Penetration of phytoecdysones through the pupal cuticle of the silkworm, Bombyx mori

The penetrability of some phytoecdysones, ecdysterone, inokosterone, ponasterone A, and cyasterone, through silkworm pupal cuticle was tested and their effect on pupal-adult development is described. The first three chemicals applied topically to fresh pupae accelerated the pupal-adult development and induced abnormal adults with aberrant legs and antennae, indicating penetration of phytoecdysones through fresh pupal cuticle. Females were more sensitive to the chemicals than males as they showed many more abnormalities. When pupae 1 day after ecdysis were treated topically with phytoecdysones, they transformed into normal adults, suggesting no penetration of ecdysones through old pupal cuticle.     

Haemolymph control of sericin gene expression studied by organ transplantation

A factor that affects synthesis of sericin mRNAs of Bombyx mori was analyzed by organ transplantation and allatectomy. When silk glands of the third instar larvae were transplanted into the abdomen of fifth instar larvae, substantial amounts of sericin mRNAs were induced in the transplant. The induced sericin gene activity was suppressed upon re-transplantation into the abdomen of fourth instar larvae and induced again when the second hosts grew up to fifth instar larvae. An allatectomy performed on fourth instar larvae promoted production of these mRNAs, suggesting that the synthesis of sericin mRNA is regulated by the titer of juvenile hormone.     

Effect of cadmium-feeding on tissue concentrations of elements in germ-free silkworm (Bombyx mori) larvae and distribution of cadmium in the alimentary canal

Silkworm (Bombyx mori) larvae were reared on an artificial diet containing cadmium (Cd) at concentrations of 5 and 80 micrograms/g wet diet from hatching to the fourth instar and then for 5 days at the fifth instar, respectively. Concentrations of Cd and other elements in the alimentary canal, Malpighian tubes, silk gland, fat body and other organs were determined simultaneously by inductively coupled argon plasma-atomic emission spectrometry. Cd was accumulated in the alimentary canal and Malpighian tubes at concentrations of 1100 and 470 micrograms/g dry wt, respectively. The distribution of Cd in the supernatants of the two highly accumulated organs were determined on an SW column by high performance liquid chromatography-atomic absorption spectrophotometry. Cd was primarily bound to inducible high molecular weight Cd-binding proteins.     

Tissue-specific and periodic changes in the nuclease sensitivity of the fibroin gene chromatin in the silkworm Bombyx mori

Nuclei of substantial purity were isolated from the middle or posterior silk glands of the silkworm Bombyx mori larvae. Both the fibroin H- and L-chain gene sequences in the isolated nuclei from the posterior silk glands of the fifth instar larvae, where the genes are transcribed actively, are extremely sensitive to the digestion with DNaseI; on the other hand, these sequences in the middle silk gland nuclei from the same larvae, where the genes are not expressed, are markedly resistant to the digestion. The H-chain gene sequences in the posterior silk gland nuclei from the fifth instar larvae are also highly susceptible to the digestion with micrococcal nuclease, HinfI, and HhaI. The digestion products with micrococcal nuclease show a continuous size distribution. The H-chain gene sequences in the middle silk gland nuclei or the posterior silk gland nuclei from the fourth molting stage are cleaved partially into nucleosome dimer to oligomer sizes upon digestion with higher concentrations of micrococcal nuclease, suggesting that the inactive forms of the H-chain gene chromatin are constructed by folding of the chromatin fiber containing a regular array of nucleosomes. Hypersensitive sites to micrococcal nuclease are present near both ends of the second exon, a major body of the fibroin H-chain gene, in both the active and inactive forms of the chromatin. The DNaseI or micrococcal nuclease sensitivity of the H-chain gene chromatin in the posterior silk gland nuclei shows periodical changes corresponding to the intermolt-molt-intermolt cycle.     

Quantitative measurements of fibroin messenger RNA synthesis in the posterior silk gland of normal and mutant Bombyx mori

The amount of newly synthesized and accumulated fibroin messenger RNA has been measured quantitatively at various stages of posterior silk gland development in Bombyx mori. The two-step method involves fractionation on a Bio-Gel column which excludes the large mRNA, followed by RNAase T₁ digestion, and fractionation of the oligonucleotides on DEAE-Sephadex. Larvae in the feeding stages of the third and fourth instar synthesize and accumulate fibroin mRNA to about 2% of cellular RNA; this corresponds to 0.2 and 2 μg per pair of posterior glands in the third and fourth instars, respectively. More than 70% of this mRNA is degraded in vivo during the third and fourth moulting stages. Fibroin mRNA synthesis resumes again within the first 24 hours of the fifth instar; the mRNA accumulates and predominates over other DNA-like RNAs as the stage proceeds until finally it comprises about 3.5% of cellular RNA in a mature larva (170 μg per pair of posterior glands). These results indicate that more than 99% of the fibroin mRNA detected in the fifth instar is synthesized during this stage.Four spontaneous mutants of B. mori which synthesize very low levels of fibroin have been analyzed for their RNA content in the middle fifth instar. The total cellular RNA of the posterior gland is reduced to 4 to 7% of normal. Fibroin mRNA is more severely reduced to 1% of normal. In three heterozygotes, which have mutant phenotypes with respect to fibroin production, only slight increases of total cellular RNA and fibroin mRNA were observed. Thus, the primary biochemical lesion in these mutants is still unknown.The presumed ancestor to B. mori, the wild silkworm B. mandarina, was also analyzed for its fibroin mRNA. The mRNA isolated from fifth instar larvae of B. mandarina is indistinguishable from that of B. mori with respect to its nucleotide sequence, molecular weight and fraction of total cellular RNA.     

Prothoracotropic action of ecdysone analogues in Bombyx mori

The prothoracotropic action of ecdysone analogues was examined, using the brainless, diapausing pupae of Bombyx mori with or without inclusion of prothoracic glands. The most effective of those hormones to stimulate prothoracic glands to secrete the moulting hormone was found to be cyasterone. The other analogues such as ponasterone A, ecdysterone, and inokosterone showed a lower activity with regard to prothoracotropic action. The female prothoracic glands were found to be more sensitive to the ecdysones than the male ones. The time lag from hormone injection to emergence indicated the dual actions of the injected ecdysones, directly on the target organs and indirectly on the prothoracic glands subsequent to secreting the moulting hormone.     

Prothoracic glands in the regulation of ecdysone titres and metabolic fate of injected labelled ecdysone in Locusta migratoria

In the absence of the prothoracic glands, fifth instar larvae of Locusta migratoria contain no demonstrable quantities of ecdysone and ecdysterone (assayed together in the Calliphora bioassay), whereas normal larvae show a high peak of ecdysone activity. The metabolic fate of injected radiolabelled ecdysone is found to be very similar in prothoracectomized larvae to that of normal larvae (hydroxylation rate, dehydrogenation of ecdysone and ecdysterone, inactivation rate). However, in the absence of the prothoracic glands, the larvae excrete radiolabelled ecdysone in their faecal material at a rate which is considerably higher than that of normal insects of the same age. These results are discussed in view of the regulation of the ecdysone titres by the prothoracic glands in L. migratoria.     

A reinvestigation of the effects of cortisol on growth in insects

Incorporation of cortisol, or various derivatives and analogues of cortisol, into the diet had no effect on the rate of growth of Tenebrio molitor. Injections of cortisol, or its derivatives, into fourth instar larvae of Schistocerca americana gregaria, had no effect on their rate of growth. The moult to fifth instar was normal. Administration in the diet, or injections of cortisol had no effect on the rate of growth of fourth and fifth instar larvae of Manduca sexta, or on the insect's uptake and utilization of food. The development of larvae to pupae and adults was unaffected. It is concluded, contrary to the findings of two previously published reports, that the vertebrate hormone cortisol does not affect the growth and development of insects.     

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.     

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.     

Regulation of prothoracic gland ecdysteroidogenic activity leading to pupal metamorphosis

The prothoracic glands of early last (fifth) instar larvae of the silkworm are inactive with regard to ecdysteroidogenesis and unresponsive to prothoracicotropic hormone (PTTH) [J. Insect Physiol. 31 (1985) 455]. In an attempt to elucidate the hormonal mechanisms that cause the inactivity, we compared the effects of PTTH, dibutyryl cyclic AMP (dbcAMP), a cAMP phosphodiesterase inhibitor (IBMX), juvenile hormone analogue (JHA) and 20-hydroxyecdysone (20E) on secretory activity of the third, fourth and fifth instar glands. Among the factors examined, feedback inhibition by 20E was indicated to be the most likely factor. Inhibition was moderate in the third and early fourth instars while 20E strongly inhibited the glands of middle fourth instar larvae. The inhibitory effect of 20E was reduced by removal of the brain and corpora allata. Once the glands were suppressed by 20E to the degree of exhibiting neither secretory activity nor responsiveness to PTTH, dbcAMP or IBMX did not elicit ecdysone secretion at all. Thus the feedback inhibition may shut down ecdysteroidogenesis although it is obscure whether it affects the intracellular transductory cascade from the PTTH receptor through cAMP. Taken together, this evidence suggests that inactivity of the gland in the early fifth instar is brought about by feedback inhibition of the glands by 20E occurring in the late fourth instar, and that this inactivity is maintained by the juvenile hormone found in the early fifth instar.     

Combined effects of allelochemicals, prey availability, and supplemental plant material on growth of a generalist insect predator

We examined the effects of the presence of plant allelochemicals in prey diet, prey availability and supplemental plant material on the growth of the generalist predator Podisus maculiventris (Hemiptera: Pentatomidae). We tested two different nymphal stages of this predator. Third to fourth instar nymphs and fifth instar nymphs were fed a diet of prey (Manduca sexta larvae, Lepidoptera: Sphingidae) without allelochemicals in their diet or prey fed maximal levels of allelochemicals (tomatine, rutin and chlorogenic acid) found in their host plant (Lycopersicon esculentum). The nymphs were fed prey ad libitum, once every three days, or once every five days. They were given either no supplemental plant material or a 2 cm slice of green bean pod (Phaseolus vulgaris). We also conducted another experiment with fifth instar nymphs using the same conditions, except that mean levels of allelochemicals found in the host plant were fed to prey instead of maximal levels and the prey were provided either once a day or once every five days. For all experiments, prey scarcity depressed developmental rate, weight gain and relative growth rate. Overall, there was no negative effect of allelochemicals in the diet of the prey on these variables when predators were supplied with an excess of prey, but allelochemicals in the prey diet negatively affected these predators when prey were scarce. The addition of plant material to the diet of third to fourth instar nymphs did not have any effect on developmental rate, final dry weight, or relative growth rate. However, for fifth instar nymphs, the addition of plant material negatively affected these variables. Thus, the addition of plant material to the diet of the nymphs did not alleviate the negative effects of prey scarcity or allelochemicals in prey diet.     

A carotenoid-binding protein (CBP) plays a crucial role in cocoon pigmentation of silkworm (Bombyx mori) larvae

We examined the role of carotenoid-binding protein (CBP) in yellow cocoon pigmentation. First, using yellow or white cocoon races, we investigated the linkage between the yellow pigmentation and CBP expression. CBP was expressed only in the silk gland of the yellow cocoon races, which utilize carotenoids for cocoon pigmentation. Furthermore, CBP expression in the silk glands of day 1-7 fifth instar larvae matched the period of carotenoid uptake into the silk gland. Finally, we gave double-stranded CBP RNA to Bombyx mori (B. mori) larvae to induce RNA interference. The significantly reduced expression of CBP in the silk gland of fifth instar larva was confirmed on day 4 and a decrease in yellow pigmentation was observed in the cocoon. We showed that CBP plays a key role in the yellow cocoon pigmentation caused by carotenoids.     

Disruptive developmental effects of benzyl-1,3-benzodioxole derivatives on unparasitized and parasitized Manduca sexta larvae

Treatment of tobacco hornworm larvae with the benzyl-1,3-benzodioxole derivative J-2710 immediately after ecdysis to the fourth instar disrupted development either during the moult to the fifth instar or shortly thereafter. Larvae given topical applications of 100 μg J-2710 in 1 μl acetone suffered 100% mortality, often after secreting moulting fluid in large pockets between the epidermis and the cuticle later in the fourth instar. Larvae that successfully ecdysed had abnormalities of the mouthparts and cervix that interfered with normal feeding, inhibiting growth in the fifth instar. Larvae of the gregarious endoparasitic wasp Cotesia congregata (=Apanteles congregatus) frequently failed to emerge from host Manduca sexta larvae treated with high doses of J-2710, particularly when the host failed to feed normally. Less potent disruptive effects on Manduca and Cotesia were seen after treatment of larvae with the derivatives J-3370 and J-2581.No anti-juvenile hormone action of J-2710 was observed. J-2710-treated M. sexta larvae showed no precocious metamorphosis and the developmental effects of J-2710 were not prevented by co-application of the juvenile hormone analogue methoprene in doses ranging from 1 to 100 μg/larva. Moreover, J-2710 had no effect on the action of methoprene in the black larval assay for juvenile hormone-like activity, unlike results reported to occur using the Galleria wax wound assay.     

Patterns and mechanisms of growth of fifth-instar Manduca sexta caterpillars following exposure to low- or high-protein food during early instars.

For many insect herbivores, variation in protein availability is a pervasive part of the environment. I explore how variable protein availability affects growth rates of fifth-instar Manduca sexta caterpillars and how growth is related to behavior and physiology. Groups of larvae were reared on low- or high-protein artificial diets (5.9% and 17.7% casein by dry weight, respectively) and then transferred in the fifth instar to the same or opposite diet. During or after the 24-h period following transfer, I measured growth rate, consumption rate, growth efficiency, midgut proteolytic activity, and masses of midgut contents and tissues. Fifth-instar caterpillars reared in earlier instars on high-protein diet grew about 20% more rapidly over 24 h than did caterpillars reared on low-protein diet. This growth pattern appears to be caused by differences in consumption and growth efficiency: caterpillars reared on high protein consumed more food, and used it more efficiently, than did caterpillars reared on low-protein diet. Over the short term (24 h), in contrast, fifth instars that received low-protein diet grew as rapidly as caterpillars that received high-protein diet. Increased (compensatory) consumption appears to be the primary mechanism by which caterpillars consuming low-protein food maintained growth rates.     

Anopheles stephensi and Toxorhynchites amboinensis: aseptic rearing of mosquito larvae on cultured cells

Aseptic larvae of Anopheles stephensi and Toxorhynchites amboinensis were reared on a continuous cell line (RU TAE 12 V) from the mosquito, T. amboinensis, that grew in suspension as multicellular vesicles. Surface-sterilized eggs were hatched in a 24-well plate containing 0.2 ml of Leibovitz's L-15 medium per well and incubated in a humidified atmosphere. Toxorhynchites amboinensis eggs of 36 hr or older were placed singly to assure hatching and avoid cannibalism. Hatching rates were over 80%. All larval instars were maintained in L-15 medium at 28 C with a 12-hr photoperiod. Anopheles stephensi larvae were reared in 25-cm2 tissue culture flasks containing 10 ml of L-15 medium with 30 to 50 first and second instar larvae or 10 third and fourth instar larvae per flask. Toxorhynchites amboinensis larvae remained in the 24-well plate in 1.5 ml of medium through the second instar; third instar larvae were kept in 12-well plates (3 ml of medium per well) and transferred to 25-cm2 flasks (10 ml per flask) when they reached the fourth instar. First and second instar A. stephensi larvae were fed cultured cells once, and third or fourth instar larvae twice a day. Toxorhynchites amboinensis larvae were fed vesicles once during the first 4 days after hatching, and every 1 or 2 days thereafter. Each A. stephensi larva consumed approximately 2 X 10(6) cells, and T. amboinensis larvae 10 times more cells before pupating. Anopheles stephensi pupated after 7 to 8 days and adults emerged during days 9 to 11. Pupation in T. amboinensis began on day 21 after hatching and adults emerged 5 days later. Cell lines isolated from A. stephensi larvae or embryos of the ticks Rhipicephalus sanguineus and Anocentor (Dermacentor) nitens supported only limited growth of A. stephensi larvae. Defibrinated hamster (Mesocricetus auratus) blood, though readily ingested, did not support the growth of A. stephensi whereas larvae reared on blood cells plus T. amboinensis cells showed limited growth.     

Régulation de la synthèse du DNA dans la glande séricigène de Bombyx mori: II. Etude de l''activité DNA-polymérasique présente dans la fraction soluble brute

In the silkglands of Bombyx mori larvae, DNA synthesis suddenly stops in the middle of the fifth larval instar, when cellular growth is over and cell activity is turned on to massive silk synthesis. Of much interest is to determine what factors are implied in the quantitative and temporal control of DNA synthesis in this tissue.Previous work on thymidine metabolism in the intact organ lead us to look for several enzymatic activities in the soluble fraction of the cells during this period.The results presented here concern the kinases of the thymidylic pathway. As measured in vitro from crude cellular extracts, thymidine-kinase is present at a lower level than thymidylate-kinase but both activities vary in connection with the pattern of in situ DNA synthesis, reaching their highest levels on the fourth day of the instar and decreasing as DNA synthesis definitively stops in the glands.     

An in vitro study on regulation of prothoracic gland activity in the early last-larval instar of the silkworm Bombyx mori

The endocrine mechanisms that regulate prothoracic gland (PG) activity in early stages of final larval instar of the silkworm Bombyx mori were investigated using a newly developed long-term cultivation system of the gland. The PGs dissected from day-0 fifth instar larvae did not secrete detectable amounts of ecdysone for the first 24 h in culture but started secretion within the next 2 days. The amount of secreted ecdysone increased day by day. When day-0 PGs were co-cultivated with corpora allata, however, they remained inactive for at least 8 days. PGs dissected from 1-day younger larvae (day-3 fourth instar larvae) secreted ecdysone for the first 24 h but stopped secretion for the next 24 h, followed by recovery of ecdysone secretory activity. By contrast, PGs from day-1 fourth instar larvae remained active throughout a cultivation period without any sign of inactivation. However, when the same glands were exposed to a high titer of 20-hydroxyecdysone for the second 24h in culture, they gradually lost their activity. These results indicate that PGs of fourth instar larvae are inactivated by ecdysteroid through a negative feedback mechanism and that thus inactivated PGs spontaneously recover ecdysone secretory activity in the early fifth instar unless inhibited by juvenile hormone.     

Hormonal effects on carotenoid uptake by the silk gland in the silkworm, Bombyx mori

The carotenoid uptake by the silk gland of the silkworm (Bombyx mori), which occurs only during the middle to late period of the last (fifth) instar in the natural condition, was studied in relation to the hormonal controls. During certain stages of the fourth and last instars, the corpus allatum hormone (JH) was found to inhibit the activation of the absorbing function of the silk gland. The absorbing activity was inactivated, if the activated silk gland was implanted into larva at the late stage of the fourth instar in the presence of the moulting hormone (MH). As more ponasterone-A (ecdysone-analogue) was injected into decapitated larvae, the pigmentation of the silk gland was increased; but injection of a high titre inhibited its activity. It seems that, through serial transplantations, the silk gland inactivated experimentally at the late stage of the fourth instar is reactivated in the presence of MH during the middle to late period of the last instar. The results indicate that MH and JH at each stage control the activity of the carotenoid uptake.     

Larval growth and development in the caddisfly Cheumatopsyche brevilineata under natural thermal regimes

Cheumatopsyche brevilineata (Iwata) is a filter‐feeding caddisfly without distinct or distinguishable cohorts. In a semi‐natural channel, we reared fourth and fifth instar larvae of C. brevilineata in individual cages with hourly recording of water temperature. We calculated the individual growth rate from the wet‐weight gain of each larva, and the development rate from the ratio of larvae that progressed to the next instar or pupal stage during each rearing experiment. We analyzed the linear regressions of growth (increase in size) and development (physiological and morphological progression toward maturity) rates against the statistical parameters of water temperatures during each rearing period, i.e. mean and given percentiles of water temperatures. We presumed that the most appropriate parameter of water temperature to explain larval growth and development would show a peak value of the determination coefficients (r²) in the linear regressions. There were highly significant regressions in the growth rates for fourth and fifth instar larvae and in development rates for fourth instar larvae against every statistical parameter of water temperature, but not in the development rates for fifth instar larvae. For the growth of fourth and fifth instar larvae, we could not specify the most appropriate parameters of water temperatures, because we observed no clear peaks in the determination coefficients. For the development of fourth instar larvae, this parameter could be the 65th percentile value, where the development zero temperature and effective degree‐days were 11.1°C and 56 degree‐days, respectively.