A Sericesthis iridescent virus infection of the hemocytes of the waxmoth,Galleria mellonella (Lepidoptera)

The cytopathology of the Seriscethis iridescent virus (SIV) infection of Galleria hemocytes was studied by phase-contrast microscopy, [³H]thymidine autoradiography, and fluorescent antibody and acridine orange staining. Of the five hemocyte classes only the oenocytoid was not infected. Infected plasmatocytes and adipohemocytes undergo nuclear and cytoplasmic hypertrophy accompanied by nuclear DNA synthesis. The timing and identity of nuclear and cytoplasmic DNA synthesis are discussed.     

Purification and characterization of a flavin-binding storage protein from the hemolymph of Galleria mellonella

The 85K storage protein that accumulates in the hemolymph of Galleria mellonella during the final larval instar was isolated and purified from newly molted pupae. The separation of fresh hemolymph proteins from larvae or pupae by different chromatographic and electrophoretic procedures indicated the native protein had a Mr of 170,000 and consisted of two identical 85K subunits. Crosslinking experiments using fresh hemolymph followed by Western blotting also indicated a dimeric structure for the native protein. Analyses of the dimer purified from pupal hemolymph indicated that 85K was a glycoprotein, containing approximately 6.5% neutral sugar and about 1.9% amino sugar. Like other insect flavin-binding proteins, 85K has a relatively high histidine content but an uncharacteristically high arginine content. The purified 85K dimer did not bind riboflavin, suggesting that the integrity of the molecule had been altered during purification. However, 85K purified in low yield by Affi-Gel Blue chromatography, did bind riboflavin, indicating that under certain, undefined conditions the functional integrity of the protein could be retained during purification. © 1994 Wiley-Liss, Inc.

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

     

Some effects of CGA 72662 on larval development in the housefly,Musca domestica (L.)

Summary

Houseflies reared on artificial diet supplemented with a range of concentrations of CGA 72662 suffered severe developmental disruption at the larval stage. Death was preceded by the appearance of a range of cuticular lesions and abnormalities. These cuticular abnormalities have been observed under the light and electron microscopes.     

Changes in acetylcholinesterase during pupal development of Apis mellifera queen

Total, membrane, and soluble acetylcholinesterase (AchE, EC 3.1.1.7) activities increase during the pupal development of Apis mellifera queen to reach maximum values at emergence. Membrane and soluble AchE are inhibited by 10^-5 M eserine or BW284C51 except at Pr, Pdm, and Pdd stages in which soluble AchE presents eserine-sensitive and eserine-resistant fractions. At all pupal stages, AchE occurs in a major amphiphilic membrane form that represents about 98% of total AchE activity and whose sedimentation coefficient is about 5.7S, and in a minor hydrophilic form that represents about 2% of total AchE activity and whose sedimentation coefficient is about 7S. At all pupal stages, phosphatidylinositol-specific phospholipase C (PI-PLC) and glycosyl phosphatidylinositol-specific phospholipase D (GPI-PLD) convert the membrane form into soluble counterparts which electrophoretic mobilities differ from that of the soluble form. AchE exhibits a butyrylcholinesterase (BuChE) activity that represents about 14% of AchE activity. During pupal development, the BuChE/AChE ratio of the membrane fraction is relatively stable, whereas the BuChE/AChE ratio of the soluble fraction is subjected to significant variations. At early pupal stages (Pw–Pd), membrane AchE displayed a high K_m value, higher than 40 μM, that decreases to an intermediary value of about 30 μM at Pdl and Pdm stages, to reach finally about 20 μM at Pdd and emergence stages. Arch. Insect Biochem. Physiol. 36:69–84, 1997. © 1997 Wiley-Liss, Inc.     

Variations in the density of lipophorins during late larval and early pupal development of Manduca sexta

The density of lipophorin was determined in individual Manduca sexta during development from the second day of the fifth larval instar to the second day of the pupal stage. Lipophorin formed defined bands when subjected to density gradient ultracentrifugation. All lipophorin observed was high density lipophorin; however, the densities varied from 1.100 to 1.184 g/ml, and 40% of the animals had more than one density form of lipophorin. The lipophorins were divided into five density classes: class 1 from 1.100 to 1.113 g/ml, class 2 from 1.114 to 1.132 g/ml, class 3 from 1.133 to 1.145 g/ml, class 4 from 1.146 to 1.162 g/ml, and class 5 from 1.163 to 1.184 g/ml. In feeding larvae, classes 2 and 3 were the most abundant. Larvae of the first day of wandering had either lipophorin in class 2 or in classes 2 and 5. Later during wandering the variation increased, but on the third day most of the lipophorin was in class 2. In first day pupae, only lipophorins of classes 4 and 5 were detected, while on the second day of the pupal stage, classes 2 and 3 were predominant. Class 1 lipophorin was abundant in larvae injected with Manduca adipokinetic hormone (M-AKH), and rare in young feeding larvae. In no other stage was class 1 lipophorin observed. Our results show that the density of lipophorin is much more variable than previously reported which makes it difficult to ascribe any lipophorin density to a developmental stage. These results also show that adipokinetic hormone decreases the density of lipophorin in larvae. © 1996 Wiley-Liss, Inc.     

Changes in heat shock protein synthesis and heat sensitivity during mouse thymocyte development

Heat shock protein synthesis was examined in mouse thymocytes at three stages of development: early embryonic thymocytes, which are CD4^?CD8^?, adult thymocytes, which are primarily CD4⁺CD8⁺, and mature spleen T cells, which are CD4⁺CD8^? or CD4^?CD8⁺. After either a 41°C or 42°C heat shock, the synthesis of the maior heat-inducible protein (hsp68) was elevated during the first hour of recovery but then decreased abruptly in thymocytes from adult mice. In contrast, the synthesis of hsp68 continued for up to 4 h after heating embryonic mouse thymocytes or mature spleen T cells. The more rapid termination ofthe heat shock response in the adult thymocytes was not the result of eitherless heat damage or more rapid repair since the recovery of general protein synthesis was more severely delayed in these cells. As well, the double positive CD4⁺CD8⁺ cells were more sensitive to hyperthermia than either the double negative CD4^?CD8^? or single positive CD4⁺CD8^? or CD4^?CD8⁺ cells. Exposure of fetal thymus organ cultures to elevated temperature revealed that the double negative thymocytes were able to survive and differentiate normally following a heat shock treatment that was lethal for the double positive thymocytes. Exposure of thymocytes from adult mice to elevated temperatures induced apoptotic cell death. This was evident by the cleavage of DNA into oligonucleosome-sized fragments. Quantitation of the extent of DNA fragmentation and the number of apoptotic cells by flow cytometry demonstrated that the extent of apoptotic cell death was related to the severity of the heat stress. Double positive (CD4+CD8+) thymocytes are selected on the basis of their T-cell antigen receptor (TCR). Most of these cells are negatively selected and die within the thymus by an active process of cell deletion known as apoptosis. Restricting hsp synthesis in response to stress might be essential during developmental processes in which cell maturation is likely to result in death rather than functional differentiation. © 1993Wiley-Liss, Inc.     

Growth-blocking peptide titer during larval development of parasitized and cold-stressed armyworm

Growth-blocking peptide (GBP) has been isolated for the first time from the haemolymph of the host armyworm Pseudaletia separata whose development was halted in the last larval instar stage by parasitization with the parasitoid wasp Cotesia kariyai. Recent studies demonstrated that GBP not only exists in the plasma (haemolymph without cells) of parasitized last instar larvae, but also in the plasma of nonparasitized penultimate (5th) instar larvae. Monoclonal antibodies were prepared to measure the titers of GBP in nonparasitized and parasitized larval plasma. One of three monoclonal antibodies raised against GBP, which is the most specific for GBP, was used to quantify the concentration of plasma GBP. As this antibody recognized two plasma peptides other than GBP in crude plasma fractions, each plasma peptide fraction was separated by a reversed phase HPLC, and then plasma GBP level was measured by ELISA. The highest level of plasma GBP detected on Day 0 of the penultimate instar larvae was gradually decreased throughout the larval growth except for the temporary increase on Day 0 of last larval instar. After parasitization on Day 0 of last larval instar, two peaks of plasma GBP titer were detected during the last larval instar, one day and six days after parasitization. This characteristic increase and decrease in plasma GBP level was also observed by transferring last instar larvae of the armyworm from 25 to 10°C, as a result of which larvae delayed pupation by more than 15 days. From these results, it is reasonable to propose that plasma GBP in lepidopteran larvae might control certain upstream steps in a cascade of events leading to pupation; thus, an elevated level of plasma GBP interferes with normal metamorphosis from larvae to pupae.     

Changes in biomass and chemical composition during lecithotrophic larval development of the southern king crab, Lithodes santolla (Molina)

Changes in biomass and elemental composition (dry mass, W; carbon, C; nitrogen, N; hydrogen, H) were studied in the laboratory during complete larval and early juvenile development of the southern king crab, Lithodes santolla (Molina), formerly known as Lithodes antarcticus (Jacquinot). At 6±0.5 °C, total larval development from hatching to metamorphosis lasted about 10 weeks, comprising three demersal zoeal stages and a benthic megalopa, with mean stage durations of 4, 7, 11 and 47 days, respectively. No differences in development duration or mortality were observed in larvae either fed with Artemia sp. nauplii or unfed, indicating that all larval stages of L. santolla are lecithotrophic. First feeding and growth were consistently observed immediately after metamorphosis to the first juvenile crab stage. Regardless of the presence or absence of food, W, C, N and H decreased throughout larval development. Also the C:N mass ratio decreased significantly, from 7.7 at hatching to 4.1 at metamorphosis, indicating that a large initial lipid store remaining from the egg yolk was gradually utilized as an internal energy source, while proteins played a minor role as a metabolic substrate. In total, 56-58% of the initial quantities of C and H present at hatching, and 20% of N were lost during nonfeeding larval development to metamorphosis. Nine to ten percent of the initially present C, N and H were lost with larval exuviae, half of these losses occurring in the three zoeal stages combined and another half in the megalopa stage alone. Metabolic biomass degradation accounted for losses of about 47-50% in C and H but for only 10% in N. Hence, most of the losses in C and H reflected metabolic energy consumption (primarily lipid degradation), while about half of the losses in N and two thirds of those in W were due to larval exuviation. Complete independence from food throughout larval development is based on an enhanced maternal energy investment per offspring and on energy-saving mechanisms such as low larval locomotory activity and low exuvial losses. These traits are interpreted as bioenergetic adaptations to food-limited conditions in Subantarctic regions, where a pronounced seasonality of day length limits the period of primary production, while low temperatures enforce a long duration of pelagic development.     

Effects of starvation,chilling, and injury on endocrine gland function in Galleria mellonella

Starvation, chilling, and injury of last instar Galleria mellonella larvae typically elicit extra larval molts or a delay in pupation. The primary sites of action and the nature of the signals by which these treatments affect development are not known. However, since the connections of the brain to the nerve cord are crucial for the effects of starvation and chilling, these signals apparently affect the brain-centered program of developmental regulation via the nerve cord. Chilling, and occasionally starvation, cause extra larval molts in last instar larvae treated prior to the nervous inhibition of their corpora allata; release of a cerebral allatotropin, which stimulates the production of juvenile hormone, appears to be involved in this effect. After this time, a delay in pupation is the principal effect of starvation and chilling, and is apparently due to a temporal inhibition of the release of the prothoracicotropic hormone. Chilling also appears to inhibit unstimulated ecdysteroid production by the prothoracic glands. The effect of injury is not mediated by the nerve cord, but appears to involve an inhibitory humoral factor that affects either the brain or the prothoracic glands themselves. Injury also stimulates juvenile hormone production, an effect which is enhanced when the brain is separated from the nerve cord and which is evidenced by a delay of ecdysis and the occasional retention of some larval features in the ecdysed insects. None of the effects of these various treatments on the brain and the endocrine glands persist when the brains or glands are implanted into untreated hosts.     

Changes in the distribution of gap junctions inDrosophila melanogaster wing discs during the third larval and early pupal stages of development

Summary Developmental changes in the distribution of gap junctions in early, mid and late third larval stage wing discs and in pupariation+6 h and pupariation+24 h stage wing discs fromDrosophila melanogaster were analyzed by quantitative electron microscopy. Gap junctions occur in all 12 intradisc regions examined in each of the five developmental stages. Their distribution is non-random and changes during development which suggests that they are developmentally regulated. The gap junctions are not static structures, rather they grow and regress during development. The changes tend to be gradual ones without sudden increases or decreases. Gap junctions continuously form and grow in size throughout the third larval stage and during the first 6 h following pupariation. Their surface density, number, percent of the lateral plasma membrane area, and absolute area as well as the lateral plasma membrane surface density all increase during this time. Between pupariation+ 6 h and pupariation+24 h all but one of these parameters decrease indicative of gap junctional breakdown. Gap junctions are most numerous and change least during development in the apical cell regions where intercellular contacts are close and stable. They change most in the basal cell regions where intercellular contacts tend to be looser and change during development. The most dramatic change is in the absolute area which increases by a factor of 23 between the early third larval stage and pupariation+24 h. At pupariation the rate of gap junction growth undergoes a transient increase before the phase of disassembly begins. Developmental changes in gap junction surface density are closely coupled with changes in the lateral plasma membrane surface density which suggests that these may be coregulated. Evidence from mutants suggests that when the number and density of gap junctions fail to increase in proportion to lateral plasma membrane growth, wing disc development will be abnormal. Our results support the idea that some minimum gap junction density is required for normal development and that this must increase as development proceeds. The results are consistent with the notion that gap junctions are involved in pattern formation and growth control and are discussed with respect to the acquisition of competence for metamorphosis, disc growth, disc morphogenesis and changes in the hormonal environment.     

Insect acetyl-CoA carboxylase: Activity during the larval,pupal and adult stages of insect development

• 1.1. The activity of the lipogenic enzyme, acetyl-CoA carboxylase, was investigated in four insect species; Bombyx mori (Lepidoptera), Tenebrio molitor (Coleoptera), Glossina morsitans and Sarcophaga nodosa (Diptera).
• 2.2. Acetyl-CoA carboxylase activity in larval, pupal and adult forms was compared with the saponifiable lipid mass at each stage of the life-cycle, and found to follow similar patterns except for Tenebrio molitor.
• 3.3. The results are examined in relation to known metabolic requirements for each insect.

     

Changes in protein synthesis during the development of Agrostemma githago seeds

The transition from the growth to the maturation phase in developing seeds of Agrostemma githago L. was found to coincide with major changes in the rate of protein synthesis, the kinds of proteins synthesized, and the composition of the non-proteinbound amino acid pool. Coincident changes were observed in viability and the ability to withstand desiccation. Desiccated mature Agrostemma seeds are dormant and need at least three months of after-ripening. In imbibed dormant and after-ripened seeds no synthesis of storage proteins was observed with the exception of one particular set of storage proteins. Dormant and after-ripened seeds synthesized the same kinds of proteins during early imbibition, indicating an almost identical metabolic state which differs considerably from that of developing seeds.     

Changes in vitellin and other yolk proteins during embryonic development in the silkworm, Bombyx mori

Changes in the amounts of vitellin and other yolk proteins of the eggs of the silkworm, Bombyx mori were investigated during embryonic development using polyacrylamide gel electrophoresis and immunotitration techniques. In the newly laid eggs, soluble proteins were separated into at least nine bands after electrophoresis. The major band was identified as vitellin, accounting for about 40% of the total proteins. The four predominant bands including vitellin exhibited the same mobility as the proteins of haemolymph, but one other major band was specific to the eggs, accounting for about 20% of the proteins.During embryonic differentiation 6–7 days after oviposition, the total protein content did not decrease and the banding patterns and their relative concentrations remained unchanged as a whole. However the concentration of the egg specific protein steadily decreased. During subsequent larval differentiation until hatching, the total proteins were utilized to about 50% of the initial levels: the rapid degradation was observed in almost every species of proteins.An immunotitration experiment further demonstrated that vitellin was not utlilized during embryonic differentiation but was consumed markedly during larval differentiation. However, about 30% of initial level was reserved in the newly hatched larvae. Such a prolonged persistence of vitellin is discussed in relation to protein metabolism during embryonic development in silkworms.     

Ecdysteroid-dependent protein synthesis in caste-specific development of the larval honey bee ovary

In the honey bee, Apis mellifera, the fifth larval instar is a critical period for caste differentiation. During this premetamorphic phase the hormonal milieu shows pronounced caste differences and several organs, particularly the ovaries, enter different developmental pathways leading to highly fertile queens and nearly sterile workers. Developmental profiles of total protein synthesis in larval ovaries showed marked caste differences starting with the early fifth instar. By two-dimensional electrophoresis, caste-specific patterns could be detected in the synthesis of a 29 kDa/pI 4.6 and two 24 kDa/pI 5.2–5.5. proteins (pI=isoelectric point). A marked decrease in the expression of these proteins was found to coincide with caste-specific differences in the haemolymph ecdysteroid titer. In vitro exposure of larval worker ovaries to physiological (10^–7 m) concentrations of synthetic makisterone A elicited an identical response. Juvenile hormone did not affect protein synthesis patterns in larval ovaries, and also did not inhibit or reverse the ecdysteroid-induced effects. Heat shock experiments revealed that the 29 kDa/pI 4.6 ecdysteroid-regulated protein belongs to the class of small heat shock proteins.     

Changes in soluble protein and isoenzymes in normal and opaque-2 Zea mays endosperm during grain development

Electrophoretic patterns of soluble proteins, pH 5 enzyme fraction, peroxidase, glutamic dehydrogenase, leucine aminopeptidase in developing endosperm of normal and opaque-2 were studied. Multiple forms were found for all the enzymes studied. The GDH pattern showed considerable differences in normal and opaque-2 maize; the soluble protein pattern also differed, both qualitatively and quantitatively. The leucine-amino-peptidase pattern was identical and the peroxidase pattern showed slight differences.