolymph ecdysteroid titre during the last larval instar of the tobacco hornworm, Manduca sexta

During the fifth larval instar of Manduca sexta the commitment of the epidermis to the synthesis of pupal cuticle is presumably affected by a small increase in ecdysteroid titre when juvenile hormone levels are minimal. Two sequential rounds of DNA synthesis without an intervening mitosis occur at about this time, resulting in polyploidy of the epidermis. There is a definite temporal correlation between the first peak of ecdysone and the second round of DNA synthesis and indirect evidence has been presented which suggests that this small increase in ecdysteroid titre actually initiates the second period of DNA synthesis. Further, it appears that large doses of ecdysteroids do not elicit the same response as smaller doses at a specific developmental stage, indicating that the different physiological effects of ecdysteroids (reprogramming and apolysis) may be dependent upon the relative concentration of the hormone. Following mitosis which takes place on approximately day 6 of the last instar, the epidermis undergoes apolysis and secretes pupal cuticle, expressing the commitment made 4.5 days earlier. These results support the ‘quantal mitosis’ theory of cytodifferentiation since the covert differentiative event occurs during a period of DNA synthesis and since mitosis precedes the expression of that event.     

Ecdysone metabolism in Pieris brassicae during the feeding last larval instar

Ecdysone metabolism in Pieris brassicae during the feeding last larval stage was investigated by using ³H-labeled ecdysteroid injections followed by high-performance liquid chromatographic (HPLC

1 Abbreviations: 3DE = 3-dehydroecdysone; 3D20E = 3-dehydro-20-hydroxyecdysone; 2026E = 20,26-dihydroxyecdysone; E = ecdysone; Eoic = ecdysonoic acid; 2026E′ = 3-epi-20,26-dihydroxyecdysone; E′ = 3-epiecdysone; E′oic = 3-epiecdysonoic acid; E′8P = 3-epiecdysone 3-phosphate; 20E′ = 3-epi-20-hydroxyecdysone; 20E′3P = 3-epi-20-hydroxyecdysone 3-phosphate; FT = Fourier transform; HPLC = high-performance liquid chromatography; 20E = 20-hydroxyecdysone; 20Eoic = 20-hydroxyecdysonoic acid; NMR = nuclear magnetic resonance; NP-HPLC = normal phase HPLC; RP-HPLC = reverse phase HPLC; TFA = trifluoroacetic acid; Tris = tris(hydroxymethyl)-aminomethane.

) analysis of metabolites. Metabolites were generally identified by comigration with available references in different HPLC systems. Analysis of compounds for which no reference was available required a large-scale preparation and purification for their identification by ¹H nuclear magnetic resonance spectrometry. The metabolic reactions affect the ecdysone molecule at C-3, C-20, and C-26, leading to molecules which are modified at one, two, or three of these positions. At C-20, hydroxylation leads to 20-hydroxyecdysteroids. At C-26, hydroxylation leads to 26-hydroxyecdysteroids which can be further converted into 26-oic derivatives (ecdysonoic acids) by oxidation. At C-3, there are several possibilities: there may be oxidation into 3-dehydroecdysteroids, or epimerization possibly followed by phosphate conjugation. Thus, injected 20-hydroxyecdysone was converted principally into 20-hydroxyecdysonoic acid, 3-dehydro-20-hydroxyecdysone, and 3-epi-20-hydroxyecdysone 3-phosphate. Labelled ecdysone mainly gave the same metabolites doubled by a homologous series lacking the 20-hydroxyl group.     

Cellular events in the prothoracic glands and ecdysteroid titres during the last-larval instar of Spodoptera littoralis

Changes in prothoracic gland morphology were correlated to developmental events and ecdysteroid titres (20-hydroxyecdysone equivalents) during the last-larval instar in Spodoptera littoralis. After ecdysis to the last-larval instar the haemolymph ecdysteroid titre remained at about 45 ng/ml, when the prothoracic glands appeared quiescent. The first signs of distinct gland activity, indicated by increased cell size and radial channel formation, were observed at about 12 h prior to the cessation of feeding (36 h after the last-larval moult), accompanied by a gradual increase in ecdysteroid titre to 110 ng/ml haemolymph, at the onset of metamorphosis. During this phase ecdysteroid titres remained at a constant level (140–210 ng/ml haemolymph) and prothoracic gland cellular activity was absent for a short period. The construction of pupation cells occurred when haemolymph ecdysteroids titres increased to 700 ng/ml. A rapid increase in ecdysteroids began on the fourth night (1600 ng/ml haemolymph) reaching a maximal level (4000 ng/ml haemolymph) at the beginning of the fourth day. In freshly moulted pupae a relatively high ecdysteroid titre (1100 ng/ml haemolymph) was still observed, although during a decrease to almost negligible levels. The increase in ecdysteroid level during the third and the fourth nights of the last-larval instar was correlated with the period when almost all the prothoracic gland cells showed signs of high activity. Neck-ligation experiments indicated the necessity of head factors for normal metamorphosis up to the second to third day of the instar. The possibility that the prothoracic glands are under prothoracicotropic hormone regulation at these times is discussed.     

Adaptation and constraint in the evolution of environmental sex determination

When environments differentially influence male and female performance, environmental sex determination (ESD) might evolve. The conclusion from several previous theoretical models was that reaction norms for sex determination should have a single, sharp threshold, with only females being produced in some environments and only males in others. These reaction norms can be disadvantageous in fluctuating environments, however, because they lead to sex-ratio fluctuations. We analysed the evolution of ESD, looking for equilibrium strategies in unconstrained as well as constrained strategy spaces. We identified situations where a single-threshold reaction norm is not evolutionarily stable. In these cases, we found stable strategies in the form of complex reaction norms, showing an oscillatory pattern of sex determination with respect to variation in an environmental variable. Considering that constraints could prevent such phenotypes from being realized, we found that certain randomized reaction norms, with probabilistic sex determination for a range of environments, would achieve nearly the same fitness. We also investigated reaction norms constrained to have a single threshold and found that genetic polymorphism in the environmental threshold value could evolve, producing a similar effect as a randomized reaction norm. We argue that the appearance of genetic variation can be regarded as an alternative outcome when constraints prevent the evolution of a more complex or a randomized strategy.     

Morphogenesis of the wing Anlagen in the mealworm beetle tenebrio molitor during the last larval instar

The wing Anlagen of Tenebrio develop from epidermal cells located on the lateral margins of meso- and metathoraces. Three to four days after larval ecdysis, these cells start to proliferate slowly, continuing to do so until day 13 which corresponds to the period of the pupal commitment of the remaining epidermis. The wing Anlagen cells then proliferate rapidly until day 18.5. Three days before pupal ecdysis, the mitotic index falls suddenly while 40% of the Anlagen cells disappear owing to cell degeneration. The sudden changes observed in the mitotic index are correlated with two hemolymphatic peaks in ecdysteroid levels. Anlagen of the forewings and hindwings show similar development except for cuticular secretion at the end of the instar which is greater on the upper face of the forewings. A comparison is made with imaginal discs and histoblasts described in other holometabolous insects.     

Regulation and modulation of electric waveforms in gymnotiform electric fish

Weakly electric gymnotiform fish specialize in the regulation and modulation of the action potentials that make up their multi-purpose electric signals. To produce communication signals, gymnotiform fish modulate the waveforms of their electric organ discharges (EODs) over timescales spanning ten orders of magnitude within the animal’s life cycle: developmental, reproductive, circadian, and behavioral. Rapid changes lasting milliseconds to seconds are the result of direct neural control of action potential firing in the electric organ. Intermediate-term changes taking minutes to hours result from the action of melanocortin peptides, the pituitary hormones that induce skin darkening and cortisol release in many vertebrates. Long-term changes in the EOD waveform taking days to weeks result from the action of sex steroids on the electrocytes in the electric organ as well as changes in the neural control structures in the brain. These long-term changes in the electric organ seem to be associated with changes in the expression of voltage-gated ion channels in two gene families. Electric organs express multiple voltage-gated sodium channel genes, at least one of which seems to be regulated by androgens. Electric organs also express multiple subunits of the shaker (Kv1) family of voltage-gated potassium channels. Expression of the Kv1 subtype has been found to vary with the duration of the waveform in the electric signal. Our increasing understanding of the mechanisms underlying precise control of electric communication signals may yield significant insights into the diversity of natural mechanisms available for modifying the performance of ion channels in excitable membranes. These mechanisms may lead to better understanding of normal function in a wide range of physiological systems and future application in treatment of disease states involving pathology of excitable membranes.     

Adenylate cyclase in prothoracic glands during the last larval instar of the silkworm, Bombyx mori

We have previously reported that the absence of prothoracicotropic hormone (PTTH) signal transduction during the early last larval instar of Bombyx mori plays a role in leading to very low ecdysteroid levels in the hemolymph, inactivation of the corpora allata, as well as larval-pupal transformation. In the present study, adenylate cyclase was characterized in crude preparations of prothoracic gland cell membranes in an effort to localize the cause of refractoriness to PTTH. It was found that cyclase activity of the prothoracic glands from the day 6 last instar showed activation responses to fluoride, a guanine nucleotide analogue, as well as calmodulin (CaM) in dose-dependent fashions. The additive effects of day 5 prothoracic gland adenylate cyclase stimulation by fluoride and CaM imply that there may exist Gs protein-dependent and CaM-dependent forms of adenylate cyclase. For day 1 last instar prothoracic glands, which showed no response to stimulation by PTTH in either cAMP generation or ecdysteroidogenesis, adenylate cyclase activity exhibited far less responsiveness to Ca(2+)/CaM than did that from day 5 glands. These findings suggest that day 1 prothoracic glands may possess some lesions in the receptor-Ca(2+) influx-adenylate cyclase signal transduction pathway and these impairments in PTTH signal transduction may be, at least in part, responsible for decreased ecdysteroidogenesis.     

Models of coral growth: spontaneous branching,compactification and the Laplacian growth assumption

In stony corals it is often observed that specimens collected from a sheltered growth site have more open and more thinly branched growth forms than specimens of the same species from more exposed growth sites, where stronger water currents are found. This observation was explained using an abiotic computational model inspired by coral growth, in which the growth velocity depended locally on the absorption of a resource dispersed by advection and diffusion (Kaandorp and Sloot, J. Theor. Biol 209 (2001) 257). In that model a morphological range was found; as the Péclet-number (indicating the relative importance of advective and diffusive nutrient transport) was increased, more compact and spherical growth forms were found. Two unsatisfactory items have remained in this model, which we address in the present paper. First, an explicit curvature rule was responsible for branching. In this work we show that the curvature rule is not needed: the model exhibits spontaneous branching, provided that the resource field is computed with enough precision. Second, previously no explanation was given for the morphological range found in the simulations. Here we show that such an explanation is given by the conditions under which spontaneous branching occurs in our model, in which the compactness of the growth forms depends on the ratio of the rates of growth and nutrient transport. We did not find an effect of flow. This suggests that the computational evidence that hydrodynamics influences the compactness of corals in laminar flows may not be conclusive. The applicability of the Laplacian growth paradigm to understand coral growth is discussed.     

The ecdysteroid titres during the last-larval instar of Ephestia kuehniella Z. (Lepidoptera: Pyralidae)

The ecdysteroid titre and the body weight during the last-larval instar of Ephestia kuehniella were determined. Slightly elevated ecdysteroid titres occur during the first 12 h following the last larval-larval ecdysis (38 ng/g) and again some 120 h later, lasting about 48 h (33 ng/g). A high ecdysteroid peak (750 ng/g) with a maximum in prepupae of the eye-class A4 precedes the larval-pupal ecdysis. The basal levels between these increased ecdysteroid titres are between 13 ng/g and 15 ng/g. Compared with the body weight, the first sligtly increased ecdysteroid titre 12 h after ecdysis is associated with the beginning of food intake, the second increase at 144 h after ecdysis with reduced gain in body weight. The prepupal ecdysteroid peak occurs whilst the body weight remains constant. Correlations between the varying ecdysteroid titre and morphological and physiological events accompanying the progress in larval-pupal development are discussed.     

Motor neurone responses during a postural reflex in solitarious and gregarious desert locusts

Desert locusts show extreme phenotypic plasticity and can change reversibly between two phases that differ radically in morphology, physiology and behaviour. Solitarious locusts are cryptic in appearance and behaviour, walking slowly with the body held close to the ground. Gregarious locusts are conspicuous in appearance and much more active, walking rapidly with the body held well above the ground. During walking, the excursion of the femoro-tibial (F-T) joint of the hind leg is smaller in solitarious locusts, and the joint is kept more flexed throughout an entire step. Under open loop conditions, the slow extensor tibiae (SETi) motor neurone of solitarious locusts shows strong tonic activity that increases at more extended F-T angles. SETi of gregarious locusts by contrast showed little tonic activity. Simulated flexion of the F-T joint elicits resistance reflexes in SETi in both phases, but regardless of the initial and final position of the leg, the spiking rate of SETi during these reflexes was twice as great in solitarious compared to gregarious locusts. This increased sensory-motor gain in the neuronal networks controlling postural reflexes in solitarious locusts may be linked to the occurrence of pronounced behavioural catalepsy in this phase similar to other cryptic insects such as stick insects.     

Separate effects of macronutrient concentration and balance on plastic gut responses in locusts

It is well established that animal guts are phenotypically plastic, adjusting inter-alia to diet quality. However, the relative contributions due to the two principal dimensions of diet “quality”—nutrient concentration and nutrient balance—remain to be teased apart. We report an experiment using synthetic foods in which the balance and overall concentration (in relation to indigestible cellulose) of protein and digestible carbohydrate were varied orthogonally, and the effects on the dry mass of locust guts measured. There were three principal results: (1) larger guts were associated with dilute compared with concentrated diets, suggesting a compensatory response to ameliorate the impact of reduced diet quality; (2) there was, by contrast, an anti-compensatory response to nutrient imbalance, where larger guts were associated with surplus protein intake; (3) the experimental group given the food that contained low protein and low cellulose, the composition that predicted the smallest guts, showed a bimodal response in which half of the insects had guts that were larger than expected for their cellulose intake, suggesting that they were able to respond to a protein-related cue in the absence of significant dietary fibre. We discuss these results in relation to regulatory theory.     

Life stage-related differences in hardening and acclimation of thermal tolerance traits in the kelp fly, Paractora dreuxi (Diptera, Helcomyzidae)

It is widely appreciated that physiological tolerances differ between life stages. However, few studies have examined stage-related differences in acclimation and hardening. In addition, the behavioural responses involved in determining the form and extent of the short-term phenotypic response are rarely considered. Here, we investigate life stage differences in the acclimation and hardening responses of the survival of a standard heat shock (SHS) and standard low temperature (or cold) shock (SCS), and the crystallization temperature (or supercooling point, SCP) of adults and larvae of the sub-Antarctic kelp fly, Paractora dreuxi. These stages live in the same habitat, but differ substantially in their mobility and thus environmental temperatures experienced. Results showed that neither acclimation nor hardening affected the lower lethal limits in larvae or adults. Adults showed an increase in survival of upper lethal limits after low temperature acclimation, whilst larvae showed a consistent lack of response. The acclimation × hardening interaction significantly affected the SCP in adults, but no response to either acclimation or hardening was found in the larvae. This study further demonstrates the complexities of thermal tolerance responses in P. dreuxi.     

Differences in plastic responses to defoliation due to variation in the timing of treatments for two species of Sesbania (Fabaceae)

BACKGROUND AND AIMS: Plastic responses to stress in components of reproduction can have important effects on plant fitness and can vary both within and between species. Responses may also depend on when in the life cycle stress occurs. Here, it is predicted that the timing of initiation of a stress, defoliation, would affect the pattern of plastic responses. These differences should occur because some components of reproduction, such as flower number, are determined earlier in a plant's life than others, such as individual seed mass. METHODS: To test this prediction, 50 % artificial defoliation treatments were initiated at four different times for Sesbania macrocarpa and S. vesicaria. Responses were measured in plant size, number of flowers, number of flowers/plant size, fruit set, number of seeds per fruit, individual seed mass and total seed mass per plant. KEY RESULTS: For S. vesicaria, changes in the timing of stress changed the severity, but not the pattern of response. For S. macrocarpa, plastic responses to defoliation varied strikingly between early and late treatments. Late treatments resulted in over-compensation in this species. Sesbania macrocarpa was generally more plastic than S. vesicaria and the species showed opposite responses for most components of reproduction. CONCLUSIONS: While there were effects of timing of defoliation and differences between species, the nature of these effects did not precisely fit our predictions. Our results suggest that differences in the length and flexibility of the life cycles of the two species allowed for unexpected variation in responses. For example, because flower production continued after the last treatment in S. vesicaria, responses were not constrained to reductions in individual seed mass.     

The effects of herbivory and resource variability on the production of a second inflorescence by the desert lily, Pancratium sickenbergeri

We investigated the effects of herbivory by the dorcas gazelle, Gazella dorcas, on the production of a second inflorescence in a desert lily, Pancratium sickenbergeri (Amaryllidaceae), after the first inflorescence was eaten. In three populations exposed to different levels of herbivory, we conducted an inflorescence-clipping experiment in the flowering seasons of 1997, 1998 and 1999. The experiment was performed on inflorescences at both the emerging stage and the anthesis stage. Plants in each control group were undamaged. Clipped plants had a greater probability of producing a second inflorescence stalk than unclipped plants. In all the populations, the probability of producing a second inflorescence was greater when the first inflorescence was cut in the emerging stage than when it was cut in anthesis, indicating that there was a cost of compensation. In the population with the highest level of herbivory, the production of a second inflorescence stalk was related to resource availability in all 3 years. Plants produced as many flowers or fruits on the second stalk as on the first, indicating that potential reproductive output is double that usually achieved. Although we found no evidence for a trade-off between investment in first and second inflorescence stalks, the lower probability of producing a second inflorescence if heavy investment had been made in the first inflorescence indicates that compensation is costly. These results contradict the mutualism hypothesis that herbivory is beneficial to plants and provide support for the trade-off hypothesis that benefits of herbivory are proximal and are attained at an evolutionary cost.     

Phenotypic responses of the twining vine Ipomoea purpurea (Convolvulaceae) to physical support availability in sun and shade

Vines depend on external support to prevent shading by neighbouringplants. Hence, it is important to determine whether shading enhances thephenotypic responses of vines to support availability. I evaluated theconsequences of support availability (a vertical stake) on shoot and leaftraitsof the morning glory Ipomoea purpurea (Convolvulaceae)under full sunlight and extreme shade. It was hypothesised that phenotypicresponses of vines to support availability should be greater in the shade. Inaddition, to investigate possible constraints to such phenotypic responses, thecorrelations among phenotypic traits and the plasticity of such correlationswere evaluated. The phenotypic variation of the main stem length and of thenumber of branches was consistent with the hypothesis, i.e. greater responsestosupport availability in the shade. In contrast, both internode length and leafarea (two traits that showed a significant and positive correlation) decreasedin the sun and increased in the shade with support availability. Petiole lengthdecreased with support in the sun but had no response in the shade. On theotherhand, the number of significant trait correlations found in plants in the sunand supported plants was higher than those of shade and non-supported plants,respectively. Several of the correlations were significantly sensitive to theenvironment. Flowering only occurred in the sun treatment. Whereas no shoot orleaf trait was significantly correlated with flower number in supported plants,both petiole length and shoot biomass showed a significant correlation withsuchestimate of plant fitness in non-supported plants.