Stadium Larvae Research Articles

Effects of a New Juvenile Hormone Mimic, NC-170, on Metamorphosis and Diapause of the Small Brown Planthopper, <i>Laodelphax striatellus</i>

Effects of a new jevenile hormone mimic (JHM), NC-170 [4-chloro-5-(6-chloro-3-pyridylmethoxy)-2-(3,4-dichlorophenyl)-pyridazine-3(H)-one], on the metamorphosis and diapause of the small brown planthopper were examined in comparison with those of Juvenile Hormone(JH)-1, 2 and 3. NC-170 had a strong metamorphosis-inhibiting activity with a ID50 of 0. 42 pg/larva when topically applied to nondiapause mid-4th stadium larvae. The three JHs showed no signs of metamorphosis inhibition not only by single topical application to mid-4th stadium larvae at a maximal dosage (300 ng/larvae), but also by daily repeated topical applications to larvae in the early 4th to 5th stadium (30 ng/day/larva). When the planthoppers were reared at 20C under short-day (8L/16D) photoperiods throughout the embryonic and larval stages, the development was considerably retarded in the 4th larval stadium, which is apparently the state of diapause. They resumed normal (nondiapause) development when 20-hydroxyecdysone was topically applied. Contact applications of 100 ppm NC-170 through the larval stage prevented larvae from entering diapause, and topical applications of the chemical terminated diapause and induced 4th larval molts about 8 days after treatment. JH-1 also terminated diapause in the same manner at the same dosages as NC-170.

The effect of host age ofLymantria dispar larvae [Lep.: Lymantriidae] on the development ofGlyptapanteles liparidis [Hym.: Braconidae

The endoparasitic development ofG. liparidis was examined in 3 different host stages of gypsy moth larvae. Hatching ofG. liparidis-larvae occurred 3 to 5 days after oviposition in hosts parasitized during their premoulting period, and after 5 to 7 days in those parasitized in the 3rd midinstar state. The parasites generally moulted to the 2nd larval instar between the 11th and 13th day in the first group, and between the 13th and 15th day in the latter, when they had reached a volume of 0.04–0.05 mm3. The positive correlation between host ecdysis and the ecdysis of 1st stadium larvae to L2 suggested that host moulting influenced the development of the parasitoid larvae. Emergence from the host larvae occurred at 20°C after 27 days on average, and coincided with the parasites moulting to the 3rd instar. Five to 7 days after spinning their cocoons near the developmentally arrested host larva, the male, and 1 to 2 days later the female wasps eclosed. Due to the variation in the number of parasites per host, no difference was observed between the hosts parasitized at various stages; however, a tendency for later parasitized hosts to contain more parasite larvae was evident. The nutritional conditions of the moth parental generation influenced both host and parasite development. On the other hand no influence of host age was observed on emergence dates of larvae and wasps.

Effect of larval age and dosage of nuclear polyhedrosis virus on the susceptibility of diamondback moth, Plutella xylostella

AbstractA laboratory experiment was conducted to study the efficacy of nuclear polyhedrosis virus (NPV) with nine concentrations against all stadia of Plutella xylostella (L). The susceptibility of the larvae was correlated negatively with the period of development of the larvae and positively with the virus concentrations. The highest mortality of 84% was recorded in first stadium larvae compared to lowest mortality of 38% in fourth stadium larvae. The LC50 was 5.5 × 101 and 4.0 × 104 polyhedral inclusion bodies (PIB)/ml for first and fourth stadium larvae, respectively.

Biochemical ecology of the forest tent caterpillar: responses to dietary protein and phenolic glycosides.

Interactions between quaking aspen (Populus tremuloides) and the forest tent caterpillar (Malacosoma disstria) are likely to be influenced by leaf protein and phenolic glycoside levels, and insect detoxication activity. We investigated the direct and interactive effects of dietary protein and phenolic glycosides on larval performance and midgut enzyme activity of forest tent caterpillars. We conducted bioassays with six artificial diets, using both first and fourth stadium larvae. Four of the diets comprised a 2×2 factorial design-two levels of protein, each with and without phenolic glycosides. Additionally, we assayed high protein diets containing S,S,S-tributylphosphorotrithioate (DEF, an esterase inhibitor) and DEF plus phenolic glycosides. Enzyme solutions were prepared from midguts of sixth instars and assayed for β-glucosidase, esterase and glutathione transferase activities. First instar mortality and development times were higher for larvae on diets low in protein or containing phenolic glycosides. Effects of phenolic glycosides were especially pronounced at low protein levels and when administered with DEF. Fourth instar development times were prolonged, and growth rates reduced, in response to consumption of low protein diets. Effects of phenolic glycosides on growth were less pronounced, although the effect for larvae on the low protein diet was nearly significant. Activity of each of the enzyme systems was reduced in larvae reared on low protein diets, and esterase activity was induced in larvae fed phenolic glycosides. Our results suggest that larval performance may be strongly affected by levels of protein and phenolic glycosides commonly occurring in aspen foliage, and that these factors may play a role in differential defoliation of aspen by forest tent caterpillars.

Ecdysteroid binding sites localized by autoradiography in the central nervous system of precommitment fifth-stadium Manduca sexta larvae

Brains and subesophageal ganglia from day 3.5 fifth stadium larvae of Manduca sexta were incubated in vitro with 4 nM tritiated ponasterone A, a 20-hydroxyecdysone analog, to determine whether uptake and specific binding of ecdysteroids occur at a cellular level. These tissues, which were taken just prior to the commitment peak in the hemolymph-ecdysteroid titer, showed saturable uptake of 3H-ponasterone A after 40-60 min of incubation. Uptake was blocked by the addition of 400 nM unlabelled ponasterone A, or of 500 nM or 1000 nM 20-hydroxyecdysone. RH 5849, a synthetic 20-hydroxyecdysone agonist with a long half-life, for which ecdysteroid receptors have low affinity, also reduced ponasterone A uptake at a concentration of 10 microM. Autoradiographs of 4 microns sections of brains revealed distinct nuclear concentrations of silver grains over cell populations in the pars intercerebralis, pars lateralis, and ventral tritocerebrum. Nuclear labelling was also found in many small cells around the mushroom bodies and the neuropil, and between the inner and outer larval optic lobes. Nuclear labelling of cells in the subesophageal ganglion was observed in the fronto-medial and lateral regions, in small cells around the neuropil, and caudally in a few large neurons. In addition to cells with nuclear labelling, both brains and ganglia at this development stage contained cells with exclusively cytoplasmic or both nuclear and cytoplasmic labelling. None of these apparent binding sites were observed in the competition experiments, suggesting that the binding is specific.

Reversed-phase liquid chromatographic separation of juvenile hormone and its metabolites, and its application for an in vivo juvenile hormone catabolism study in Manduca sexta

A convenient reversed-phase liquid chromatographic method was developed to separate juvenile hormone (JH) and its metabolites. The known metabolites including JH acid, JH diol, and JH acid-diol, as well as an unknown metabolite, were efficiently separated within 25 min on a 50 × 4.6 mm polymer column using a linear gradient of acetonitrile:5 m m Hepes (pH 7.4) buffer. Use of the polymer column diminished tailing observed for the diol metabolite on a C 18 silica column, and allowed use of slightly basic buffers without concern of column instability. Use of buffer was essential to give good peak shape and reproducible retention behavior for the acidic metabolites. Using this method, an in vivo JH catabolism study was performed in fifth stadium larvae of Manduca sexta. Injected (10 R)-[ 3H]JH III was rapidly converted to JH acid-diol and to an unknown compound(s) indicating that, in addition to JH esterase, epoxide hydrolase and other reactions play an important role in the catabolism of JH.

Regulation of juvenile hormone esterase gene expression in the tobacco budworm ( Heliothis virescens)

The tissue distribution, developmental control, and induction of juvenile hormone esterase (JHE) mRNA was examined in Heliothis virescens using an 800-base pair fragment of a JHE cDNA clone. Northern hybridization analysis of poly(A) +RNA from fat body and integument of fifth stadium larvae indicated the presence of a single JHE mRNA species having an estimated length of 3 kilobases. On Day 2 of the fifth stadium (L5D2), basal JHE mRNA levels were 3-fold higher in the integument than the fat body, which correlated with the higher specific activity of the enzyme in the integument at this time. However, JHE mRNA levels in the fat body on Day 4 of the fifth stadium were 9-fold higher than on Day 2, while mRNA levels in the integument remained the same. This endogenous increase in JHE mRNA and activity in the fat body occurred at the time of peak hemolymph JHE activity. JHE mRNA was not detected in third stadium larvae which have very low levels of JHE activity. Treatment of L5D2 larvae with the juvenile hormone mimic epofenonane resulted in a 7- and 14-fold increase in the level of JHE mRNA in the integument and fat body, respectively. The mRNA induced in both tissues was of the same estimated length as the constitutively expressed message. The data indicate that the developmental regulation and induction of JHE can occur at the level of mRNA. There is evidence that the fat body secretes more JHE than does the integument and could be the major source of hemolymph JHE.

The juvenile hormone titre during the penultimate and ultimate larval stadia of Trichoplusia ni

The juvenile hormone titre in penultimate and last-stadium larvae of Trichoplusia ni (Hübner) was determined by a method utilizing gas chromatography-mass spectrometry with selected ion monitoring. Juvenile hormone II was the predominant homologue detected; highest levels were only ⋍400 pg/g. The juvenile hormone titre was high during the fourth stadium and during apolysis of the last stadia then declined to barely detectable levels just before cocoon spinning, and peaked again during the prepupal stage. Prepupal levels of this hormone were similar to those detected in 4th and 5th larval stadia suggesting an important biological role for juvenile hormone in the prepupa. Starvation of day-1, 5th stadium larvae or inhibition of in vivo juvenile hormone esterase activity with O-ethyl- S-phenyl-phosphoroamidothioate (EPPAT) resulted in higher levels of juvenile hormone in mid last-stadium larvae. T. ni larvae undergoing precocious development caused by parasitization with Chelonus sp. showed greatly reduced levels of juvenile hormone II compared to control larvae, but contained a considerable level of juvenile hormone III. Separate analysis of the host carcass and the parasite larva supported the hypothesis that the hymenopterous parasite was the major source of the juvenile hormone III detected.

In vivo and in vitro effects of epofenonane on juvenile hormone esterase activity in tissues of last stadium larvae of Trichoplusia ni

Topical application of the juvenoid, epofenonane, to last stadium postwandering larvae of Trichoplusia ni caused a precocious elevation of juvenile hormone esterase (JHE) activity that was tissue speific and time dependent. This increase in enzyme activity over controls was most dramatic in the hemolymph, whereas increases in the fat body were lower. Antibodies raised against JHE reacted on Western blots with a fat body and hemolymph protein present in epofenonane treated and untreated last stadium day 3 larvae. The abundance of this protein, which comigrated with JHE, closely coincided with the temporal increases in JHE catalytic activity that occurred in response to treatment in vivo with epofenonane. The presence of epofenonane (5–10,000 nM) in the medium at the start of fat body incubations failed to shift the temporal appearance of JHE activity or boost activity levels significantly over those of controls. If larvae were treated in vivo with epofenonane before fat body tissue was removed, only a small, but significant increase in JHE activity was found in vitro. The rate of enzyme secretion was insufficient to account for the rapid increases in enzyme activity that occur in the hemolymph in response to epofenonane, even though tissue held in vitro was deemed viable by monitoring lactate dehydrogenase activity in the medium, fat body intracellular ATP, and the incorporation of [ 35S]methionine into fat body protein. Fat body tissue removed from various aged last stadium larvae released enzyme at different rates in vitro.

Microplitis croceipes teratocytes cause developmental arrest of Heliothis virescens larvae

AbstractMicroplitis croceipes teratocytes placed into nonparasitized Heliothis virescens larvae survived in the absence of a parasitoid larva and caused developmental changes in the host. Expressions of these changes included delayed larval mortality, incomplete larval‐pupal ecdysis, or delayed pupation. Two day old 4th stadium H. virescens larvae were more sensitive to injected teratocytes than were 5th stadium larvae. Three day old teratocytes were more effective than were 6 day old teratocytes. The degree of response was related to the number of injected teratocytes. For example, 750 three day old teratocytes (the approximate number from a single parasitoid egg) caused delayed larval mortality in 96% of the treated larvae whereas 175 three day old teratocytes caused delayed larval mortality in only 33% of the treated larvae. Even a dose of 80 teratocytes resulted in 15% incomplete larval‐pupal ecdysis compared to 0% for controls. Treatment with hemocyte‐and teratocyte‐free hemolymph from parasitized larvae, hemocytes from nonparasitized H. virescens, unfertilized M. croceipes eggs, Cotesia congregata teratocytes, or Micrococcus lysodeikticus cells all had very little effect either on larval growth or development time.

Kinetics of binding and hydrolysis of juvenile hormone II in the hemolymph of Trichoplusia ni (Hübner)

Juvenile hormone II (JH II) was studied for the kinetic behavior in its reaction with JH-esterase and the binding protein from the hemolymph of the last instar larvae of the cabbage looper, Trichoplusia ni. Steady state kinetics revealed 70.6 nM and 65 nmol substrate hydrolyzed min −1 ml, respectively, for the K m and V max for JH hydrolysis and 175 nM and 8.1 μM, respectively, for the equilibrium dissociation constant ( K d) and the total concentration of the binding sites for the interaction with the binding protein. The rate constant for the dissociation of bound JH II was experimentally evaluated to be 0.095 min −1. This in turn enabled the second order rate constant of association to be calculated as 5.41 × 10 5 M −1 min −1. The slow tight binding inhibitor of JH-esterase, 3-octylthio 1,1,1-trifluoropropan-2-one, was applied to the enzyme for the Ackermann-Potter kinetic analysis. The data from this treatment indicated the compound acts as pseudo-irreversible and stoichiometric inhibitor and allowed determination of k cat (31.8 min −1) and the enzyme molar equivalency (1.5 μM) in whole hemolymph. The above determination helped compare the interaction of JH II with the two major components that regulated JH titer following biosynthesis. We conclude that degradation of JH in the hemolymph of prewandering last stadium larvae is described by the kinetic dissociation constant of the hemolymph binding protein. A model based on mass action is presented which may help to describe the distribution of JH in the last larval stadium.

In vivo fluctuation of JH, JH acid, and ecdysteroid titer, and JH esterase activity, during development of fifth stadium Manduca sexta

Juvenile hormone (JH), JH acid, and ecdysteroid titer, and JH esterase activity, were measured in hemolymph from synchronous last stadium larvae of Manduca sexta. JH and JH acids were identified and quantified by GC-MS: JH I and II (and the corresponding acids) were the predominant JH homologs detected in males or females. Maximum levels of JHs and JH acids were observed just following ecdysis to the fifth (last) stadium (day 0, 0 hr) and at the prepupal stage (day 6–day 7). JH titer (≥ 1 ng JH I or II/ml) was higher than JH acid titer (∼0.7 ng JH I acid or JH II acid/ml) in very early fifth stadium larvae. However, this was reversed at the prepupal stage when higher titers of JH acids than JH were observed. JH acid titer began to rise prior to JH titer at the prepupal stage. JH esterase activity rose significantly only after JH or JH acid titers had begun to decline; maximum JH esterase activity was observed at day 3 and day 8. Ecdysteroid titer (measured by RIA) decreased during the last larval molt to a low level by day 0 (0 hr) and to undetectable levels at day 0 (12 hr) of the fifth stadium, by which time JH and JH acid levels had also declined substantially. Just prior to wandering, a small ecdysteroid peak was noted and a slightly elevated level of ecdysteroid was maintained for a further 2 days before a surge in ecdysteroid titer occurred at the prepupal stage, in synchrony with JH and JH acid titer maxima. There was no sexual dimorphism in timing or magnitude of JH, JH acid, and ecdysteroid titer or JH esterase activity.

The evaluation of anti juvenile hormones using last stadium larvae of the cabbage looper, Trichoplusia ni (Hübner)

Treatment of prepupae of the cabbage looper, Trichoplusia ni (Hübner), with the anti juvenile hormone (AJH) fluoromevalonolactone (FMev) reduces juvenile hormone esterase (JHE) activity and either delays or inhibits the normal larval-pupal ecdysis in a dose-dependent manner, resulting in the formation of pupae with a suite of characteristic abnormalities. A variety of putative AJHs were evaluated using these teratogenic effects and changes in JHE activity as indicators of AJH activity. Like FMev, the compactin analog L-643,049-01K01 and, at very high doses, 3,3-dichloro-2-propenyl hexanoate (DPH) behaved as AJHs. FMev and L-643,049-01K01 were most effective when larvae were treated at gut purge, while DPH was the most effective after the initiation of spinning behavior.

The effect of juvenoids, anti juvenile hormone agents, and several intermediates of juvenile hormone biosynthesis on the in vivo juvenile hormone levels in Manduca sexta larvae

JH levels in Manduca sexta larvae were suppressed in a dose dependent fashion following topical treatment with either the potent juvenoids fenoxycarb or hydroprene, or the anti juvenile hormone (AJH) agent (3R,S)- fluoromevalonolactone (FMev>). We determined ED 50 values, expressed as the amount of substance (applied topically to early third stadium larvae) required to lower JH titer (determined in the early fourth stadium) by 50%; these values were 0.001, 0.1 and 3 μg for fenoxycarb, hydroprene, and FMev>, respectively. Combined enzymatic and chemical methods were used to prepare a sample containing predominatly (−)FMev>. Comparison of dose-response curves obtained following treatment of M. sexta larvae with (−)FMev>;, versus treatment with racemic FMev>f, confirmed that the (−)enantiomer is the active principle. Similarly, we showed that the (−)optical isomer of the “mixed” JH agonist/antagonist ethyl 4-[2-(t- butylcarbonyloxy) butoxy] benzoate (ETB) is the active principle of the racemic mixture. Injection or topical application of several intermediates of JH biosynthesis (mevalonlactone, homomevalonolactone or farnesoic acid) had little effect on resultant JH titer profiles in M. sexta larvae.

Hydrocarbon accumulation and lipid biosynthesis during larval development in the cabbage looper, Trichoplusia ni

Larvae of the cabbage looper, Trichoplusia ni, were analyzed for the accumulation and biosynthesis of cuticular and internal hydrocarbon at closely spaced and accurately timed intervals during the fourth and fifth stadia. Large differences in the incorporation of [1- 14C]acetate into hydrocarbon were observed at different times during larval development. Much higher incorporation was observed during feeding stages as compared to wandering stages, while lowest rates of biosynthesis occurred just prior to ecdysis. Fourth stadia wanderers accumulated increased amounts of internal hydrocarbon, which is apparently used to cover the newly forming cuticle. During the fourth to fifth stadium moult insects lost all cuticular hydrocarbon that was present on the old cuticle (about 8 μg/insect) and had about 8 μg/insect on the surface of the newly exposed cuticle. During the fourth stadium incorporation of [1- 14C]acetate into total lipid declined between feeding and wandering stages from 24% of injected radiolabel to 7%. Similar decreases in lipid biosynthesis were observed between feeders and wanderers in fifth stadium larvae with the greatest decrease found in the triacylglycerol fraction. These results document dramatic changes in the accumulation and biosynthesis of hydrocarbon and other lipids during larval development.

Juvenile hormone esterase activity in developmentally synchronous ultimate stadium larvae of the migrant insect, Anticarsia gemmatalis

Growth and development of cumulatively aged velvetbean caterpiller ( Anticarsia gemmatalis Hübner) larvae were characterized on the basis of weight, stadium and gate. Criteria were developed for the selection of synchronous ultimate (5th)-stadium larvae before they entered the ultimate stadium. Using synchronous larvae, precise determinations were made of haemolymph juvenile hormone III esterase activity, α-naphthyl acetate esterase activity and protein concentration. The first of two juvenile hormone III esterase activity peaks coincided with the time of maximal weight gain while the second peak coincided with formation of the prepupa. Juvenile hormones (5 × 10 −6 M) I, II and III were hydrolyzed at similar rates. The α-naphthyl acetate esterase activity and protein patterns differed from the pattern of juvenile hormone III esterase activity. A major peak of α-naphthyl acetate esterase activity was observed during the wandering phase of the penultimate and ultimate stadia. Haemolymph protein concentration increased to a peak, which coincided with the initiation of wandering, and then decreased through pupation.

Tyrosine and tyrosine glucoside titres in whole animals and tissues during development of the tobacco hornworm Manduca sexta (L.)

Tyrosine glucoside first appeared in two-day-old fifth stadium larvae and reached maximum whole body titres in pharate pupae (2.5 mg/g). A rapid decline occurred shortly before larval-pupal ecdysis as the free tyrosine pool increased to peak titres at ecdysis (1.4 mg/g). Both tyrosine and tyrosine glucoside decreased to low levels (0.5 mg/g) as the pupal cuticle tanned. Tyrosine again increased in one-day-old pupae and remained at high levels (1.5 mg/g) until the beginning of darkening of the pharate adult. Tyrosine glucoside remained at relatively low titres (0.5 mg/g) during pharate adult development. Both tyrosine and tyrosine glucoside increased slightly during adult eclosion and decreased to very low levels after adult tanning. Nearly all the tyrosine glucoside and two-thirds of the tyrosine occurred in the haemolymph where the concentrations during development paralleled those in whole body. Much lower amounts were present in fat body, integument, and gut.

The haemolymph juvenile hormone esterase of Manduca sexta (L.)—inhibition and regulation

A number of compounds were screened as potential juvenile hormone esterase (JHE) inhibitors, of which O- ethyl-S- phenyl phosphoramidothiolate was found to be the most potent ( I 50 = 4 × 10 −9 M). Gel filtration of the haemolymph resolved only one peak of enzyme activity capable of rapidly hydrolyzing juvenile hormone (JH). JHE titres were monitored during the last instar in wild type and black mutant larvae of Manduca sexta (L.). In wildtype larvae the increase in JHE activity was most rapid during the first 18 h and then became a function of weight until mid-day three. Although the prewandering peak of the JHE activity occurred on day three in both strains, the JHE activity peak was greatly reduced in black mutant and allatectomized larvae. JHE activity was induced in freshly ecdysed final stadium larvae by juvenoid applications. When pharate fifth stadium larvae were either neck- or abdomen-ligated, or when freshly ecdysed larvae were starved, the levels of JHE activity remained low and could not be induced by juvenoids. Thus, JH may complement the action, or stimulate the production of an unknown factor(s) responsible for increasing JHE activity during the prewandering phase. Both strains also possessed a second smaller JHE activity peak of identical size just prior to pupation (day eight). Just prior to pupation the haemolymph JHE activity can be induced by epofenonane to higher than normal levels in black mutant and in allatectomized larvae, as well as in untreated insects, suggesting that JH directly stimulates the second JHE peak.

Detection of nuclear polyhedrosis virus infection in Heliothis spp. by agar gel double diffusion

Nuclear Polyhedrosis virus infection was detected in single Heliothis zea larvae by the agar gel double diffusion technique using antiserum to alkali-solubilized polyhedra. Virus bands were observed in 2nd-5th stadium larvae following homogenization in 0.1 M Na 2CO 3-0.05 M NaCl, pH 11.0, in approximately one-half the time necessary for mortality to occur. In a field test, virus infection was detected by this method as early as 3 days after virus treatment.