Penultimate Instar Larvae Research Articles

Hormonal control of cuticular melanization in the common cutworm, Spodoptera litura

The penultimate-instar larvae of Spodoptera litura raised under crowded conditions deposited melanized cuticle, which could be labelled by injected [ 14C]dopamine, while those reared in isolation deposited non-melanized cuticle. In crowded culture, melanization-and-reddish-colouration hormone was secreted between 6 and 9 h after head-capsule slippage, followed by melanization about 4 h later. Extirpation of the suboesophageal ganglion from the crowded larvae totally blocked melanization, while removal of the brain allowed 50% of the melanization as after denervation between the two organs. This suggests that the melanization-and-reddish-colouration hormone is secreted from the suboesophageal ganglion, and the brain regulates its secretion from that organ. Injection of melanization-and-reddish-colouration hormone of Bombyx mori into an isolated abdomen induced melanization, while this treatment combined with methoprene blocked it. Application of methoprene to crowded larvae up to 2 h before head-capsule slippage greatly reduced the black pigmentation. Allatectomy of the larvae reared in isolation could induce full melanization. These results indicate that a decrease in juvenile hormone titre at around the time of head-capsule slippage is essential for exhibiting the action of melanization-and-reddish-colouration hormone.

Developmental regulation of juvenile hormone esterase in Trichoplusia ni: Its multiple electrophoretic forms occur during each larval ecdysis

Juvenile hormone esterase activity has been found during the intramoult period of each larval stadium in Trichoplusia ni. The activity is indistinguishable from that occurring during the final larval stadium, on the basis of its four isoelectric forms and kinetic data. The 4th- to 5th-instar intramoult peak in activity also occurs in other Lepidoptera ( Heliothis virescens, Spodoptera exigua, Manduca sexta and Hyphantria cunea). Further, the final species also possessed a peak of activity during the intramoult period to the penultimate larval instar. The findings have important implications for the current concept that the function of juvenile hormone esterase is, by reason of its anti-juvenile hormone action, an enzyme of the last larval instar which enables metamorphosis to begin.

Juvenile hormone esterase activity in the plasma and body tissue during the larval and adult stages of the house cricket

Superimposed peaks of juvenile hormone esterase (JHE) on electrofocusing gels indicate that the same JHE occurs in the tissues and plasma of both last instar larvae and adults of Acheta domesticus. Electrofocusing resolved two peaks of JHE activity, pI 5.0 and 4.5. The apparent K m of the plasma JHE was 1.1 × 10 −7 M. Acheta JHE hydrolyzed racemic JH III twice as fast as racemic JH I, which is consistent with previously published evidence that the endogenous JH homolog will be the homolog most quickly hydrolyzed. JHE was essentially lacking in both tissues and plasma of the penultimate instar larvae. There was a single mid-instar peak of both plasma (16 nmol/min-ml) and tissue JHE activity during the last instar. A second prepupal JHE peak (characteristic of Lepidoptera) was lacking in Acheta, possibly because orthopterans lack a pupal stage. The distinct differences in the developmental activity patterns for alpha-naphthyl acetate esterase (ANAE) and JHE in Acheta emphasized that JH hydrolysis is not likely to be the result of carboxyl esterases. We confirmed the JH dependent degeneration of the dorsal longitudinal flight muscles [Bocharova-Messner et al. (1970) Ontogenez 1, 209–212; Bocharova-Messner et al. (1974) Ontogenez 5, 130–138], which indicates a peak of JH on day 3 following adult emergence. Plasma JHE decreased from 5.5 nmol/min-ml on the day of emergence to 3.2 on day 3 and remained low in mated females that were permitted to oviposit. When eggs were caused to accumulate in the females so that vitellogenesis-oogenesis was supressed, as consistently happened in virgins > 14 days old or in mated females not afforded oviposition sites, the plasma JHE level increased to 8.8 nmol/min-ml. Within 24 hr of ovipositing, oogenesis restarted and the JHE activity was halved. The evidence indicated that JHE activity is correlated with a reduction in the JH titer.

Role of low ecdysteroid titre in acquisition of competence for pupal transformation in Bombyx mori

Penultimate-instar larvae of Bombyx mori were neck-ligated or ligated posterior to the prothoracic glands. Repetitive injections of 20-hydroxyecdysone every 3 or more hours elicited the gut purge in thorax-ligated animals. Single injections of 20-hydroxyecdysone up to 40 μg failed to induce the gut purge. However, a single injection of 20-hydroxyecdysone together with juvenile hormone analogue, resulted in larval moulting of thorax-ligated animals. Once the thorax-ligated larvae showed the gut purge, a single injection of 20-hydroxyecdysone was enough to provoke pupation. The change in ecdysteroid titre in those animals receiving repeated injections was compared with that observed in neck-ligated larvae that spontaneously underwent the gut purge followed by precocious pupation. These data indicate that the very low ecdysteroid titre found before the gut purge is important for the acquisition of competence to undergo the gut purge in response to a small ecdysteroid surge just before the gut purge.

Differential responses of tiger swallowtail subspecies to secondary metabolites from tulip tree and quaking aspen.

Two subspecies of the eastern tiger swallowtail butterfly, Papilio glaucus, exhibit reciprocal inabilities to survive and grow on each other's preferred foodplant. P. g. canadensis R. & J. performs well on quaking aspen (Populus tremuloides Michx.) but not on tulip tree (Liriodendron tulipifera L.); P. g. glaucus L. performs well on tulip tree but not on quaking aspen. This study was designed to test the hypothesis that secondary metabolites in tulip tree and quaking aspen are responsible for these differential utilization abilities. We extracted and fractionated leaf constituents into different chemical classes, applied them to a mutually acceptable diet (black cherry, Prunus serotina, leaves), and bioassayed them against neonate larvae (survival) and penultimate instar larvae (survival, growth, digestibility and conversion efficiencies). For each plant species, one fraction in particular showed activity against the unadapted subspecies. One tulip tree fraction dramatically reduced survival of P. g. canadensis neonates, and reduced consumption rates, growth rates, and ECI's of fourth instar larvae. The tulip tree constituents most likely responsible for these effects are sesquiterpene lactones. One quaking aspen fraction greatly lowered survival of P. g. glaucus neonates, and decreased survival, consumption rates, growth rates and ECD's of fourth instar larvae. The compounds responsible for these results are probably simple phenols or phenolic glycosides. Surprisingly, P. g. glaucus and P. g. canadensis showed slightly poorer performance on the active tulip tree and quaking aspen fractions, respectively, indicating that even adapted insects incur a metabolic cost in the processing of their host's phytochemicals.

Regulation of prothoracicotropic hormone release by juvenile hormone in the penultimate and last instar larvae of Mamestra brassicae

The penultimate instar larvae of Mamestra brassicae have one ecdysteroid peak 2 days after ecdysis which induces larval ecdysis. Allatectomy deleted this peak and caused a larger peak 7 days later just before precocious pupation, in a pattern similar to the ecdysteroid titer of the last instar. Application of juvenile hormone (JH) immediately after allatectomy restored the normal larval pattern and larval ecdysis. The brain of the normal Day 1 or 2 penultimate instar larva had higher prothoracicotropic hormone (PTTH) activity than that of the allatectomized larva. Therefore, JH in the penultimate instar activates the brain to secrete and/or produce PTTH. Allatectomy of last instar larvae prolonged the period from gut purge to pupation by causing a delay of the ecdysteroid peak necessary for pupation. Application of JH restored the normal timing of ecdysteroid release. Thus, JH is important for the occurrence of the ecdysteroid surge on Day 7, since its absence delayed this surge by a day.

Effects of hydrolyzable and condensed tannin on growth and development of two species of polyphagous lepidoptera: Spodoptera eridania and Callosamia promethea.

The effects of tannins on survival, growth, and digestion were compared in two polyphagous species of Lepidoptera (one, the southern armyworm, a forb-feeder; and the other, the promethea silkmoth, a tree-feeder). Two different types of tannins (hydrolyzable and condensed) were incorporated into artificial basal diets in order to determine whether or not differential survival and growth would result between the forb feeder, which normally does not encounter tannins in its natural diet, and the tree-feeder, whose host species include many tanniniferous plants from several different families.Neonate larvae of the forb-feeding armyworms exhibited significantly suppressed 10-day growth rates at all tannin concentrations tested (0.5, 1.0, 1.5, 2.0, 2.5, and 3.0% of wet weight) for both the hydrolyzable and the condensed tannin compared to the control diet, however no dose-effect was detectable. In contrast, there were no detectable differences in neonate survival or growth through the first 10 days for the tree-feeding promethea silkmoth larvae fed diets with either tannic acid or quebracho tree condensed tannin.In order to determine the physiological mechanisms of action of these tannins against armyworms, we conducted detailed physiological bioassays of biomass and nitrogen utilization by penultimate instar larvae. Standard gravimetric feeding studies with both tannic acid and the quebracho tree condensed tannin demonstrated that reduced relative growth rates (RGR's) of Spodoptera eridania Cram. were due to the suppressed relative consumption rates (RCR's) and decreased conversion efficiencies (ECD's) rather than due to digestibility-reduction (as reflected by approximate digestibility, AD). As with the neonate larval growth rate suppression, there were no detectable dose responses at the different concentrations of tannic acid (0.25, 0.50, 0.75, 1.00, 2.50, and 5.0 percent) and condensed tannins from quebracho (0.25, 0.50, 0.75, 1.0, and 2.5 percent) in our penultimate instar studies.

Morphological and physiological changes in central auditory neurons following unilateral foreleg amputation in larval crickets

1. In view of the surprising recent demonstration that developmentally one-eared female crickets can track sound sources (Huber et al. 1984), we have looked for correlates in the morphological and physiological properties of auditory interneurons of these animals. One foreleg was amputated in the 3rd/4th or penultimate (8/9th) larval instar; in both cases the leg regenerated without developing a functional ear. In the adult stage, these animals were studied first for their phonotactic behavior and then by intracellular recording and staining; three types of auditory interneurons in the prothoracic ganglion were identified: the omega neuron ON1, and the ascending neurons AN1 and AN2. 2. Of these three neuron types, those that normally receive excitatory input from the side now deafferented send dendrites across the midline of the ganglion, along specific pathways, to end in the auditory neuropil of the intact side (Figs. 1–4). 3. The new connections are functional, as shown by the responses of the neurons to synthesized calling songs presented to the remaining ear. With respect to the copying of chirp structure, threshold curves and intensity characteristics, these neurons respond like cells in intact animals that are presented with the same stimulus on the side ipsilateral to the main input region of the neurons (Figs. 2–4). The implication is that in animals with one ear missing, functional pathways within the central nervous system are reorganized, resulting in better orientation of one-eared animals.

MIXED‐FUNCTION OXIDASE ACTIVITY IN LARVAE OF HELIOTHIS PUNCTIGER WALLENGREN (LEPIDOPTERA: NOCTUIDAE)

AbstractThe O‐demethylation of 4‐nitroanisole was used to evaluate mixed‐function oxidase (MFO) activity in last and penultimate instar larvae of H. punctiger. The specific activity of tissues of early fifth instar larvae in decreasing order were midgut, integument, fatbody, head capsule, and whole larva. Specific activity of the midgut rose rapidly from the beginning of the instar and then returned to a relatively low level when the insect stopped feeding. Fatbody specific activity increased until early in the last instar and remained at about this level until pupation. Specific activity of the integument was highest at the beginning and end of the instar and lowest in the middle. O‐demethylase activity per larva revealed that although midgut activity was low late in the fifth instar, the insect still possessed relatively high levels of MFO's in the integument and particularly the fatbody.

Predation efficiency and capacity of the. larvae of three libellulid dragonflies (Anisoptera: Libetluidae)

The predation efficiency of the final and penultimate instar larvae of Urothemis assignata Selys, Palpoleura lucia (Drury) and Acisoma panorpoides Selys on six prey types was studied in the laboratory. Predation efficiency on tadpoles, zygopteran larvae ephemeropteran larvae and ostracods was high (40 to 92%); it was highest on ephemeropteran larvae (76 to 92%) and lowest on zygopteranlarvae (40 to 720/0). Predation efficiency was also low on fish fry (0 to 56%) and anisopteran larvae (24 to 50%). The prey type, the size of prey, the movement pattern of prey, the size of the libellulid larvae and their agility influenced predation efficiency. Predation capacity was high; prey were still killed even after the larvae were satiated. It seems that a regulation of food intake exists in the larvae of the three Iibellulid larvae.

Temperature as a Factor in the Development and Feeding Ecology of Tiger Swallowtail Caterpillars, Papilio glaucus (Lepidoptera)

The influence of temperature upon the development and feeding ecology of the eastern tiger swallowtail butterfly, Papilio glaucus L. (Lepidoptera: Papilionidae) was investigated under natural and controlled conditions. The developmental threshold upon black cherry, Prunus serotina Ehrh., was determined to be 11.7'C. Accumulated thermal units, calculated using this base, and meteorological weather data were similar for the June-July larvae and the August-September larvae feeding on black cherry trees in spite of significant differences in actual larval durations for these periods (36.3 ? 3.4 d and 47.0 ? 4.0 d, respectively). A detailed analysis of mass and nitrogen budgets of penultimate instar larvae fed black cherry leaves under laboratory conditions revealed an increase in both mass and nitrogen consumption rates (per mg of larval tissue) across all temperatures tested (15, 22, 26, 30, and 37TC). Significantly increased respiratory expenditures at the highest temperature resulted in suppressed mass accumulation rates and poor survival. The digestibility of the cherry leaves was largely independent of temperature. It thus appears that faster growth at higher temperatures is achieved by faster consumption (i.e. a power-function) rather than by increased efficiency.

Juvenile hormone and regulation of dichotomous spermatogenesis during the larval diapause of the codling moth

Spermatogenesis ceases during diapause and resumes after diapause. A possible causal relationship between the regulation of these phenomena and the fluctuations of the juvenile hormone titre connected with the diapause was examined in the codling moth. Spermatocyte lysis and spermatogenesis arrest are directly and reversibly related to these fluctuations. They are induced in the last-larval instar by sole application of the juvenoid Altosid to the penultimate-instar larvae predetermined not to enter diapause. Conversely, allatectomy of early last-instar larvae predetermined to diapause induces renewal of spermatogenesis and disruption of diapause. However, the continuation of the arrest of the spermatogenesis during diapause is not directly related to the presence of a high titre of juvenile hormone. The arrest extends throughout diapause which may continue for months, although no juvenile hormone could be detected in the species after 35 days of larval diapause.

The epidermal cell cycle during the metamorphosis of Tenebrio molitor L. (Insecta Coleoptera)

The main features of epidermal cell cycle during the metamorphosis of Tenebrio molitor have been investigated by cell density study, [ 3H]thymidine incorporation, and cytophotometry. G 1, S, and M phases are restricted to short periods during each larval stage. The pupal mitotic wave is relatively longer than the larval ones. Outside of each mitotic period, 95 to 100% of the epidermal cells are blocked in G 2 (4C DNA content). The cell density study shows that during the penultimate larval instar, numerous cells bypass the mitotic period without dividing while during the mitotic period of the last larval instar and of the pupal stage, each cell divides once and twice for some cells. After the pupal mitotic period, numerous adult cells are definitely blocked in G 2 but 20 to 30% of cells present in old adults are in G 1. Adult epidermal oenocytes differentiate during the last larval mitotic period, becoming 8 to 32C. From these results, it can be concluded that the G 2 block and the release of G 2-blocked cells into M are two critical events of each molting cycle. A possible hormonal control of these events is discussed.

Precocious reprogramming of eupyrene-apyrene spermatogenesis commitment induced by allatectomy of the penultimate larval instar of the moth Actias selene

A possible causal relationship between the switch-over from eupyrene to apyrene spermatogenesis and pupation in Lepidoptera was examined in Actias selene. Precocious pupation, 11 days earlier than in the controls, was induced by allatectomy on the first day of the penultimate larval instar. The course of the eupyrene spermatogenesis, until nuclear elongation in the spermatid, is not related to pupation. In both allatectomized and control individuals, eupyrene metaphases appear 8 days after ecdysis of the fourth larval instar. Nuclear elongation, however, is triggered in the allatectomized individuals earlier than in the controls, probably by the premature decline of the juvenile hormone titre following allatectomy. Apyrene commitment, on the other hand, is directly related to pupation, as apyrene spermatogenesis begins 2 days after pupation in both control and allatectomized individuals.

Ecdysteroid Titres during Autonomous Metamorphosis in a Dermestid Beetle

The larvae of Dermestes maculatus are able to undergo several moults in complete absence of the cephalic and prothoracic neuroendocrine system, showing thus spontaneous metamorphosis. Measurements of ecdysteroid contents by radioimmunoassay methods have revealed distinctive larval, prepupal and pupal ecdysteroid peaks, as found in other species where development requires frequent stimulation by the centrally produced hormones. Mass fragmentographic analysis has indicated that more than 80% of the ecdysteroids detected by the radioimmunoassay during the peaks consists of ecdysterone. The prothoracic glands (PG) of D. maculatus larvae are located in the head capsule and in the cervical region. They can be separated from the body by neck-ligation. Only the larval peak of ecdysterone can be correlated with PG function. The prepupal and pupal peaks of ecdysterone are not produced by the central neuroendocrine system. The isolated abdominal fragments of larvae and pupae positively produce ecdysteroids in the course of their independent development, but this synthesis has been subnormal and occasionally delayed in time. In contrast, the isolated thoracic fragments produce well synchronized peaks of ecdysterone and this does not depend on the PG. It thus appears that some thoracic tissue other than PG is able to maintain the physiological concentration of ecdysterone by means of a concentration-dependent feed-back mechanism. Due to spontaneous metamorphosis and extremely good survival we have succeeded to induce in this species: a) premature formation of the prepupal cycle of ecdysterone synthesis in the penultimate larval instar by ligation; b) inhibition and delay of the prepupal peak by means of juvenoid treatments; c) reappearance of the prepupal peak in the inhibited “permanent larvae” by exogenous administration of ecdysterone, or; d) modification of the pupal peak by a qualitatively new pupal-pupal one after juvenoid treatments. These experimental transformations of the ecdysterone peaks have suggested that the kind and nature of the peaks are closely related to nature and stage of the ontogenetic development. We therefore believe that the process of larval-pupal reprogramming has occurred in this species long before the increase of ecdysterone titer in the body. Both the prepupal and pupal peaks do not represent a cause but they show to be rather consequences of the developmental programming. Further features associated with possible physiological role of ecdysteroids in insect development have been briefly discussed.

Osmeterial secretions of papilionid larvae in the genera Luehdorfia, Graphium and Atrophaneura (lepidoptera)

The osmeterial secretions of Luehdorfia larvae (two species and one subspecies) are chemically characterized as monoterpene hydrocarbons consisting mainly of α-pinene, β-myrcene (major component), limonene and terpinolene, whereas those of Atrophaneura larvae (one species) are marked by sesquiterpenic compounds (the principal component being δ-cadinene). In contrast, the osmetrical secretions of Graphium larvae (two species) predominantly consist of a mixture of aliphatic acids and esters ( iso-butyric acid, 2-methylbutyric acid and their methyl and ethyl esters). In all the species examined of the three genera, the secretions of the last and the penultimate larval instars were of similar composition, which is unlike the results reported previously for larvae of Papilio protenor.

Uric acid excretion in larval Manduca sexta

The characteristic coating of frass of last-instar tobacco hornworms, Manduca sexta, reared on artificial diet, proved to be uric acid. Results indicated that uric acid is the major nitrogenous excretory product; during most of the larval feeding stage, 5–7% of the excreta (dry weight basis) was uric acid; only minute quantities of allantoic acid were present. The rate of uric acid excretion was linear for the periods when coated pellets were observed. Abrupt increases in uric acid resulted from delays in pellet expulsion associated with delays in feeding activity. A distinctive coating was not generated by penultimate instar larvae, but abrupt changes in uric acid content did occur, which suggests that the phenomenon of coated frass is directly related to a differential in uric acid concentration. The source of uric acid in the frass was the Malpighian tubule system. The transition period between feeding and the wandering stage was a time of rapid decrease in uric acid excretion; there were only low levels in the last fecal pellets and none in Malpighian ampullae of wandering-stage larvae. Since the first appearance of coated fecal pellets preceded the release of ecdysone by about 24 hr, the involvement of this hormone was not indicated.

Life history patterns of some species of Hydropsyche (Trichoptera: Hydropsychidae) in southern Ontario

Life history patterns are described for five species of Hydropsyche in the Credit and Humber rivers. Larvae of univoltine species matured in May or June. Bivoltine species had a fast-growing summer generation of larvae maturing in July or August and a slow-growing winter generation of larvae maturing in April or May. Hydropsyche betteni, H. dicantha, H. slossonae, and H. sparna were bivoltine at some sampling stations, univoltine at others. Hydropsyche scalaris was exclusively univoltine. Most species were represented by many instars concurrently during summer. Overwintering occurred in a smaller number of instars. Species that overwintered in the final or penultimate larval instar were more likely to mature early and to be bivoltine than species overwintering in younger instars. High summer water temperatures and (or) food quality increased growth rates and the probability that a summer generation could mature before fall. Smaller species were more likely to be multivoltine than larger species.Differences among the life cycles of the five species did not suggest effective ecological segregation in summer in terms of larval size. Different overwintering stages among the species may prove to be important factors controlling the number of generations per year.

Juvenoid action on the total body metabolism in larvae of a noctuid moth

A juvenoid compound known as methoprene has no effect on growth and respiratory metabolism in penultimate instar larvae that contain endogenous juvenile hormone. In the last larval instar the juvenoid induces enormously large somatic growth and postpones pupal ecdysis although it does not increase the overall metabolic intensity beyond certain limits. The metabolic changes caused by the juvenoid were more pronounced when connected with formation of the supernumerary larval instars. After this developmental change the larvae further continued to grow on and turned finally into the conspicuous giants. The overall metabolic capacity of the supernumerary larval tissues surpassed the above limitations determined by the body of a normal last instar larva. However, unlike in some other species, there was no hypermetabolic response to juvenoid treatments in this material.

Disturbance of larval orientation in Spodoptera frugiperda (Lepidoptera : Noctuidae) by microsurgical removal of endocrine centres

The larvae of S. frugiperda showed differences in orientation depending largely upon their physiological states. A preference for wet conditions manifested itself in the orientation of the penultimate instar larvae, whereas the larvae of the final stage exhibited preference neither for wet nor for dry conditions. However, the ‘light level’ proved to be of prime importance in the final stage, such that it drove the larvae almost exclusively to the dark, while it appeared to be of secondary importance in the preceding stage where the larvae only responded photopositively if deprived of the humidity-preference. Microsurgical removal of the medial neurosecretory cells in the pars intercerebralis resulted in a preference for dry conditions in both larval instars concomitant with an appreciable increase in the total water content of the larvae. The normal situation could be easily restored by implantation of some active cells. Allatectomy failed to alter the normal preferences of the final instar larvae, whereas it induced a humidity-preference in the larvae of the penultimate stage, a response exhibited normally by intact larvae of the final stage. This result might suggest the implication of the morphogenetic hormones in some way or another in the control of larval response to the ‘humidity level’. However, photopreference in both larval instars appeared to be genetically controlled.