Production of [14C]-Labeled 3-Hydroxy-L-Kynurenine in a Butterfly,Heliconius charitonia L. (Heliconidae), and Precursor Studies in Butterfly Wing Ommatins

A method was developed to produce radiolabeled 3-hydroxy-L-kynurenine by injection of [¹⁴C]-L-tryptophan into pupae of the heliconid butterfly, Heliconius charitonia, which was converted into [¹⁴C]-3-hydroxy-L-kynurenine and deposited as a wing pigment. Extractions of 3-hydroxykynurenine (3-OHK) with 60% methanol from wings yielded in 14.4 μg per mg dry weight. In extracts from yellow wing areas, 3-OHK represented 100% of detectable amino acids. Resulting specific radioactivity of [¹⁴C]-3-OHK was between 0.05 and 0.07 mCi/mmol when 0.5 μCi [¹⁴C]-tryptophan was injected into pupae 1 or 2 days before emergence of the butterfly. Incorporation of [¹⁴C]-3-OHK into wing ommochromes was studied in nymphalid butterflies, Araschnia levana and Precis coenia. After injection into pupae [^I4C]-3-OHK as well as [¹⁴C]-tryptophan were specifically incorporated into red and red-brown wing scales as shown by autoradiography. The same incorporation occurred in isolated wings after incubation in Grace's medium containing [¹⁴C]-3-OHK. In Araschnia levana, [¹⁴C]-3-OHK offered to left wing pairs was incorporated into dihydroxanthommatin six times more effectively than [¹⁴C]-tryptophan offered to right wing pairs from the same specimen. Therefore, 3-OHK seems to be the ultimate precursor of wing ommatins.     

Lipophorin as a yolk precursor in Hyalophora cecropia: uptake kinetics and competition with vitellogenin

Vitellogenic follicles of Hyalophora cecropia were incubated in metabolically radiolabeled, high-density lipophorin isolated from pharate adult hemolymph by KBr density gradient centrifugation. The follicles transferred this probe from the incubation medium to the cortical yolk spheres in the oocyte by an energy-dependent and saturable mechanism. Vitellogenin and high-density lipophorin competed with each other for uptake, and are therefore concentrated by the follicle with a common mechanism. Microvitellin and lipophorin, in contrast, did not compete for uptake. The K(uptake) for the accumulation of high-density lipophorin was substantially higher than the value estimated earlier for vitellogenin (133 microM vs. 18 microM). This relationship helps explain why the shared concentrating mechanism does not deplete the lipid transport capacity of the hemolymph, and how a low vitellogenin: lipophorin molar ratio in the hemolymph yields a high ratio in the mature egg.     

Selective endocytosis,in vitro,by ovarian follicles from Hyalophora cecropia

Ovarian follicles of Hyalophora cecropia, incubated in vitro with isolated and radiolabelled hemolymph and yolk proteins, provided a satisfactory model of in situ vitellogenesis. Uptake of proteins was specific. The follicles accumulated vitellogenin and microvitellin at constant rates for 6 hr, depositing them in the protein yolk spheres of the oocyte. Uptake of these two proteins was saturable by high concentrations of homologous protein and inhibited by p-dinitrophenol. In contrast, two other abundant hemolymph proteins, arylphorin and flavoprotein, were taken up at lower rates, and become concentrated primarily in the basement lamina of the follicle. Their accumulation was not saturable and not inhibited by p-dinitrophenol. The two yolk precursors were accumulated only by follicles at stages known to be vitellogenic, and the rates of uptake were shown to approximate the rates of accumulation of these proteins in situ. The uptake of vitellogenin, but not microvitellin, was enhanced 2- to 3-fold by hemolymph ultrafiltrates. Vitellin from mature eggs was not distinguishable from vitellogenin by the endocytotic apparatus. Finally, endocytotic uptake was not affected by inhibition of protein synthesis. This finding supports the concept of membrane and receptor recycling in yolk formation, and argues against an essential role of the follicle cell product paravitellogenin in the mechanism of hemolymph protein uptake.     

Distribution and accumulation of storage protein-1 in ovary of Hyphantria cunea Drury

Storage protein-1 (SP-1) is a major storage protein found in the hemolymph and fat body of Hyphantria cunea. In this study, the uptake and accumulation of SP-1 into the ovary of H. cunea was investigated using biochemical and immunocytochemical methods. SP-1 in H. cunea has a high methionine content (4.6%) but is not female-specific, like other high methionine storage proteins. In the 6-day-old pupal ovary, SP-1 was detectable in trace amounts but accumulated to significant levels toward the end of the pupal stage. After adult emergence, SP-1 rapidly decreased in the ovarian follicles and remained low in the egg. This suggest that SP-1 is either extensively modified or degraded, causing a loss of its antigenic property in the ovary after adult emergence. During vitellogenesis, SP-1 is present in the hemolymph and penetrates through the tunica propria to reach the perioocytic space. From there, SP-1 is incorporated into yolk bodies. These results clearly show that SP-1 is taken up by the developing oocyte. Its disappearance suggests that SP-1 might be an amino acid reservoir for providing precursors for egg formation, in contrast to yolk proteins, which are utilized during postembryonic development. Arch. Insect Biochem. Physiol. 37:115–128, 1998. © 1998 Wiley-Liss, Inc.     

Comparative analysis of proteome maps of silkworm hemolymph during different developmental stages

Background  

The silkworm Bombyx mori is a lepidopteran insect with four developmental stages: egg, larva (caterpillar), pupa, and adult. The hemolymph of the silkworm is in an open system that circulates among all organs, and functions in nutrient and hormone transport, injury, and immunity. To understand the intricate developmental mechanisms of metamorphosis, silkworm hemolymph from different developmental stages, including the 3^rd day of fifth instar, the 6^th day of fifth instar, the 3^rd day of pupation, the 8^th day of pupal stage and the first day of the moth stage, was investigated by two-dimensional electrophoresis and mass spectrometry.     

体内体外培养下飞蝗雄性生殖细胞的分化

在单一TCl99或GRACE培养液中培养的四龄三天东亚飞蝗(Locusta mtgratoria mani lensis精小管,其精子发生只发育至初级精母细胞期,培养液中添加10%小牛血清或飞蝗精巢匀浆液可促使其发育至次级精母细胞期,添加10%分别取自东亚飞蝗蝗蝻、柞蚕蛹及蓖麻蚕蛹的血淋巴可促进其产生约20%的精子。 蜕皮激素及保幼激素对精子的产生无显著影响。移植培养的精小管在受体飞蝗体内不能发育产生精子,注射20μg/虫蜕皮激素可促使其产生大量精子。完整精巢无需注射蜕皮激素即可在受体飞蝗体内发育产生精子。结果表明,昆虫血淋巴内可能含有促细胞分化类因子,此(类)因子可能无种属特异性,外源蜕皮激素可能对精子发生无直接作用,但精子发生同时需要蜕皮激素和血淋巴因子,精巢本身可能有自己的蜕皮激素来源。     

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.     

An autoradiographic analysis of vitellogenin synthesis and secretion in the fat body of the stick insect Bacillus rossius

The fat body of the stick insect Bacillus rossius was studied with a view to clarifying the metabolic pathway leading to secretion of vitellogenin (VG). Electrophoretic analysis of ovarian follicles and hemolymph from egg-laying females showed that the two tissues shared a common polypeptide composition consisting of five major polypeptides with molecular weights ranging from 60-180 Kd. Following in vivo exposure to [(35)S]-methionine for up to 24 h, these polypeptides were labeled in a stage- and time-dependent manner, suggesting that they were transferred from the hemolymph to the oocyte during vitellogenesis. Fat body pulse-labeled with [(35)S]-methionine for up to 240 min and immunoprecipitated with an anti-yolk serum was labeled only in a fraction containing high molecular weight polypeptides. We presume these polypeptides to be VG precursors bearing a precursor-product relationship with the five major polypeptides of the hemolymph and developing ovarian follicles. Fat body exposed in vivo to [(3)H]-leucine for time intervals ranging from 20-240 min were processed for EM autoradiography. The results of this analysis showed that incorporated radioactivity was progressively transferred from the endoplasmic reticulum to the Golgi apparatus and from there to the composite granules. The data provided in this study are consonant with previous findings by which composite granules were shown to contain two compartments differing both in content and origin. In addition, the autoradiographical data of in vivo labeled fat body demonstrate that only the material partitioned into the electron-dense compartment of these granules is exocytosed.     

Transcytosis in thyroid follicle cells

Inside-out follicles prepared from pig thyroid glands were used for studies on endocytosis. endocytosis. In this in vitro system, only the apical plasma membranes of follicle cells were exposed to tracers added to the culture medium. Cationized ferritin (CF) bound to the apical plasma membrane and was transferred first to endosomes and to lysosomes (within 5 min). Later, after approximately 30 min, CF was also found in stacked Golgi cisternae. In addition, a small fraction of endocytic vesicles carrying CF particles became inserted into the lateral (at approximately 11 min) and the basal (at approximately 16 min) plasma membranes. Morphometric evaluation of CF adhering to the basolateral cell surfaces showed that the vesicular transport across thyroid follicle cells (transcytosis) was temperature-sensitive; it ceased at 15 degrees C but increased about ninefold in follicles stimulated with thyrotropin (TSH). Thyroglobulin-gold conjugates and [3H]thyroglobulin (synthesized in separate follicle preparations in the presence of [3H]leucine) were absorbed to the apical plasma membrane and detected mainly in lysosomes. A small fraction was also transported to the basolateral cell surfaces where the thyroglobulin preparations detached and accumulated in the newly formed central cavity. As in the case of CF, transcytosis of thyroglobulin depended on the stimulation of follicles with TSH. The observations showed that a transepithelial vesicular transport operates in thyroid follicle cells. This transport is regulated by TSH and includes the transfer of thyroglobulin from the apical to the basolateral plasma membranes. Transcytosis of thyroglobulin could explain the occurrence of intact thyroglobulin in the circulation of man and several mammalian species.     

Trans-order yolk protein can stimulate endocytic activity in Drosophila oocytes

Vitellogenins (Vgs) and mature yolk from some non-Dipteran insects can be recognized by Drosophila melanogaster oocyte Vg receptors and incorporated via receptor-mediated endocytosis into nascent yolk spheres (NYS). It had previously been assumed that only Vgs of Drosophila or other Dipterans could be so endocytosed. Drosophila ovarian follicles from 4-day old females were incubated in the presence of physiological salt solution (PSS) containing some fluorescent TexasRed-Dextran (Dex-red) or PSS-Dex-red in which either female hemolymph, or vitellin (mature yolk) from lysed oocytes was present from any of the following: (1) Drosophila (Diptera); (2) Oncopeltus (milkweed bug, Hemiptera); (3) Acteaus (luna moth, Saturniidae Lepidoptera); (4) Papilio (swallowtail butterfly, Papilionidae Lepidoptera); or (5) Xylocopa (carpenter bee, Hymenoptera). Under incubation conditions, any NYS would become fluorescent due to non-specific fluid-phase uptake. Ovarian follicles incubated in PSS-DexRed alone or in PSS with hemolymph from males did not carry out endocytosis detectable by this technique, but all other treatments listed above did.     

N-β-Alanyldopamine levels and synthesis in integument and other tissues of Manduca sexta (L.) during the larval-pupal transformation

N-β-Alanyldopamine (NBAD) and other diphenols in tissues of the fifth larval instar of the tobacco hornworm, Manduca sexta (L.), were analyzed by HPLC with electrochemical detection. NBAD accumulated in the integument during the intermolt feeding period, with maximal levels in the wandering stage (6 mmol/g). It then declined to a low level during apolysis and endocuticle digestion, while hemolymph NBAD increased during the same interval to a peak concentration (3 mM) shortly before pupal ecdysis. Trachea and foregut contained lesser amounts of NBAD (0.5 mmol/g), perhaps associated with cuticle, whereas fat body, muscle, midgut and hindgut had 0.1 mmol/g or less. Dopamine (DA), N-acetyldopamine and 3,4-dihydroxyphenylalanine (DOPA) were at least 10-fold less abundant than NBAD in the integument. NBAD synthase, which catalyzes the formation of NBAD from DA and β-alanine, was assayed in both integument and fat body. Highest activity was detected in the integument, where two peaks were observed, one at day 3 near the end of larval feeding and the other at day 9 as pupal cuticle tanning was initiated. Fat body enzyme was substantially less and was detected only in the pharate pupa. Maximal NBAD synthesis by integument cultured in vitro was dependent upon DA supplementation of at least 1.4 mM. 20-Hydroxyecdysone did not alter NBAD synthesis in vitro in either the integument or the fat body, even though injection of this hormone into isolated larval abdomens induced synthesis and/or transport of integumental NBAD back into the hemolymph. The rate-limiting steps in the NBAD biosynthetic pathway appear to be the production of DOPA and DA, because β-alanine occurs in the hemolymph at relatively high levels throughout larval-pupal development.     

The ontogeny of multiple hemoglobins in Chironomus thummi (Diptera): The effects of a compound with juvenile hormone activity

The multiple hemoglobins (Hbs) of Chironomus thummi show distinct and significant ontogenetic changes during development from the third instar through the fourth instar and metamorphosis into the pupa. A total of nine Hbs are resolved by 12.7% acrylamide gel electrophoresis (pH 8.65). Hbs 2 and 3, which are stage specific for the fourth instar, are first detected on the fourth day of this stage by electrophoresis and immunoprecipitation. Hb 4 is the predominant Hb species in the early and middle fourth instar, but during the late fourth instar and prepupa, Hb 1 predominates. The concentrations of Hbs 5–9 remain relatively constant in middle instars and decrease during later development. The Hb content of larval hemolymph exhibits changes that coincide with developmental stages; molting is characterized by low Hb content, whereas, the hemolymph of intermolt animals contains relatively high levels of Hbs. Treatment of fourth instars with a juvenile hormone analog, Altosid, prolongs this stage and inhibits the progress of normal development resulting in the formation of larval-pupal intermediates. Altosid also appears specifically to inhibit the accumulation of soluble hemolymph proteins related to pupation and metamorphosis, without affecting the concentration of Hb. Most significantly, it induces the precocious appearance of Hbs 2 and 3, which remain elevated above control levels in the late larval and prepupal stages. The present results strongly suggest that Altosid stimulates the appearance and accumulation of larval-specific proteins in vivo, while it inhibits the appearance of pupation-related proteins.     

Ecdysteroid profiles for hemolymph and testes from larvae,pupae, and pharate adults of the European corn borer,Ostrinia nubilalis hubner

Total ecdysteroid levels as well as concentrations of several individual ecdysteroids were determined for hemolymph and testes of fifth instars, pupae, and pharate adults of the European corn borer, Ostrinia nubilalis (Hubner). For total levels, the patterns of fluctuation in hemolymph and testes were similar, but the concentrations in testes were lower than those in hemolymph. In both hemolymph and testes there were two ecdysteroid peaks: the first just prior to the formation of the pharate pupa, the second just prior to the formation of the pharate adult. An examination of ecdysteroid profiles revealed some important differences. Ecdysone was either absent or present at extremely low levels in larval testes, whereas in hemolymph there was a premolt ecdysone peak. In pupal testes, ecdysone was present, but levels of 26-hydroxyecdysone were much lower than those in hemolymph. Thus, in regard to ecdysteroids, testes have the ability to control their own internal milieu.     

Manduca sexta lipid transfer particle: Synthesis by fat body and occurrence in hemolymph

Lipid transfer particle (LTP) is present in hemolymph of the tobacco hornworm Manduca sexta. Biosynthesis of LTP, occurrence in hemolymph, and the role of LTP-apoproteins in the lipid transfer reaction were investigated using antibodies specific for LTP or for each of the apoproteins. In vitro protein synthesis followed by immunoprecipitation demonstrated that LTP is synthesized by the fat body and secreted into the medium. In contrast to apolipophorin III, an exchangeable apoprotein of lipophorin (the major lipid transport protein in hemolymph), apoLTP-III could not be detected free in hemolymph. LTP concentrations in the hemolymph were measured by a sandwich ELISA using a mouse monoclonal antibody against apoLTP-III as capturing antibody and rabbit polyclonal antibody against apoLTP-I as detecting antibody. LTP concentration increased during the late fifth instar larval stage, followed by a decrease in the wandering stage. Subsequently, LTP concentrations were strongly increased in hemolymph of adult moths. The role of the three apoproteins of LTP in the lipid transfer reaction was analyzed using apoprotein-specific antibodies. All three, apoLTP-I, -II, and -III, appeared to be important for lipid transfer activity, as shown by inhibition of lipid transfer by antibodies specific for each of the three apoproteins. © 1996 Wiley-Liss, Inc.     

Development of male-incubated ovaries in the gypsy moth,Lymantria dispar

Ovaries from Lymantria dispar females were transplanted into an environment lacking vitellogenin, the male milieu, in order to determine how the presence of vitellogenin in the hemolymph affects the process of protein uptake by gypsy moth oocytes. When undeveloped ovaries from newly ecdysed last instar females were transplanted into males of the same stage, follicles detached from the germarium and increased in size, but the growth of oocytes proceeded more slowly than those from female controls. Although chorion fromation was delayed in male-grown ovaries, scanning electron microscopy of chorionated eggs recovered from adult males showed that a chorion with normal surface architecture was formed by the adult stage. SDS-PAGE analysis of the male-grown ovaries and hemolymph from males receiving ovaries showed that vitellogenin production was not stimulated by the organ transplant and only male hemolymph proteins were internalized by the male-incubated ovaries. Thus, in the absence of vitellogenin, endocytosis of male hemolymph proteins occurred, but the rate of oocyte growth was slowed.     

Retinal is the essential form of retinoid for storage and transport in the adult of the ascidian Halocynthia roretzi

Retinoids in the organs (gonad [GND], body wall muscle [BWM], hepatopancreas [HP], gill, hemolymph cells and hemolymph plasma) of the adult ascidian Halocynthia roretzi were analyzed by high performance liquid chromatography. Retinal (RAL) occurred in every organ examined, and most of RAL (≥99%) was localized in the GND and BWM. None of the organs contained significant amounts of retinol (ROL) or retinyl ester (RE). Lipid droplets, which are characteristic of stellate cells (RE-storing cells of vertebrates), could not be found in the GND, BWM and HP by microscopic observations. These results indicate that this ascidian lacks the RE-storing mechanism, which is ubiquitous in adult vertebrates. The amount and localization of RAL showed the annual change in relation to the reproductive cycle. During summer, the growing season, RAL was present in both GND and BWM at a ratio of about 3:2. From summer to winter, RAL in the GND gradually increased, concomitant with the decrease of RAL in the BWM. In winter, the spawning season, most of RAL was present in the GND (ca. 98%). RAL appears to be accumulated first in the BWM and transported to oocytes accompanying yolk accumulation. ROL and RE were not implicated in the storage and transport of retinoids. The results in the present research strongly suggest that retinoic acid (RA) is produced by the two-step enzymatic reaction: carotenoid cleavage to RAL followed by RAL oxidation to RA and that the prevertebrate chordate lacks ROL-metabolizing systems.     

Die motorische aktivität der endoparsitischen Larven vonPimpla turionellae L. undPimpla flavicoxis Ths. [Hym., Ichneum.] in der wirtspuppe

Zusammenfassung Die endoparasitischen Larven der beiden nahe verwandten PuppenparasitenartenPimpla turionellae undPimpla flavicoxis legen w?hrend ihrer Larvenentwicklung in der Wirtspuppe eine charakteristische Wanderung zurück. Unabh?ngig von der Lage des Parasiteneies in der Puppe wandert die Junglarve sofort nach dem Verlassen des Eies zum Kopfende der Puppe und zerst?rt dort das Gehirn und die umliegenden Organe. Anschliessend kriecht sie ins Abdomen, w?hrend sie gleichzeitig die Wirtsgewebe aufl?st und Nahrungsbrei zu sich nimmt. Im Abdomen dreht sie sich abermals um, so dass ihr Kopf zum Vorderende der Puppe weist. In dieser Lage verzehrt sie den Rest des verflüssigten Puppeninhalts und verpuppt sich. Die Parasitenimagines schlüpfen durch das Vorderende der Wirtspuppe. —In superparasitierten Puppen wandern alle Junglarven zum Kopfende der Wirtspuppe und tragen dort ihre Konkurrenzk?mpfe aus, indem die überz?hligen Larven durch Bissverletzungen ausgeschaltet werden. In der Regel bleibt eine Larve am Leben und entwickelt sich weiter. — Die Junglarven werden wahrscheinlich durch chemische oder Str?mungsreize zum Vorderende der Puppe gelenkt. Dieses ausgepr?gte Orientierungsverhalten gestattet die Ausschaltung gleichalteriger Nahrungskonkurrenten zum frühest m?glichen Zeitpunkt. Die Wanderung der Larven bringt den Parasiten allem Anschein nach noch andere Vorteile.

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Summary The endoparasitic larvae of two pupal parasites,Pimpla turionellae L. andPimpla flavicowis Ths. (Hym., Ichneumonidae) move inside the host pupa (Galleria mellonella L.) in a characteristic manner during their development. Soon after hatching from the eggs the young larvae arrive in the head of the pupa (tab. 1 and 6) and destroy the organs situated here by histolysis. As is shown in tables 2, 3, 4 and in figure 2 the movement of the larvae to the head region is independent of the site of the egg in the pupa. One or two days afterwards the larvae change their position by moving into the abdomen, liquefying its contents (fig. 3). They turn again in the abdomen but remain there feeding on the rest of the liquified host tissues. The fullgrown larva lies in the abdomen occupying nearly the whole cavity of the pupa. Its head is directed towards the head part of the pupa. Pupation is usually performed in this position so that the adult emerges from the anterior end of the host pupa. When the host pupa is superparasitized byP. turionellae, all young larvae move to its anterior end. The competing larvae fight each other, using their mandibles for wounding the soft body of their competitors. These wounds seem always to be fatal and can be recognized by their rapid melanization (fig. 4). Since all young larvae have met in the head region of the pupa, the dead competitors can usually be found there in full number, the victor having moved into the abdomen next day (tab. 5). Only a single larva survives. The young larvae seem to be directed by a chemical attractant or by rheotaxis in the circulating hemolymph, since gravity and light can be excluded as factors of orientation. The behaviour of the larvae enables the parasites to eliminate competitors of the same age at the earliest possible moment and in this way to conserve all the food for the surviving larva. Further advantages are probably gained by the circulating movement of the larvae, i.e. the early elimination of the hormonal control system of the host and the final position of the fullgrown larva, which leads the emerging adult to openings in the cocoon or gallery preformed by the host larva.

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Pimpla flavicoxis Гhs=Pimpla aquiloniaCress (Aubert, 1969).     

In vitro lipid transfer between lipoproteins and midgut-diverticula in the spider Polybetes pythagoricus

It has been already reported that most hemolymphatic lipids in the spider Polybetes pythagoricus are transported by HDL1 and VHDL lipoproteins. We studied in vitro the lipid transfer among midgut-diverticula (M-diverticula), and either hemolymph or purified lipoproteins as well as between hemolymphatic lipoproteins. M-diverticula and hemolymph were labeled by in vivo ¹⁴C-palmitic acid injection. In vitro incubations were performed between M-diverticula and either hemolymph or isolated lipoproteins. Hemolymph lipid uptake was associated to HDL1 (67%) and VHDL (32%). Release from hemolymph towards M-diverticula showed the opposite trend, VHDL 75% and HDL1 45%. Isolated lipoproteins showed a similar behavior to that observed with whole hemolymph. Lipid transfer between lipoproteins showed that HDL1 transfer more ¹⁴C-lipids to VHDL than vice versa. Only 38% FFA and 18% TAG were transferred from M-diverticula to lipoproteins, while on the contrary 75% and 73% of these lipids, respectively, were taken up from hemolymph. A similar trend was observed regarding lipoprotein phospholipids. This study supports the hypothesis that HDL1 and hemocyanin-containing VHDL are involved in the uptake and release of FFA, phospholipids and triacylglycerols in the spider P. pythagoricus. The data support a directional flow of lipids from HDL1 and VHDL suggesting a mode of lipid transport between lipoproteins and M-diverticula.     

In vitro culture of ovaries of a viviparous gall midge

Summary Ovaries of the viviparous pedogenetic gall midgeHeteropeza pygmaea can be cultured in hemolymph obtained from X-ray-sterilized larvae of the same species. In this culture medium, formation of follicles is essentially the same as in vivo, and sometimes female larvae develop from these follicles. The ovaries of such larvae, in their turn, have been cultured in vitro to produce larvae. In this way, in vitro development from oogonium to larva has been maintained for several generations. When using hemolymph obtained from larvae grown under different conditions, the in vitro cultured ovaries produce a second type of egg which probably is male-determined. Ovarian development in vitro has been studied with differential interference contrast optics and time-lapse cinemicrography. This work was supported by the Swiss National Science Foundation Grant No. 3.2010.73.     

Neuroendocrine control of diapause hormone secretion in the silkworm,Bombyx mori

To clarify the control mechanism of diapause hormone (DH) secretion in the silkworm Bombyx mori a series of anatomical and pharmacological experiments were carried out. The arrangement of 'diapause' and 'non-diapause' eggs in the ovarioles of the moths was determined by the coloration method to estimate the accumulation of 3-hydroxykynurenine in the eggs. The females destined to lay non-diapause eggs (non-diapause producers) had diapause eggs in their ovaries if their subesophageal ganglions (Sg) had been surgically removed at 2days after larval-pupal ecdysis or later. In contrast when the surgical extirpation extended to the brain and the corpora cardiaca (CC)-corpora allata (CA) complex in addition to the Sg, the non-diapause producers had no diapause eggs. When the Sg was removed from the females destined to lay diapause eggs (diapause-producers), diapause eggs appeared in response to the treatment at 2days after larval-pupal ecdysis, but the appearance of diapause eggs was delayed by 2days when the brain-CC-CA complex was included among the organs removed. These observations suggested that DH is produced in Sg and transferred to the CC-CA complex, and that the secretion of DH from the complex is suppressed in non-diapause producers. The pattern of diapause and non-diapause eggs induced by the transection of the subesophageal connective in diapause and non-diapause producers suggested a regenerative and secretory capacity of the neurosecretory cells after the operation. The appearance of diapause eggs in non-diapause producers with transected protocerebrum of the brain confirmed that there was an inhibitory center in the protocerebrum. Changes in parts of the ovarioles containing diapause and non-diapause eggs with time of injection of gamma-aminobutyric acid (GABA) and picrotoxin suggested that a GABAergic inhibitory mechanism in DH secretion may be active in non-diapause producers but inactive in diapause producers throughout the pupal stage.