Influence of Pyriproxyfen on the Expression of Haemolymph Protein Genes in the Colorado Potato Beetle,Leptinotarsa decemlineata

Pyriproxyfen, a potent juvenile hormone analogue for the Colorado potato beetle, Leptinotarsa decemlineata, was applied topically to last-instar larvae and short-day adults at different times after moulting. The effect of the hormone analogue on concentration and composition of protein in the haemolymph was studied at different intervals after pyriproxyfen application. The hormone analogue had little effect on total protein concentration of the haemolymph, but affected protein composition. Diapause protein 1 was prevented from being synthesized if pyriproxyfen was applied before the gene was activated and disappeared from the haemolymph if applied after the gene had been expressed. It therefore inactivated the gene for diapause protein in both larvae and adults. Pyriproxyfen also induced appearance of vitellogenin at both stages, indicating induction of expression of the vitellogenin gene. It also affected the stability of mRNA for diapause protein. The analogue caused mRNA for diapause protein 1 to disappear untimely compared to controls in last-instar larvae and short-day adults. The response of adults to the JHA was much more pronounced than that of larvae, although the analogue had a strong biological effect on last-instar larvae because it prevented metamorphosis at low doses. Copyright 1997 Elsevier Science Ltd. All rights reserved     

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.     

Developmental and sex-dependent regulation of storage protein synthesis in the silkworm,Bombyx mori

The mechanism of sex-dependent expression of a major plasma protein, referred to as storage protein 1 (SP-1) was studied during development of the silkworm, Bombyx mori. SP-1 occurred in the hemolymph of the female as well as in the male larvae until the end of the fourth larval instar. In the last instar larvae, the amount of SP-1 in the hemolymph greatly increased in females, but markedly declined in males. The level of fat body mRNA for SP-1 reflected the developmental and sex-dependent changes in the hemolymph concentration of SP-1. The developmental patterns of hemolymph proteins in the third and the fourth instar larvae of sex-mosaic individuals were quite analogous to those observed in normal larvae at the same developmental stages. The hemolymph concentration of SP-1 at the last larval instar of the sex mosaics varied among individuals irrespective of the gonad compositions. In vitro culture of the fat body cells dissected from several locations of a sex-mosaic larva provided evidence that each fat body cell in a common hemolymph milieu synthesizes a high (female type) or a low (male type) level of SP-1 depending on the sex chromosome composition. The amount of vitellogenin in the hemolymph of the sex-mosaic pupae was in proportion to that of SP-1 at the last larval instar. From these results, it is suggested that the sex-dependent expression of SP-1 and vitellogenin in B. mori is genetically determined and developmentally regulated without participation of the reproductive organs or any sex-specific humoral factors.     

Endocrine changes associated with metamorphosis and diapause induction in the yellow-spotted longicorn beetle, Psacothea hilaris

At 25 degrees C and under a long-day photoperiod, all 5th instar Psacothea hilaris larvae pupate at the next molt. Under a short-day photoperiod, in contrast, they undergo one or two additional larval molts and enter diapause; the 7th instar larvae enter diapause without further molt. The changes in hemolymph juvenile hormone (JH III) titers, JH esterase activity, and ecdysteroid titers in pupation-destined, pre-diapause, and diapause-destined larvae were examined. JH titers of the 5th instar pupation-destined larvae decreased continuously from 1.3 ng/ml and became virtually undetectable on day 13, when JH esterase activity peaked. Ecdysteroids exhibited a small peak on day 8, 1 day before gut purge, and a large peak on day 11, 2 days before the larvae became pre-pupae. The two ecdysteroid peaks are suggested to be associated with pupal commitment and pupation, respectively. JH titers of the 5th instar pre-diapause larvae were maintained at approximately 1.5 ng/ml for 5 days and then increased to form a peak (3.3 ng/ml) on day 11. JH esterase activity remained at a low level throughout. Ecdysteroid levels exhibited a large peak of 40 ng/ml on day 18, coincident with the larval molt to the 6th instar. JH titers of the 7th instar diapause-destined larvae peaked at 1.9 ng/ml on day 3, and a level of approximately 1.1 ng/ml was maintained even 30-60 days into the instar, when they were in diapause. Ecdysteroid titers remained approximately 0.02 ng/ml. Diapause induction in this species was suggested to be a consequence of high JH and low ecdysteroid titers.     

A larval specific lipoprotein: purification and characterization of a blue chromoprotein from Heliothis zea

The green color of the hemolymph of last instar larvae of the corn earworm, Heliothis zea (Boddie), is caused by the appearance of a blue chromoprotein. This protein (Mr approximately 560,000) is composed of 4 subunits (Mr approximately 150,000). It contains 8.4% lipid and has an equilibrium density of 1.26 g/ml. This protein is absent during all early larval stages, in pupae and in adult animals, but is a major hemolymph protein in 5th instar larvae. Although its physiological function remains unclear, this blue chromoprotein represents the first member of a new class of larval-specific insect lipoproteins.     

Purification,characterization and developmental changes in the titer of a new larval serum protein of the silkworm,Bombyx mori

A new protein was found as a major component of hemolymph proteins up to day 1 of the last larval instar of Bombyx mori, and was named Bombyx mori larval serum protein (BmLSP). The BmLSP was purified to homogeneity by ammonium sulfate precipitation, CM-cellulose column chromatography and gel permeation chromatography. The molecular weight of BmLSP was estimated to be 30,000 by SDS-PAGE and 25,000 by gel permeation chromatography. The amino acid composition of BmLSP was similar to that of 30 kDa proteins which are the major serum proteins in the older last (fifth) instar larvae. The 20 NH₂-terminal amino acids were sequenced and found to be quite different from those of the 30 kDa proteins. Developmental changes in BmLSP titer were followed throughout post-embryonic life by Western blotting using a specific antiserum against BmLSP. Within 1 day after larval hatching, BmLSP appeared in the hemolymph and remained at an almost constant level until day 1 of the last instar. On day 2 of the last instar, the BmLSP level suddenly fell and then gradually decreased toward larval-pupal metamorphosis. Thus, BmLSP is a true larval serum protein and is different from proteins stored for metamorphosis.     

Storage proteins of the fall webworm,Hyphantria cunea drury

Two storage proteins, storage protein-1 (SP1) and storage protein-2 (SP2), were found in hemolymph and fat body during the development of Hyphantria cunea, the fall webworm. Both storage proteins show similiar quantitative changes during development in males and females; however, SP1 is more abundant. The hemolymph of last instar larvae contains high concentrations of the storage proteins. However, following pupation, the storage proteins accumulate in fat bodies. SP1 peaks in the hemolymph of males and females late in last instar larvae (8-day-old 7th instar larvae). SP1 has a native molecular weight of 460,000 and consists of six identical subunits (Mr = 76,700), while SP2 has a molecular weight of 450,000 and is composed of two different subunits (Mr = 74,100 and 72,400). Both SP1 and SP2 are hexamers and are phosphorylated glycolipoproteins. The pl values of SP1 and SP2 were determined to be 5.70 and 5.50, respectively. Antibodies raised against SP1 react positively with vitellogenin and ovary extract, as well as with proteins in the hemolymph from last instar larvae and proteins in pupal fat bodies. Storage protein synthesis starts in fat bodies of a 4-day-old 7th instar larvae and in female peaks at 6–8 days of the 7th instar.     

Properties of Orange-Pupa-Inducing Factor (OPIF) in the swallowtail butterfly, Papilio xuthus L

Diapause pupae of the swallowtail butterfly Papilio xuthus L. exhibit diapause-green, orange and brownish-orange color polymorphism. Development of orange pupae involves a neuroendocrine factor inducing orange pupa (Orange-Pupa-Inducing Factor, OPIF), which is secreted from the head-thoracic region during late pharate pupal stages, in particular from the ganglia of short-day animals located posteriorly from the second thoracic ganglion2 (TG2). This report describes certain properties of OPIF using bioassays involving ligated abdomens of short-day pharate pupae. Localization of OPIF in the central nervous system of short-day larvae indicated that it was present predominantly in TG2, thoracic ganglion3 (TG3) and abdominal ganglion1 (AG1) complexes. OPIF activity in TG(2,3)-AG1 complexes was over two times higher than in the more posteriorely located ganglia. The developmental profile of OPIF in last instar short-day larvae revealed that OPIF activity in larval ganglia posterior to TG2 became gradually higher as larval growth proceeded, suggesting that OPIF might be accumulated in TG(2,3)-AG(1-7) complexes as larvae prepare for pupal molting. Furthermore, ligated abdomens of short-day larvae developed into pupae of an orange type when a 2% NaCl extract containing OPIF prepared from TG(2,3)-AG(1-7) complexes of long-day larvae was injected into ligated abdomens of short-day pharate pupae, indicating that OPIF is also present in long-day larvae. Additionally, a biochemical investigation using gel filtration chromatography showed that the molecular weight of OPIF was about 10 kDa.     

Stage-dependent effects of starvation on the growth, metamorphosis, and ecdysteroidogenesis by the prothoracic glands during the last larval instar of the silkworm, Bombyx mori

The stage-dependent effects of starvation on the growth, metamorphosis, and ecdysteroidogenesis of the prothoracic glands during the last larval instar of the silkworm, Bombyx mori, were studied in the present study. When last instar larvae were starved beginning on day 1 of that instar, all larvae died between days 5 and 7 of the instar. Although the prothoracicotropic hormone (PTTH) release from the brain-corpus cardiacum-corpus allatum (BR-CC-CA) did not significantly change during starvation, a deficiency in PTTH signal transduction was maintained, which led to very low levels of hemolymph ecdysteroids after the beginning of starvation. However, when starvation began on day 3 of the last larval instar, the major hemolymph ecdysteroid peak, preceding larval-pupal transformation, occurred 1 day earlier than that in control larvae. Protein content of the prothoracic glands in day 3-starved larvae was maintained at a low level as compared to that of control larvae. The secretory activity of the prothoracic glands in day 3-starved larvae was maintained at a level similar to that of control larvae. However, the rate of ecdysteroidogenesis, expressed per microgram of glandular protein, was greatly enhanced in these starved larvae, indicating that upon starvation, larvae increased the ecdysteroid production rate to enhance the rate of survival.     

Precocious expression of the final larval instar developmental pattern in larvae of Trichoplusia ni pseudoparasitized by Chelonus spp

The present study has used a number of electrophoretic approaches to analyze the proteins in normal Trichoplusia ni larvae and those pseudoparasitized by Chelonus spp. A number of feeding-stage, hemolymph proteins appear or increase dramatically only during the final larval stadium. Other proteins highly abundant only during the penultimate stadium disappear or decrease dramatically during the final stadium. The comparative protein profiles of penultimate instar, pseudoparasitized larvae are very similar to those of last instar larvae. These changes in hemolymph proteins are seen on gels resulting from electrophoresis, isoelectric focusing (wide range and narrow range Ampholine and very narrow range Immobiline gels) and SDS-disc electrophoresis. It is concluded that the entire last instar developmental pattern of protein gene products is occurring precociously in pseudoparasitized larvae.     

Ni2+对不同发育阶段斜纹夜蛾虫体血淋巴能量物质的影响

研究了饲料中不同浓度的Ni2+(140mg/kg)对连续3个世代斜纹夜蛾Spodoptera litura Fabricius6龄末幼虫、蛹和成虫血淋巴中能量物质总糖、蛋白质和脂肪含量及血淋巴中热量值的影响。结果表明,Ni2+胁迫1个世代,6龄末幼虫、蛹和成虫血淋巴中的总糖含量均低于对照,而血淋巴中的蛋白质含量则在低浓度Ni2+胁迫下增高,在高浓度Ni2+胁迫下降低;只有受120mg/kg Ni2+胁迫的6龄末幼虫血淋巴的脂肪含量高于对照,而蛹和成虫血淋巴中的脂肪含量及热量值均低于对照。在Ni2+胁迫的第2、3代,6龄末幼虫、蛹和成虫血淋巴中的总糖、蛋白含量和热量值与饲料中Ni2+浓度的反应关系均表现为低浓度Ni2+胁迫增加而高浓度Ni2+胁迫降低的趋势;然而,血淋巴中的脂肪含量随饲料中Ni2+浓度的增加而降低。因此,重金属Ni2+对S.litura能量物质的影响与虫体内能量物质的种类和虫体的发育阶段有一定的关联。     

Purification and characterization of epidermis-origin hemolymph protein in Galleria mellonella

Epidermis-origin hemolymph protein (EOHP) was identified and purified from the last instar larval hemolymph of Galleria mellonella by anion exchange chromatography, chromatofocusing chromatography, and Sephadex G-100. The EOHP has a native molecular mass of 47 kDa and is composed of one subunit. The isoelectric point of the EOHP was determined to be 5.3. The amino acid composition of the EOHP was rich in aspartic acid, glutamic acid and lysine, but poor in tyrosine, methionine, and tryptophan. EOHP is present in hemolymph over the period from the 4th instar larvae to the adult stage examined. Concentration of EOHP is high during the larval stage but gradually decreased during the developmental stage from pupal to adult stage. EOHP is present in the cuticle, fat bodies and trachea but not in hemocytes, fore gut, mid gut and hind gut.     

Humoral stimulation of the larval and adult prothoracic glands in Schistocerca gregaria

Fluctuations in ecdysteroid production by explanted prothoracic glands (PG) during the penultimate and last larval instars parallel changes in ecdysteroid titer in the hemolymph. The in vitro output of ecdysteroids increases up to 30-fold when PG are co-cultured with the brain. Maximal amounts of ecdysteroids are produced when both PG and brain are taken from larvae at the time of the molt-inducing ecdysteroid peaks (days 2–3 in the penultimate and days 5–6 in the last instar), and also from day 3 last instar larvae that exhibit a small rise of hemolymph ecdysteroids. Detailed investigations on penultimate instar larvae revealed that their PG become sensitive to the stimulation on day 1 (about 24 h after ecdysis), but the stimulatory brain potential is restricted to days 2 and 3. Both the stimulatory capacity of the brain and the sensitivity of PG are lost on days 4 and 5, i.e., after the ecdysteroid surge on day 3. PG explanted from young adults do not secrete appreciable amounts of ecdysteroids but can be stimulated to ecdysteroid production with active larval brains. Arch. Insect Biochem. Physiol. 36:85–93, 1997. © 1997 Wiley-Liss, Inc.     

Isolation and characterization of a larval hemolymph protein in Locusta migratoria

A high-molecular-weight protein, M_r 500,000, has been isolated and characterized from the hemolymph of the migratory locust, Locusta migratoria. It is composed of six seemingly identical subunits of apparent M_r 78,000. It contains low concentrations of carbohydrate and lipid, but high percentages of aspartate and glutamate as well as high proportions of hydrophobic amino acid residues. An antiserum, developed against this purified hemolymph protein, does not react in the double-diffusion test or after immunoblotting with purified lipophorin or cyanoprotein, two other major proteins in locust hemolymph. The concentration of this larval specific protein in the hemolymph of Locusta was examined during the last larval instar and in adult males by quantitative rocket immunoelectrophoresis. Its concentration increases in the second half of the fifth instar, concommitant with an increase in total protein. The protein is detectable by immunological techniques in adults, although its concentration is very low at this stage.     

Parasite regulation of host insect metamorphosis: a new form of regulation in pseudoparasitized larvae ofTrichoplusia ni

Summary When eggs ofTrichoplusia ni (Lepidoptera) are stung by a parasitic wasp,Chelonus sp., the developing host larvae precociously initiate metamorphosis ten days later. Precocious initiation of metamorphosis occurs even in ‘pseudoparasitized’ stung hosts which contain no living parasites at the time of symptoms of host regulation by the parasite. In feeding, penultimate instar, pseudoparasitized hosts, the corpora allata activity, hemolymph juvenile hormone esterase activity, in vivo rates of juvenile hormone metabolism and changes in hemolymph protein composition all follow the pattern of the normal last instar. This and other evidence suggests the entire developmental pattern of the last larval instar is precociously expressed in penultimate instar, pseudoparasitized hosts. The cause of precocious expression of the developmental program leading to metamorphosis is a significant decrease in the critical size parameter that, in normal larvae, signals attainment of the last instar. The induction, in preultimate instar larvae, of the entire feeding stage developmental program leading to metamorphic commitment, using either biochemical, surgical or parasitic experimental probes, has not been previously reported. The results have important implications for the study of host-parasite endocrine interaction, of normal insect metamorphosis and even of human puberty.     

In vitro synthesis, release and uptake of storage proteins by the fat body of Manduca sexta: putative hormonal control

1. Two major proteins (P1 and P2) are synthesized by the fifth instar larval fat body of Manduca sexta and then released into the hemolymph. 2. These proteins are later sequestered by the pre-pupal fat body. 20-Hydroxyecdysone does not appear to affect the synthesis of either protein. 3. When day 2 fifth instar larvae are neck-ligated there is an excessive synthesis (supersynthesis) of P2 (arylphorin). 4. Juvenile hormone I (JH I) applications to ligated animals had no effect, but brain homogenate injections resulted in the inhibition of P2 synthesis. 5. Neck ligations of larvae between days 5 and 6 revealed a head critical period between day 5 + 12 hr and day 5 + 18 hr, after which the head is unnecessary for the sequestration of either protein by the fat body. 6. JH I and JH III applications to ligated larvae before the head critical period do not restore the ability of the fat body to sequester the storage proteins. 7. P1 and P2 appear to be synthesized differentially and P2 is sequestered by the fat body to a much lesser extent than P1. 8. P2 is the hemolymph storage protein of both larval and pupal stages, whereas P1 appears to be the storage protein of the pupal fat body. 9. The data indicate that the synthesis of arylphorin and the resorption of both proteins are controlled by a putative head factor(s).     

A diapause associated protein of the pink bollworm Pectinophora gossypiella Saunders.

A diapause associated protein was electrophoretically isolated from the hemolymph of diapausing last instar larvae of the pink bollworm Pectinophora gossypiella. This protein (M(r) approximately 490,000, glycolipoprotein) was given the name Pectinophora diapause protein (PDP). It is composed of one subunit (M(r) 103,000). The concentration of PDP increased dramatically in the hemolymph of diapausing larvae from 17.4% in prediapause (PD) phase to 29.2% in early diapause (ED) phase reaching a level of 38.6% in larval hemolymph of middiapause (MD) phase. The concentrations of total proteins in the hemolymph of active feeding (A), PD, ED, and MD larvae were 69.8, 106,6, 113.3, and 118 mg/ml, respectively, while those in the fat body of the same larvae were 7.1, 7.4, 8.8, and 4.5 mg/g, respectively. In Pectinophora a drop in the concentration of fat body proteins coincided with a corresponding increase in hemolymph proteins, which suggests an active release of protein from the fat body into the hemolymph during the development of diapause. A partial amino acid sequence of pectinophorin showed the first 15 amino acids starting from the amino terminus of the peptide chain: N-ALA-LYS-THR-ILEU-VAL-GLU-ASN-MET-PRO-PRO-THR-PRO-LEU-ASN-ALA-C.     

Decreased JH biosynthesis is related to precocious metamorphosis in recessive trimolter (rt) mutants of the silkworm, Bombyx mori

In recessive trimolter (rt) mutants of the silkworm, Bombyx mori, that have four larval instars rather than five larval instars of normal B. mori, a decrease after a small increase in the hemolymph ecdysteroid titer during the early stages of the last (fourth) larval instar appeared to be a prerequisite for larvae to undergo precocious metamorphosis. The present study was carried out to investigate the possible mechanism underlying this decrease in the ecdysteroid titer. It was found that juvenile hormone (JH) biosynthetic activity of the corpora allata (CA) increased during the first day of the last larval instar, but its absolute JH biosynthesis activity was relatively lower compared to that of normal fourth-instar larvae in tetramolters. This lowered JH biosynthetic activity appeared to be related to a decrease in prothoracic gland ecdysteroidogenesis during the second day of the last instar, because hydroprene application prevented this decrease in prothoracic gland ecdysteroidogenesis, leading to the induction of a supernumerary larval molt. The in vitro incubation of prothoracic glands with hydroprene showed that hydroprene did not directly exert its action on prothoracicotropic hormone (PTTH) release. Further study showed that the application of hydroprene enhanced the competency of the glands to respond to PTTH. From these results, it was supposed that the lowered JH biosynthesis of the CA during the first day of last instar in rt mutants was related to decreased ecdysteroidogenesis in the prothoracic glands during the second day, thus playing a role in leading to precocious metamorphosis.