Rates of juvenile hormone synthesis control caste differentiation in the stingless bee Scaptotrigona postica depilis

Summary In social insects the expression of caste-specific characters is controlled by juvenile hormone (JH) during definite sensitive periods in preimaginal development. For a number of stingless bee species the existence of such a JH-sensitive period has already been demonstrated. Queen development can be induced by topical JH applications during the cocoon spinning phase of the last larval instar. Neither JH titers nor rates of JH synthesis were known so far for this subfamily of eusocial bees distinguished by a pronounced caste dimorphism. As the pantropically distributed stingless bees with approximately 400 recent species are the largest group of social bees, JH synthesis was studied in one of the species that can be kept under laboratory conditions. An in vitro radiochemical assay was used to measure stage- and caste-specific activities of the corpora allata (CA). For the first time in a eusocial hymenopteran species it was demonstrated how the endocrine system is reacting to trophogenic stimuli capable to induce caste differentiation during larval development. Generally JH synthesis in queen CA was found to be 30–80% higher than in workers during the penultimate and last larval instar, but a strong and distinct caste-specific modulation of JH synthesis was only observed right before the onset of a JH-sensitive period in the cocoon spinning phase of the fifth instar.     

Effect of heat shock on protein synthesis in Locusta migratoria epidermis

Heat shock induced by an increase in temperature from 30°C to 47°C led to changes in protein synthesis in wing pads of the fifth larval instar of Locusta migratoria. Synthesis of heat shock proteins in the molecular weight range of 85,000, 70,000 and 18,000–22,000 was first detected at a threshold temperature of 45°C and was found to be highest at 47°C. A marked decline in the synthesis of many other proteins was also evident at 47°C. Recovery of general protein synthesis was observed when wing pads were shifted back to 30°C after a 2-h heat shock at 47°C. Heat shock protein patterns in Locusta and Drosophila were compared.     

Caste-specific modulation of juvenile hormone III content and ecdysteroid titer in postembryonic development of the stingless bee,Scaptotrigona postica depilis

Summary Juvenile hormone III content and ecdysteroid titer were analyzed for larval and pupal development of the stingless bee,Scaptotrigona postica depilis. Castespecific differences in juvenile hormone III content were detected at three developmental phases: at the transition from the fourth to the fifth larval stadium, in the spinning phase of the fifth larval stadium, and shortly after the imaginal moult. During the fifth larval stadium, juvenile hormone content closely reflects corpora allata activity. Juvenile hormone synthesis may thus be responsible for the elevated hormone titer in spinning-phase queen larvae, a phase of known sensitivity for induction of queen characters by exogenous juvenile hormone. For ecdysteroids, two phases of caste-specific differences were found: in the pre-pupal phase, and shortly after the imaginal moult. In both periods the titer in queens is distinctly higher compared to workers.Abbreviations Im imago 1 day after eclosion - L3, L4, L5 larval instars 3, 4, and 5 - L5F1, L5F2 substages of feeding phase in fifth larval instar - L5S1, L5S2, L5S3 substages of spinning phase in fifth larval instar - PP1, PP2 substages of prepupal phase - Pw white eyed pupa - Pp pink eyed pupa - Pr red eyed pupa - Pd dark eyed pupa - Pdl, Pdm, Pdd dark eyed pupa with progressive tanning of cuticle - RIA radioimmunoassay     

Impact of heat shock on heat shock proteins expression, biological and commercial traits of Bombyx mori

We report the thermotolerance of new bivoltine silkworm, Bombyx mori strains NB₄D₂, KSO₁, NP₂, CSR₂ and CSR₄and differential expression of heat shock proteins at different instars. Different instars of silkworm larva were subjected to heat shock at 35°C, 40°C and 45°C for 2 hours followed by 2 hours recovery. Heat shock proteins were analyzed by SDS‐PAGE. The impact of heat shock on commercial traits of cocoons was analyzed by following different strategies in terms of acquired thermotolerance over control. Comparatively NP₂ exhibited better survivability than other strains. Resistance to heat shock was increased as larval development proceeds in the order of first instar > second instar > third instar > fourth instar > fifth instar in all silkworm strains. Expression of heat shock proteins varies in different instars. 90 kDa in the first, second and third instars, 84 kDa in the fourth instar and 84, 62, 60, 47 and 33 kDa heat shock proteins in fifth instar was observed in response to heat shock. Relative influence of heat shock on commercial traits that correspond to different stages was significant in all strains. In NB₄D₂, cocoon and shell weight significantly increased to 17.52% and 19.44% over control respectively. Heat shock proteins as molecular markers for evaluation and evolution of thermotolerant silkworm strains for tropics was discussed.     

Germ cell cluster formation and cell death are alternatives in caste-specific differentiation of the larval honey bee ovary

Summary

Caste-specific differentiation of the female honey bee gonad takes place in the fifth larval instar. In queen larvae most ovarioles exhibit almost simultaneous formation of numerous germ cell clusters within the first 20 h after the last larval molt. Ultrastructurally distinctive fusomal cytoplasm connects these cystocytes. Germ cell differentiation is accompanied by morphological changes in somatic components of the ovarioles, the follicle and the terminal filament cells. Subsequently, queen ovarioles elongate and differentiate basal stalks that coalesce in a basal calyx. A second round of mitotic activity was found to occur in the late prepupal and early pupal queen ovary. This round may elevate germ cell numbers composing each cluster to levels observed in follicles of adult honey bee queens. In contrast, germ cell cluster formation does not occur in most of the 120–160 ovarioles of the larval worker ovary, but instead many cells in such ovarioles show signs of impending degeneration, such as large autophagic bodies. DNA extracted from worker ovaries did not reveal nucleosomal laddering, and ultrastructurally, chromatin in germ cell nuclei appeared intact. In the 4–7 surviving ovarioles of the small worker ovary, germ cell clusters were found with ultrastructural characteristics identical to those in queen ovarioles. The temporal window during which divergence in developmental pathways of the larval ovaries initiates shortly after the last larval molt coincides with caste-specific differences in juvenile hormone titer which have long been considered critical to caste-specific morphogenesis.     

Evidences for a stage-specific juvenile hormone binding protein in the hemolymph of the silkworm,Bombyx mori L.: Identification and characterization by photoaffinity labeling and immunological analyses

Two molecular forms of juvenile hormone binding proteins were identified in the larval hemolymph of Bombyx mori by photoaffinity labeling. One form having an Mr of 33 kDa was present constantly in the hemolymph of the third to the fifth instar larvae while the other form having an Mr of 35 kDa was detected in the hemolymph until in the early fifth instar larvae but not in the prewandering larvae and prepupae. A 33 kDa binding protein was purified by hydrophobic interaction chromatography, gel filtration, and native PAGE. Antiserum against 33 kDa binding protein cross-reacted with 35 kDa binding protein on Western blots, suggesting that these binding proteins shared the same epitopes. From the results of saturation binding assays, it was inferred that 33 and 35 kDa binding proteins had a similar binding affinity for JH 1. It was revealed that one of these binding proteins, 35 kDa binding protein, was produced in the fat body in a stage-specific manner: fat body of the early fifth instar larvae synthesized both 33 and 35 kDa binding proteins while that of prewandering larvae synthesized only 33 kDa binding protein. © 1996 Wiley-Liss, Inc.     

Effect of High Temperature on Protein Expression in Strawberry Plants

Strawberry plants (Fragaria×ananassa Duch.) cvs. Nyoho and Toyonoka were exposed to temperatures of 20, 33, and 42 °C for 4 h, and protein patterns in leaves and flowers was analyzed by 2-dimensional polyacrylamide gel electrophoresis and immunoblotting. In leaves and flowers of both cultivars, the content of most proteins decreased, but a few new proteins appeared in response to heat stress. These heat shock proteins (Hsps) were detected in the range of 19 – 29 kDa in leaves, and 16 – 26 kDa in flowers. The intensity of a 43 kDa protein spot increased in response to heat stress in Nyoho flowers, but not in Toyonoka flowers. The peaHsp17.7 antibody recognized one band at approximately 26 kDa in leaves, and two bands at approximately 16 and 17 kDa in flowers of both cultivars. These results show that the effects of heat stress on Hsp synthesis in strawberry plants differ between plant organs and between cultivars.     

Differences in the envelope proteins ofChlamydia pneumoniae,Chlamydia trachomatis,andChlamydia psittaci shown by two-dimensional gel electrophoresis

Analysis by two-dimensional gel electrophoresis of theN-laurylsarkosinate(Sarkosyl)-insoluble envelope complexes ofl-^[35]S-cysteine-iabeled elementary bodies ofChlamydia pneumoniae strain IOL-207,Chlamydia trachomatis serovar LGV2, D, and F, andChlamydia psittaci strain 6BC showed differences in the molecular charges of chlamydial outer membrane proteins. The apparent isoelectric point (pI) of the major outer membrane protein ofC. pneumoniae strain IOL-207 was 6.4, whereas the pI of the major outer membrane protein of theC. trachomatis andC. psittaci strains differed little from one another, ranging from 5.3 to 5.5. The 60-kDa cysteinerich protein ofC. pneumoniae was the only 60-kDa chlamydial protein with a pI value (5.9) more acidic than that of the corresponding major outer membrane protein. As a general rule, the charges of both the 60-kDa and the lowmolecular-mass (12–15 kDa) cysteine-rich proteins were widely variable, depending on the strain. However, in cach individual strain, the variation of the charge of the 60-kDa protein had a compensatory change in the lowmolecular-mass cysteine-rich protein.     

Caste-specific differences in ecdysteroid titers in early larval stages of the bumblebee Bombus terrestris

Mounting evidence implicates ecdysteroids in queen-worker differentiation during the last larval instars of highly social insects. In the present study, we analyzed ecdysteroid titers in queen and worker larvae of the bumblebee Bombus terrestris from the second to the early fourth instar. B. terrestris is of particular interest because caste is already determined in the second instar, presumably by a pheromonal signal emitted by the egg-laying queen. Caste differences in the adults, however, are only expressed at the physiological and not at the morphological level, except for the distinctly larger size of the queen. In the second and third instar, ecdysteroid titers in queen larvae were generally higher than those of workers. These early caste-specific differences, however, were abolished in the fourth instar. In the early fourth instar we could detect two small ecdysteroid peaks, with the one preceding the cocoon-spinning phase presenting the characteristics of a pupal commitment peak. The synchrony of caste differences in ecdysteroid and juvenile hormone titers suggests a synergistic action of these hormones in caste determination.     

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.     

Representational Difference Analysis (RDA) reveals differential expression of conserved as well as novel genes during caste-specific development of the honey bee (Apis mellifera L.) ovary

In highly eusocial insects, such as the honey bee, Apis mellifera, the reproductive bias has become embedded in morphological caste differences. These are most expressively denoted in ovary size, with adult queens having large ovaries consisting of 150-200 ovarioles each, while workers typically have only 1-20 ovarioles per ovary. This morphological differentiation is a result of hormonal signals triggered by the diet change in the third larval instar, which eventually generate caste-specific gene expression patterns. To reveal these we produced differential gene expression libraries by Representational Difference Analysis (RDA) for queen and worker ovaries in a developmental stage when cell death is a prominent feature in the ovarioles of workers, whereas all ovarioles are maintained and extend in length in queens. In the queen library, 48% of the gene set represented homologs of known Drosophila genes, whereas in the worker ovary, the largest set (59%) were ESTs evidencing novel genes, not even computationally predicted in the honey bee genome. Differential expression was confirmed by quantitative RT-PCR for a selected gene set, denoting major differences for two queen and two worker library genes. These included two unpredicted genes located in chromosome 11 (Group11.35 and Group11.31, respectively) possibly representing long non-coding RNAs. Being candidates as modulators of ovary development, their expression and functional analysis should be a focal point for future studies.     

Protein synthesis by the milk gland and fat body of the tsetse fly,Glossina pallidipes

The patterns of protein synthesis by the milk gland and the fat body of female Glossinapallidipes during the pregnancy cycle were studied by incubation with [³⁵S]methionine both in vivo and in vitro. The pattern of protein synthesis by the milk gland changed with the stage of the larva in the uterus. Very little synthesis occurred in the milk gland until the first instar larva hatched. Then four proteins (13, 16, 24 and 72 kDa) were prominently synthesized. As the larva matured, the synthesis of 19, 38, 40 and 72 kDa proteins increased, whereas that of the 13 and 24 kDa proteins decreased. Just before larviposition, only the 16 and 72 kDa proteins were still being synthesized. The milk gland secreted into the medium primarily the 13, 16, 19 and 72 kDa proteins, all of which were found in the larval gut after a 5 hr pulse of labeled methionine in vivo. During most of the pregnancy cycle protein synthesis in the fat body was low compared to that of the milk gland and only small amounts of several low molecular weight proteins (less than or equal to 16 kDa) were released into the medium. But when a large third instar larva was present in the uterus, the fat body synthesized and secreted a 72 kDa and a 15–17 kDa complex of proteins.     

Developmental study of thermotolerance and the heat shock response inLucilia cuprina (Weidemann)

The ability to withstand thermal stress in a laboratory population of the blowflyLucilia cuprina (measured as per cent adult survival following varying periods of exposure to elevated temperature up to a maximum of 48°C) was in the order pupa > larva > adult. Pre-exposure to a mild heat shock (37°C) induced tolerance to temperatures which were otherwise lethal. An analysis of heat shock-induced protein synthesis during development at similar elevated temperatures presented patterns corresponding to the above observations on thermotolerance. The induced level of synthesis of major heat shock proteins (viz., 79, 69, 28, 20 and 19 kDa) were greater in larval tissues than in most of the adult tissues except gonads. The response varied between young (2 days) and old (30 days) adults in a tissue-specific manner. In general, heat shock protein 69 kDa was most abundant in all the tissues studied. Control as well as heat shocked Malpighian tubules of adults uniquely showed two major [³⁵S]methionine labelled bands corresponding to approximately 62 and 64 kDa. Immunoblots showed the 62 kDa protein to cross react with an antibody againstHelioihis HSP60. Although the synthesis of the 62 kDa polypeptide was prominent only in Malpighian tubules of adult blowflies, nearly equal levels of this HSP60 family polypeptide were present in all tissues (control as well heat shocked) except the larval salivary glands.     

Heat shock response in mulberry silkworm races with different thermotolerances

The thermal sensitivity and heat shock response of the different races of the mulberry silkwormBombyx mori have been analysed. The multivoltine race, strainsC. Nichi andPure Mysore showed better survival rates than the bivoltine race, strainNB4D2 exposed to 41°C and above. In general, the fifth instar larvae and the pupae exhibited maximum tolerance compared to the early larval instars, adult moths or the eggs. Exposure up to 39°C for 1 or 2 h was tolerated equally whereas temperatures above 43°C proved to be lethal for all. Treatment of larvae at 41°C for 1 h resulted in a variety of physiological alterations including increased heart beat rates, differential haemocyte counts, enlargement of granulocytes and the presence of additional protein species in the tissues and haemolymph. The appearance of a 93 kDa protein in the haemolymph, fat bodies and cuticle, following the heat shocking of larvaein vivo was a characteristic feature in all the three strains examined although the kinetics of their appearance itself was different. In haemolymph, the protein appeared immediately in response to heat shock inC. Nichi reaching the maximal levels in 2–4 h whereas its presence was noticeable only after 2–4 h recovery time inPure Mysore and bivoltine races. The fat body from bothC. Nichi andNB4D2 showed the presence of 93 kDa, 89 kDa and 70 kDa proteins on heat shock. The haemocytes, on the other hand, expressed only a 70 kDa protein consequent to heat shock. The 93 kDa protein in the haemolymph, therefore could have arisen from some other tissue, possibly the fat body. The 93 kDa protein was detected after heat shock in pupae and adult moths as well, although the presence of an additional (56 kDa) protein was also apparent in the adults. The presence of 46 kDa and 28 kDa bands in addition to the 93 kDa band in the cuticular proteins immediately following heat shock was clearly discernible. The 70 kDa band did not show much changes in the cuticular proteins on heat shock. In contrast to the changes in protein profiles seen in tissues and haemolymph following heat shockin vivo, the heat treatment of isolated fat body or haemolymphin vitro resulted in protein degradation.     

The organisation of larval feeding in bumblebees (Hymenoptera,Apidae) and its significance to caste differentiation

Summary. In pollen-storing bumblebees, the rate at which workers nourish larvae has been proposed to be the main factor influencing caste differentiation since workers feed prospective queens more frequently and longer than worker larvae during the last instars. In order to determine how the frequency of feedings is established small groups of Bombus terrestris larvae were either subjected to starvation or nourished regularly by workers. Experimentally starved larvae were fed significantly earlier and more often than control larvae. Behavioural observations provide evidence for the existence of a stimulus of larval origin that releases feeding behaviour in workers. Intentional inspections in the sense of a recognisable and functional behaviour intended to organise the feeding process were never observed. We argue that worker inspections are not required for the adequate maintenance of larvae and that a simple stimulus-response chain appears to be sufficient to regulate feeding behaviour at the individual and the colony level in bumblebees. Furthermore, hand-rearing experiments with female larvae indicate that queen rearing is not dependent on a high frequency of feedings in itself, nor is worker development induced by larval deprivation. This makes it unlikely that workers impose a caste-specific frequency of feedings on larvae in order to actively control or manipulate caste development. Since caste fate seems to be predetermined early in larval life, we propose that early caste-specific differences in development are reflected in the physiology of the larvae and the associated nutritional needs to which workers respond accordingly. Consequently, caste-specific differences in feeding frequencies are a result, but not the cause of differences in development.     

Haemolymph protein patterns in developing tobacco hornworm larvae

Quantitative changes in haemolymph proteins from each physiological phase of the last three larval instars of the tobacco hornworm, Manduca sexta, were studied by means of disk electrophoresis. Twelve anodical migrating protein bands were found, six of which occurred only sporadically. Total protein concentration increased from pharate third instar to late fifth instar larvae, then decreased slightly in the pharate pupal stages. Some individual bands showed cyclic patterns within each instar similar to the overall cyclic pattern of total protein, whereas other bands showed different patterns or no pattern.     

Stage and segment specificity of the secretory cell of the dermal glands of the tobacco hornworm, Manduca sexta

The pair of epidermally derived Verson's glands on each segment of the tobacco hornworm, Manduca sexta, secretes at ecdysis proteinaceous products which coat the epicuticle. These proteins are produced by a single secretory cell which displays both stage- and segment-specificity during development. Three major 12-kDa polypeptides are synthesized at the larval molts, while higher molecular weight (14-93 kDa) polypeptides are produced at the pupal molt. In the pupa, but not in the larva, there are three segment-specific protein patterns, each involving both qualitative and quantitative differences: (1) thoracic (T) segments 1 and 2; (2) T3 and abdominal (A) segment 1; (3) A2-A8. Larval-specific proteins were found to be synthesized in low amounts throughout the penultimate fourth instar, with enhanced synthesis occurring during the molt, coincident with the molting surge of ecdysteroids. Synthesis of the major pupal products commenced about the time of wandering, with enhanced synthesis occurring throughout prepupal development, coincident with the prepupal surge in ecdysteroids. The onset of synthesis of the major pupal products differed, both within and between segments. Culture of fifth instar Day 2 glands in vitro showed that this synthesis depended on 20-hydroxyecdysone. The differential regulation within and between segments observed in vivo was also seen in vitro.