Stimulated synthesis of the female-specific storage protein in male larvae of the silkworm Bombyx mori treated with juvenile hormone analog

Application of methoprene to fourth (penultimate) instar larvae of the silkworm Bombyx mori induced the appearance of the feeding dauer larvae at the fifth (last) instar and prevented pupal metamorphosis. Methoprene also increased the protein concentrations of hemolymph last instar larvae by preventing sequestration of storage proteins by the fat body. Usually, the female-specific storage protein 1 (SP1)* disappears from the male hemolymph at the time of the last larval instar. However, exposure of male larvae to methoprene at the penultimate instar enhanced the accumulation of SP1 in the hemolymph. The SP1 accumulated in males did not differ in molecular weight and immunoreactivity from the SP1 produced in female larvae. Both sexes of fourth instar larvae allatectomized on day 1 instantly accumulated SP1 in the hemolymph, and methoprene application after allatectomy suppressed the hemolymph accumulation of the SP1. In contrast, if allatectomy was carried out at a later stage of the fourth larval instar, SP1 concentration in hemolymph of fifth instar larvae did not increase, suggesting the different juvenile hormone action for regulation of SP1 synthesis in the penultimate instar larvae of silkworms.     

Tissue- and Stage-Specific Expression of Sericin Genes in the Middle Silk Gland of Bombyx mori

Sericin gene expression in the middle silk glands during Bombyx mori larval development was analyzed using probes from a genomic DNA clone for 10.5 kb sericin mRNA. The 10.5 kb mRNA, the most abundant in the fifth instar, is not detected in the third feeding, fourth feeding and fourth moulting stages. It becomes detectable at 2 days of the fifth instar, and accumulates rapidly. The second major mRNA in the late fifth instar, a 9.0 kb component having a similar sequence to the 10.5 kb mRNA, becomes detectable only at 6 and 7 days of the instar by use of the repetitious coding sequence probe of the sericin clone. Using the same probe about 20 kb RNAs with a fainter intensity than that of the major mRNAs are detected. They are present extremely faintly in the third and fourth feeding stages, disappear in the fourth moulting stage, and increase in the fifth instar. Two other faint poly(A)⁺ RNA components are detected by a DNA probe containing the 5' end sequence of the sericin clone. One is 4.3 kb, and appears in the third, fourth and fifth feeding stages but not in the fourth moulting stage. The other is 3.0 kb, and it becomes detectable after 1 day of the fifth instar.     

Inhibitory Action of an Imidazole Compound on Ecdysone Synthesis in Prothoracic Glands of the Silkworm, Bombyx mori

The precocious pupation was induced either by allatectomy at the time of third ecdysis or by topical application of an imidazole compound (KK-42; 1-benzyl-5-[( E )-2, 6-dimethyl-1, 5-heptadienyl] imidazole) to the fourth (penultimate) instar larvae of the silkworm, Bombyx mori. However, the critical period for KK-42 treatment in induction of precocious pupation was longer than that for allatectomy. The effects of KK-42 depended on the doses applied and a half-maximum dose was estimated to be approx. 10 μg/larva. KK-42 suppressed the increase in hemolymph ecdysteroid titres leading to larval ecdysis in controls. Ecdysteroid levels remained at low levels for about 6 days after the treatment, followed by an increase toward precocious pupation. When the prothoracic glands from the mature fifth instar larvac were incubated in vitro in Grace's medium containing various concentrations of KK-42, secretion of ecdysone into the medium was suppressed depending upon the doses of KK-42 added and a half-inhibition concentration was estimated to be approx. 1 nM. Thus, KK-42 was shown to be an inhibitory agent to ecdysteroid secretion in silkworm larvae.     

Regulation of expression of arylphorin and female-specific protein mRNAs in the tobacco hornworm, Manduca sexta

Two non-cross-hybridizing cDNA clones were isolated from a lambda gt11 cDNA library prepared from Day 2 fifth instar female fat body of Manduca sexta and shown by hybrid selection to code respectively for the two storage proteins arylphorin and female-specific protein (FSP). Analysis of the developmental expression of arylphorin showed its presence during the feeding phases of the penultimate (fourth) and final (fifth) larval instars and its absence during the molt. Abdominal ligation of larvae followed by infusion of Grace's medium showed that this amino acid-rich medium was able to maintain arylphorin expression in fourth instar larvae, but not continued high expression in fifth instar larvae. This nutrient medium however was sufficient to allow initiation of expression in newly ecdysed fifth larval abdomens. Infusion of 5 micrograms 20-hydroxyecdysone (20HE) caused a significant reduction of arylphorin RNA in ligated fourth larval abdomens, whereas 50 micrograms was required in Day 2 fifth larval abdomens to suppress this RNA. Thus, both the lack of incoming nutrients and the rising titer of ecdysteroid contribute to the loss of arylphorin mRNA at the molts and at wandering. By contrast, FSP mRNA was first detected in females on Day 2 of the fifth instar, but not in males until wandering, and then was present throughout the prepupal period. In females allatectomy caused the precocious appearance of FSP mRNA which was prevented by application of 10 micrograms methoprene, a juvenile hormone analog. Expression of FSP mRNA in males however appeared to be independent of hormonal milieu.     

Death commitment in the anterior silk gland of the silkworm, Bombyx mori

The insect steroid hormone, 20-hydroxyecdysone (20E) triggers the programmed cell death (PCD) of the anterior silk glands (ASGs) of the silkworm, Bombyx mori. We tried to determine the time of commitment to die (death commitment) by examining ASG responses to 20E and juvenile hormone analogue (JHA) in vivo as well as in vitro. The ASGs obtained late on day 6 of the fifth instar completed PCD when cultured with 20E, while the ASGs obtained on day 4 and cultured with 20E did not undergo PCD. The ASGs became competent to respond to 20E at mid-day 5. The ASGs with responsiveness to 20E were not sensitive to JHA, indicating that the ASGs were committed to die before becoming capable of responding to 20E. Topical application of JHA on day 4 suppressed 20E-induced PCD, but that on day 5 failed to do so, indicating that the death commitment might occur between day 4 and 5. We also determined the time of death commitment after allatectomy of the fourth instar larvae, a procedure that induced the precocious PCD. Timed application of JHA and culture of ASGs with 20E in the presence of JHA showed that the ASGs had lost their sensitivity to JHA between 72 and 96 h after allatectomy, i.e. 24-48 h before precocious gut purge in the allatectomized larvae. This result is similar to that obtained in the fifth instar. We conclude that the cellular commitment to die takes place one day before the ASGs become competent to respond to 20E.     

Growth and silk formation of silkworm larvae influenced by phytoecdysones

The fourth and fifth instar larvae of the silkworm were reared on artificial diets containing ponasterone A, ecdysterone, and inokosterone. The growth of the larvae and their silk glands, fibroin-synthesizing activity, and silk formation have been investigated. With a diet containing ponasterone A, the fourth instar larvae grew slowly and only a few larvae could ecdyse, while the growth of the fifth instar larvae was disturbed and they died with a darkening of the skin. Ponasterone A also inhibited the growth of the silk glands during the fifth instar. In contrast, the other two phytoecdysones did not greatly influence larval growth. The fourth instar larvae grew rapidly and their ecdysis was advanced with a diet which contained 10 μg of inokosterone/1 g of dry diet. The diet which contained 5 μg of ecdysterone or 10 μg of inokosterone/1 g of dry diet accelerated maturation, while that containing 10 or 20 μg of ecdysterone, or 40 μg of inokosterone, delayed maturation of the fifth instar larvae.Only phytoecdysones caused a decrease in growth of the silk glands in the early half of the instar, and a large amount of phytoecdysones accelerated their growth during the last part of the fifth instar. The fibroin-synthesizing activity was levelled up by feeding ecdysterone and inokosterone, and inokosterone appreciably stimulated activity. Assay of in vitro fibroin synthesis showed that ponasterone A competed with ecdysterone in a stimulative action. Silk formation was much lower in larvae fed the diet containing 5 μg of ecdysterone or 10 μg of inokosterone/1 g of dry diet and was far greater in larvae fed the diet containing 40 μg of inokosterone than in the controls.     

The source and action of head factors regulating juvenile hormone esterase in larvae of the cabbage looper, Trichoplusia ni

Brain (median or lateral regions) or suboesophageal ganglion (SOG) homogenates of Day 1 fifth instar larvae of Trichoplusia ni induced the appearance of haemolymph juvenile hormone esterase (JHE) when injected into Day 1, Day 2 or early Day 4 fifth instar ligated hosts. Brain and SOG homogenates of late fourth instars also induced JHE when injected into Day 1 hosts, whole late fifth instar and pupal tissue did not. The pattern of JHE induction by early fourth through Day 3 fifth instar brain and SOG homogenates correlated with natural haemolymph JHE activity occurring at these times. Implantation of late fourth and Day 1 fifth instar brains and/or SOG into similar age hosts similarly induced JHE activity while prothoracic and abdominal ganglia did not. The relative levels of induction following implantation were SOG<brain<brain+SOG. JHE activity which appears in the haemolymph following injection of brain homogenates appears to be largely due to a single enzyme which has an isoelectric point indistinguishable from that of the natural haemolymph enzyme. Evidence is presented which suggests that inhibitory as well as stimulatory brain factors are involved in JHE regulation.     

Induction of Non-diapause Eggs by Imidazole Derivative KK-42 in the Diapause Type of Bombyx mori Silkworm

The injection of an imidazole compound, KK-42, into fifth instar larvae of a silkworm (Bombyx mori, Daizo strain), which had been destined to produce diapause eggs, induced the moths to lay non-diapause eggs. The critical period for KK-42 injection in the induction of non-diapause eggs was 24 h to 72 h after the fourth ecdysis. Topical application of KK-42 to 48 h-old fifth instar larvae also induced non-diapause eggs.     

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.     

马尾松毛虫过氧化氢酶及过氧化物酶与耐药性的关系

马尾松毛虫Dendrolimus punctatus幼虫体内存在着过氧化氢酶(CAT)和过氧化物酶(POD)。4龄幼虫的CAT和POD活力较大,其次是6龄幼虫,5龄幼虫的CAT和POD活力较4龄和6龄幼虫低。醚菊酯(etofenprox)处理后,在兴奋期(30 min),CAT和POD活力水平上升。4龄和6龄幼虫在抑制期(50 min以后),CAT和POD活力呈波动式上升,接近死亡时下降。5龄幼虫的CAT和POD活力呈波动式上升,接近死亡时下降。5龄幼虫的CAT在抑制期保持比正常虫体高的活力。结果表明,马尾松毛虫4龄、5龄和6龄幼虫与耐药性存在一定的相关性,研制酶的抑制剂具有实用意义。根据毒力测定结果,马尾松毛虫幼虫对醚菊酯的耐药力,5龄是4龄的1.43倍,6龄是4龄的1.72倍。因此,药物防治的合理时期应掌握在4龄以前较适宜。     

Influence of juvenile hormone on growth and digestion in fifth instar larvae and adults of Locusta migratoria

Food utilization was measured in female fifth instar larvae and adults of the migratory locust by following the weight of food ingested, the weight of faeces produced, and the increase in body weight. These parameters were measured in normally developing locusts, in locusts that had been implanted with a pair of active corpora allata (CA) in the beginning of the fifth instar period, and in allatectomized locusts, operated on the first day after adult ecdysis.A high titre of corpus allatum hormone results in a considerably higher water content of the insects; allatectomy reduces this content. The dry weight of the locusts is not essentially influenced by excess or absence of juvenile hormone.Food consumption in corpora-allata-implanted locusts does not differ from that in normally developing insects. Within each developmental period the digestive capacity remains constant, but the absolute value of this capacity may differ between the two developmental periods. The approximate digestibility is somewhat higher after CA-implantation and lower after allatectomy. The efficiency of conversion of digested food into body substance is greater in normally developing larvae than in adults. CA-implantation lowers this efficiency in developing larvae. Allatectomy slightly raises the efficiency of conversion in adult locusts.In the second half period of larval development, CA-implantation raises the respiratory rate, as estimated by measuring oxygen consumption. During adult development no significant influence of CA-implantation on respiration was established. Relations between the amount of food digested, the efficiency of conversion of digested food into body substance, and the respiratory activity are discussed.     

Effects of the parasitization of a braconid, Apanteles, on the blood of its host, Pieris

Parasitization of a braconid wasp, Apanteles glomeratus, of larvae of a common cabbage butterfly, Pieris rapae crucivora, caused changes in differential haemocyte count (DHC), total haemocyte count (THC), and encapsulative capacity against dead eggs of Apanteles in the fourth and fifth instar host larvae.However, no correlation could be found between the number of Apanteles eggs deposited and THC of the middle fourth instar host larvae or between the number of parasitoid larvae and specific gravity of the haemolymph from the late fifth instar host larvae.From the changes in DHC and in THC of both non-parasitized and parasitized Pieris larvae, an increase in the number of plasmatocytes of non-parasitized Pieris larvae in the early fourth instar period was supposed to be due to transformation of prohaemocytes into plasmatocytes, and a low population of plasmatocytes of parasitized larvae in the comparable period was assumed to be due to a suppression of transformation of prohaemocytes by some factor released from the parasitoid eggs.Failure of the parasitized fourth instar Pieris larvae to encapsulate injected dead eggs of Apanteles indicated that the parasitoid embryos were, in some way, actively inhibiting the encapsulation reactions of the host.The increase in THC of the parasitized fifth instar larvae could not be ascribed to a decrease in the volume of host haemolymph. Rather it could be interpreted by a suppression of adhesive capacity of haemocytes in the host haemocoel to tissue surfaces.Reduced encapsulative capacity of the parasitized fifth instar larvae might be attributed either to a depression of the adhesive activity of plasmatocytes resulting from a depletion of energy source for haemocytes in the host haemolymph by parasitization, or from an active suppression of adhesiveness of the plasmatocytes by secretions from ‘giant cells’ (teratocytes) originated from the parasitoid.     

Ontogenetic shifts in the functional response and interference interactions of Rhyacophila dorsalis larvae (Trichoptera)

1. Ontogenetic shifts in predator behaviour can affect the assessment of food‐web structure and the development of predator–prey models. Therefore, it is important to establish if the functional response and interference interactions differ between life‐stages. These hypotheses were tested by (i) comparing the functional response of second, third, fourth and fifth larval instars of Rhyacophila dorsalis, using three stream tanks with one Rhyacophila larva per tank and one of 10 prey densities between 20 and 200 larvae of Chironomus sp.; (ii) using other experiments to assess interference within instars (two to five larvae of the same instar per tank), and between pairs of different instars (one, two or three larvae per instar; total predator densities of two, four or six larvae per tank). 2. The first hypothesis was supported. The number of prey eaten by each instar increased with prey density, the relationship being described by a type II model. The curvilinear response was stronger for fourth and fifth instars than for second and third instars. Mean handling time did not change significantly with prey density, and increased with decreasing instar number from 169 s for fifth instars to 200 s for second instars. Attack rate decreased progressively with decreasing instar number. Handling time varied considerably for each predator–prey encounter, but was normally distributed for each predator instar. Variations in attack rate and handling time were related to differences in activity between instars, fourth and fifth instars being more active and aggressive than second and third instars, and having a higher food intake. 3. The second hypothesis was partially supported. In the interference experiments between larvae of the same instar or different instars, mean handling time did not change significantly with increasing predator density, and attack rate did not change for second and third instars but decreased curvilinearly for fourth and fifth instars. Interference between some instars could not be studied because insufficient second instars were available at the same time as fourth and fifth instars, and most third instars were eaten by fourth and fifth instars in the experiments. Prey capture always decreased with decreasing attack rate. Therefore, interference reduced prey consumption in fourth and fifth instars, but not in second and third instars. The varying feeding responses of different instars should be taken into account when assessing their role in predator–prey relationships in the field.     

Corazonin reduces the spinning rate in the silkworm,Bombyx mori

The insect neuropeptide, [Arg⁷]-corazonin was injected into larvae of the silkworm, Bombyx mori to investigate its influence on development and behavior. A single injection of 50 pmol of corazonin into the fourth and fifth instar larvae induced prolongation of the spinning period in all experimental groups except for those injected on day 10 of the fifth instar. The injection also caused a prolongation of the pupal period in some experimental groups, while it had no effect on the timing of larval ecdysis and the length of feeding period of the fifth instar. The spinning period was significantly prolonged even at a low dose of 1 pmol. Both the spinning rate and the rate of increase in hemolymph ecdysteroid level during the spinning stage were reduced by injection of corazonin. However, corazonin injection during days 5-7 of the fifth instar reduced the spinning rate without influencing the ecdysteroid level until the end of day 8, thereafter the rate of increase in hemolymph ecdysteroid level was slower in the corazonin-injected larvae than in the control larvae. Therefore, the suppressed ecdysteroid level observed in the corazonin-injected larvae appears to be a result rather than a cause of the reduced spinning rate. This study is the first published report for the corazonin effect on the behavior in insects.     

Quantitative determination of the juvenile hormones in the haemolymph of Locusta migratoria during normal development and after implantation of corpora allata

Simultaneous quantitative determination of the three naturally occurring juvenile hormones in insects (JH-I, JH-II and JH-III) was performed on haemolymph samples of both normally developing locusts and locusts implanted with active corpora allata, using capillary gas chromatography with electron capture detection.In fourth instar female larvae, 24–48 hr after the third ecdysis, as well as in adult females, 18 days after the imaginal ecdysis, only JH-III was detected. In fifth instar female larvae JH-III was present in very low concentrations, if at all.After implantation of four pairs of corpora allata taken from young fourth instar female larvae or one pair or corpora allata taken from adult females into fifth instar female larvae 0–24 hr after ecdysis, an elevation of the JH-III titre was observed. Neither JH-I nor JH-II could be detected. The amount of JH-III, already elevated 2 hr after implantation, remained high for several days in comparison to that of control insects. On the third day after the subsequent moult the JH-III level was comparable to that of normally developing fifth instar larvae. Factors involved in the achievement of the haemolymph JH-titre are discussed.     

Effect of KK-42 on growth,development, molting,and metamorphosis of the european corn borer,Ostrinia nubilalis (Hübner)

KK-42 (1-benzyl-5-[(E)-2,6-dimethyl-1,5-heptadienyl]imidazole), administered by feeding, delayed the growth and development of nondiapause-bound and diapause-bound Ostrinia nubilalis larvae and increased the length of the instar. At doses of 80–240 ppm, 62–100% of nondiapause-bound fourth instars precociously pupated or remained as fourth instars, while 52–100% of diapause-bound fourth instars did not molt to the fifth instar. Injection of these nondiapause- and diapause-bound KK-42-fed fourth instars with ecdysone elicited a molt and resulted in the production of larval-pupal intermediates. When mature fourth instar controls were similarly injected, they molted into normal fifth instars. These results support the view that KK-42 delays/inhibits ecdysteroid production. Both eupyrene and apyrene spermiogenesis were prematurely initiated in nondiapause-bound fourth instars that were fed on medium containing 160 ppm KK-42. Fenoxycarb, a potent juvenile hormone mimic, rescued nondiapause-bound fourth instars from precocious pupation. All fenoxycarbtreated larvae either molted to the fifth instar or remained as fourth instars and eventually died. These results support the view that treatment with KK-42 inhibits JH production. When KK-42 treatment was begun in the third instar, a considerable number of nondiapause-bound and some diapause-bound third instars precociously molted to the fifth instar. There was a correlation between weight and the incidence of precocious molting in that third instars destined to skip the fourth instar attained a weight, as pharate fifth instars, of two to three times more than pharate fourth instar controls. Similarly, fourth instars that were destined to undergo precocious pupation attained a weight, as pharate pupae, that was approximately two times more than pharate fifth instar controls. More potent analogues of KK-42 may prove useful in controlling populations of 0. nubilalis by interfering with their growth, development, and metamorphosis. © 1995 Witey-Liss, Inc.

1 This article is a US Government work and, as such, is, in the public domain in the United States of America.

     

A granulosis virus,possible biological agent for control of Adoxophyes orana (Lepidoptera: Tortricidae) in apple orchards: I. Mass production

Young larvae of Adoxophyes orana (apple race) were infected with a granulosis virus by feeding them artificial diet containing the virus suspension. The susceptibility decreased remarkably after the fourth to fifth instar. Infected larvae survived the end of the fifth (last) instar, even when they were infected at an early developmental stage. Inoculation of egg masses by dipping them into a virus suspension gave a high percentage of infection when larvae were reared en masse.     

Biological characterization of an English granulovirus from the summer fruit tortrix moth, Adoxophyes orana

Adoxophyes orana granulovirus (AdorGV) was isolated from overwintering larvae in an orchard in Kent, in the UK. The developmental time of each A. orana instar was determined by measuring the size of the head capsule. The susceptibility of the larvae to the English isolate of AdorGV was evaluated in laboratory bioassays using inoculation by microdroplet feeding and applied dose assays. A series of bioassays were performed to determine LD(50) and ST(50) values for first, fourth and fifth instar larvae. The median lethal doses ranged from 30 occlusion bodies in first instar to 1.36 x 10(6) in fifth instar. The median survival time decreased the later the larvae were infected and ranged from 37 days in first instar to 24 days in fifth instar. Approximately half of the infected larvae released a discharge rich in occlusion bodies from their posterior end prior to death. Approximately 85% of larvae attempted pupation and died as larva-pupa intermediates.