A possible role of ecdysteroids in pre-ecdysial tanning in larvae of Sarcophaga bullata (Diptera: Sarcophagidae)

The levels of ecdysteroids in Sarcophaga bullata were determined by radioimmunoassay (RIA) from the time of larviposition (0 hr) to after the 2nd ecdysis and from late larval to pupal development. Two distinct peaks of ecdysteroid activity were recorded mid-way through the first and second stadia (14 and 34 hr) and two smaller peaks occurred a few hours prior to each ecdysis. A large release of ecdysteroids occurred from 8 hr before and up to 18 hr after formation of the white prepupa. This peak initiated the formation of the prepupa, the tanning of the puparium, larval/pupal apolysis and secretion of the pupal cuticle.Assays for the cuticle tanning hormone, bursicon, in pre-ecdysial larvae were not positive and a possible role for ecdysone in pre-ecdysial tanning of larval cuticular structures is proposed.     

Patterns of puffing activity in the salivary gland chromosomes of Drosophila

The patterns of puffing activity have been studied during the late larval and prepupal stages of Drosophila melanogaster. On the major salivary gland autosomes (chromosomes 2 and 3) 108 loci form puffs at some time during these developmental stages. The timing and pattern of activity of 83 of these puffs is found to be strictly dependent upon the age of the animals. Two major peaks in puffing activity occur. The first of these is at the time of puparium formation and the second in 8 hr. old prepupae. Both of these puffing peaks precede a moult by 4 hrs. 30 puffs are active before or at the time of both of these two moults. However, the sequence of appearance and regression of many of this group of puffs is different at the prepupal moult than at the pupal moult. 12 puffs occur only before or at the time of the prepupal moult and 13 puffs only before or at the time of the pupal moult. The functional significance of these periods of puffing activity is discussed and it is concluded that one function of this genetic activity in the salivary glands of metamorphosing Drosophila is the production of substances to be utilised during the histogenesis of the adult tissues.     

Seasonal effects on cuticular permeability and epicuticular lipid composition inCentruroides sculpturatus ewing 1928 (Scorpiones: Buthidae)

Summary Third larval instar hemolymph of the fruitflyDrosophila hydei did not metabolize juvenile hormone (JH) at all developmental stages. In contrast, prepupal and pupal body fluid showed JH-esterase activity with a maximum at 4 h after puparium formation. In body wall and fat body of all developmental stages investigated, JH-metabolic activity was found. In both tissues JH catabolism was most active in the 120,000g supernatant and pellet. The 800g and 15,000g pellet showed a lower activity. In all subcellular fractions the JH-acid was identified as the predominant metabolite. There is evidence that JH-specific esterases are responsible for ester cleavage in the 120,000g supernatant. During mid and late third larval instar development in both body wall and fat body JH-esterase activity remains relatively constant.     

Water loss and respiration of Lucilia cuprina during development within the puparium

The rate of loss of water and the rate of uptake of oxygen were measured continuously throughout the development of Lucilia cuprina within the puparium. Changes in these parameters were correlated with changes observed in morphology of cuticles and respiratory structures during development.In development at 26°C, there is, at 20–22 hr after puparium formation a major loss of water by mechanical expulsion of moulting fluid chiefly through the posterior larval spiracles after the severing of the posterior larval tracheae. This loss of water is essential to survival and is followed by an extremely low rate of water loss attributed to slow diffusion of water through the resulting air gap between the pupal cuticle and the puparium. There is an increase in oxygen consumption during the pupal movements associated with the casting of the larval tracheae followed by a sharp reduction in oxygen consumption until the pupal horns are everted a short time later. This combination of physiological events enables development to proceed over a wide range of conditions in the puparial environment.     

Effects of temperature and humidity on hydration and respiration in four strains of Drosophila

The four strains of Drosophila melanica studied were found to be most sensitive to low humidities during the first 19 hr after formation of the puparium. The mean fresh weights for the four strains were consistently different at all ages after puparium formation and at all conditions of temperature and humidity. The Forest Park strain, which showed greatest mortality at low humidity, had least body water per unit dry weight in the early pupal stage.A typical U-shaped oxygen consumption rate curve was found during the puparial-pharate adult period at all conditions of temperature and humidity. Differences among the strains were manifested at an earlier age under the more extreme environmental conditions.     

Cytochemical study of acid phosphatase activity in the functional and degenerating spinning gland cells of Pericallia ricinii (lepidoptera).

A study of the presence, distribution and activity of acid phosphatase has been carried out in the cells of the spinning gland of moth pest Pericallia ricinii in sequential larval, prepupal and pupal stages. Acid phosphomonoesterase activity at first appears in the apical and basal regions of the secretory cells of second instar larva and later in the following stages is localised in the middle region of the cells. The highest activity was found while using an incubation time from 25 to 30 minutes. Prolonged incubation resulted in artifacts. Based on the acid phosphatase activity two cell types (A and B) in the spinning gland cell layer could be distinguished. The cytochemical differences in histologically similar cells of the spinning gland of the Indian moth pest were reported for the first time. In pupal period progressive deformation could be observed in the gland cells, which is accompanied by high acid phosphatase activity, which is in contradistinction to cells with small deformations and low enzyme activity. The process of degeneration in former cells is also quicker than in the latter ones.     

Developmental studies in Drosophila

A study of the puffing patterns of the salivary gland chromosomes of D. pseudoobscura was carried out through several larval, prepupal, and pupal stages of development. A total of 176 puffs were found, 111 of which changed during the stages studied. As described in previous investigations with other Drosophila species there are two major peaks of puffing activity. These two peaks occur during puparium formation and pupation. Additionally, a minor activity-peak occurs during mid-prepupal life. Attempts have been made to establish correlations between the puffing data and those obtained from electrophoretic and ultrastructural studies.Supported in part by grants GM-16736-03 and FR-05426-09 from the U.S. Department of Health, Education, and Welfare. Ann Jacob Stocker was a holder of a University of Texas predoctoral fellowship.     

Changes in dry weight, protein, and nucleic acid content during diapause and normal development of the blowfly, Lucilia sericata

Dry weight (D.W.), protein, RNA, and DNA have been determined in the blowfly, Lucilia sericata, throughout all stages of normal development in long and short photoperiod regimes and during larval diapause. During normal development protein/D.W. levels fluctuate markedly during the larval and puparial stages, increased levels being correlated with the synthesis of new cuticle, etc. prior to ecdysis and the histogenesis of adult tissues prior to emergence. Protein levels remain relatively high and constant during adult life to senescence. RNA/D.W. levels are highest in first instar larvae but decline rapidly during larval development until just before puparium formation. Sharp increases are found prior to pupation and then again prior to adult emergence. In the adult stage, the levels decline steadily throughout the life span. DNA/D.W. levels are very low in the egg but rach their highest levels in early first instar larvae. They then decline during larval development, with small increases being found prior to puparium formation and adult emergence. Adult levels remain relatively constant throughout the life span. The ratio has extremely high values in the egg, indicating the high degree of synthetic activity that takes place during embryogenesis. There is a steady decline in values during adult life to senescence in both sexes, suggesting that physiological ageing in L. sericata is accompanied by a decrease in protein synthesis potential.During larval diapause all parameters, with the exception of the ratio, are maintained at low and constant levels, reflecting the fact that diapause is a period when synthetic and mitotic activity are minimal. The great variations in levels, however, indicate that individuals within a group of larvae can terminate diapause spontaneously at 24°C and return to the normal processes of morphogenesis.     

Effect of tomato fruit cultivar and ripening stage on Bactrocera tryoni (Froggatt) egg and larval survival

In studies of frugivorous tephritids, determining when offspring (i.e. egg and three larval instars) mortality occurs within the fruit can greatly improve the mechanistic understanding of the fly/host interaction. Previous research has demonstrated that the Queensland fruit fly, Bactrocera tryoni, has differential offspring performance in two tomato cultivars Cherry and Roma, but when juvenile mortality was occurring was not determined. We examined B. tryoni egg and larval survival in three different ripening stages (immature-green (IG), colour-break (CB) and fully-ripe (FR)) of Cherry and Roma tomato cultivars through destructive fruit sampling at 72 and 120 hr for eggs, and 48 (1st instar), 96 (2nd instar) and 120 hr (3rd instar) after fruit inoculation with neonates for larvae. Cultivar and ripening stage had no significant effect on egg survival, nor larval survival at 48 hr: the overall percentage of egg survival was at least 80% across all treatments, while 1st-instar larval was less than 52% across all treatments. In immature-green tomatoes of both varieties, nearly all mortality occurred during the first and second instars, but at 96 and 120 hr, there were significant interaction effects between cultivar and ripening stage on larval survival. In both colour-break Cherry and Roma tomatoes, there was significant larval mortality between 96 and 120 hr. However, in fully-ripe Cherry, no further significant larval mortality happened after 48 hr, while in fully-ripe Roma significant larval mortality occurred between the first and second larval instars but not thereafter. The difference in timing of larval mortality with ripening stage provides indirect evidence of active fruit defence which is strongest in immature-green fruit, less in colour-break fruit and absent in fully-ripe fruit.     

Morphogenesis and cuticular markers during the larval-pupal transformation of the medfly Ceratitis capitata

Changes in morphology during early metamorphosis of the medfly, Ceratitis capitata (Wied.) (Tephritidae) were correlated with biochemical differentiation events. Protein profiles were studied both in the 3rd instar larval cuticle further transformed into puparium and the newly synthesized pupal cuticle. Beta-alanine incorporation into the puparium (0–20 h) correlates with concomitant pigmentation (completed by 16 h) and sclerotization phenomena. This early tannification program seems to be followed by deposition of a layer of substances, probably ecdysial fluid remnants, into the puparium. Their deposition ends approximately at +46 h. Simultaneously, pupal cuticle material starts to be deposited. Synthesis and deposition of the main pupal cuticle protein was detected 48 h after pupariation. At that time, eversion of the pupal head occurs. The definitive profile of pupal cuticle proteins was attained at around +72 h together with the establishment of adult body proportions.     

Ecdysteroid synthesis by the ring gland of Drosophila melanogaster during late-larval,prepupal and pupal development

Radioimmunoassay has been used to determine the characteristics of ecdysteroid synthesis by ring glands and brain-ring gland preparations from late 3rd-instar larvae of Drosophila melanogaster cultured in vitro. The rate of synthesis and secretion is linear for at least 4 hr in culture. Using a 4-hr culture period, variation in the rate of ecdysteroid synthesis by brain-ring gland preparations during larval, prepupal and pupal development has been examined. The rate of synthesis and secretion is highest in late 3rd-instar larvae and decreases after puparium formation. During pupal development, at a time when the endogenous ecdysteroid titre is again increasing, the rate of ecdysteroid synthesis by brain-ring gland preparations remains low and is only 10% of that prior to puparium formation. It is, therefore, likely that the ring gland is not a major source of ecdysteroids during this period.     

Proliferation pattern of postembryonic neuroblasts in the brain of Drosophila melanogaster.

The spatio-temporal proliferation pattern of postembryonic neuroblasts in the central brain region of the supra-esophageal ganglion of Drosophila melanogaster was studied by labeling DNA replicating cells with 5-bromo-2'-deoxyuridine (BrdU). There are five proliferating neuroblasts per hemisphere in larvae just after hatching: one in the ventro-lateral, and the other four in the postero-dorsal region of the brain. Dividing neuroblasts increase during the late first-late second instar larval stages, reaching a plateau of about 85 neuroblasts per hemisphere. Most neuroblasts cease dividing 20-30 hr after puparium formation (APF), while only four in the postero-dorsal region continue making progenies until 85-90 hr APF. The four distinct neuroblasts proliferating in the early larval and late pupal stages are identical; they lie in the cortex above the calyces of the mushroom bodies (corpora pedunculata), proliferating over a period twice as long as that for the other neuroblasts. Their daughter neurons project into the mushroom body neuropile, and hence are likely to be the Kenyon cells. The cell-cycle period of the four neuroblasts (named mushroom body neuroblasts: MBNbs) is rather constant (1.1-1.5 hr) during the mid larval-early pupal stages and is longer before and after that. The total number of the MBNb progenies made throughout the embryonic and postembryonic development was estimated to be 800-1200 per hemisphere.     

Reversal of pupal diapause in Sarcophaga argyrostoma by temperature shifts after puparium formation

Puparia from Sarcophaga argyrostoma larvae reared under short days were collected daily within 24 hr of their formation and divided into two groups: one which remained at the larval rearing temperature, and one which was transferred to a different temperature. Such temperature shifts after puparium formation can modify the subsequent incidence of pupal diapause. Temperature step-ups decrease the percentage of diapause; temperature step-downs increase it. The degree of this effect increases with the size of the temperature step. The effectiveness of a temperature step-up declines with increasing time after puparium formation.The percentage of diapause finally achieved in any group is a function of both the number of inductive (short-day) photoperiods experienced during larval life and the magnitude and direction of the subsequent temperature step. Temperature step-ups can permit the expression of photoperiodic information which would otherwise be masked. A model is presented to account for these findings.     

Metamorphosis-associated proteolysis in Ceratitis capitata

The induction of several biochemical indicators of larval tissue histolysis in the Medfly, Ceratitis capitata (Diptera: Tephritidae) (Wiedemann) was studied. Using synchronized third instar larvae, we have determined the time of occurence of gut evacuation (12 h before puparium formation, bpf), disappearance of digestive enzymes (10 h bpf), and jumping from the food (8 h bpf). We can also correlate these events temporally with other early landmarks of metamorphosis.The decrease in protein content between 0 hours and 144 hours after puparium formation (0–144 h apf) corresponds to a sharp increase of total acid proteolytic activity measured in vitro from 0 to 44 h apf. This activity appears to be lysosomal, judging by the activation of other lysosomal markers, such as acid phosphatase and -glycosidases. The maximum proteolytic activity occurs during the pre-pupa to pupa transition, i.e. during morphogenesis from the cryptocephalic to the phanerocephalic pupa. The results of endopeptidase inhibitor assays indicate that in dipterans, members of the aspartic and cystein proteinase families are responsible for the degradation of larval tissues.     

The hormonal control of salivary gland secretion in Drosophila melanogaster: Studies in vitro

The larval salivary gland of Drosophila melanogaster synthesises a complex secretion, known as ‘glue’. which is secreted at puparium formation and then cements the puparium to its substrate. This secretion is made during the third larval instar and is stored in the gland cells as large granules. A few hours before puparium formation it is secreted into the gland's lumen by exocytosis. This process is induced by ecdysone and can be studied in vitro. Secretion is initiated about 3.5 hr after exposure of glands to ecdysone and is complete by 8 hr. The effects of varying the ecdysone concentration, of inhibitors of RNA or protein synthesis, and of withdrawing the hormone at various times after initial exposure on the process of secretion have been studied. We conclude that some event(s) occurring during the first 3 hr exposure to ecdysone is necessary to initiate secretion of the glue into the gland lumen. The possible relationship between this event(s) and the ecdysone induced changes in gene activity (puffs) which occur in the salivary glands at the same time is discussed.     

Seasonal phenology and stage-specific parasitism of the apple ermine moth, Yponomeuta malinellus Zeller, in Korea

The apple ermine moth, Yponomeuta malinellus Zeller (Lepidoptera: Yponomeutidae), is a tent caterpillar that feeds on Malus spp. in Korea. Populations of the moth in native areas appeared to be regulated by the assemblage of parasitoids. Phenological associations between host stages and parasitoids, susceptible stage(s) of the host for each parasitoid, and stage‐specific parasitism were studied. The egg larval parasitoid Ageniaspis fuscicollis (Dalman) had highest parasitism of first instar larvae (24%), with 14% parasitism of other larval stages. Dolichogenidea delecta (Haliday) was recovered from all larval instars with the highest parasitism rate of second instar larvae (20.1%), followed by 19.9% parasitism of mid‐larval hosts. Herpestomus brunicornis Gravenhorst was reared from second instar larvae through to pupal collection, and had the highest parasitism rate (29.9%) at the pupal stage. The larval pupal parasitoid Zenillia dolosa (Meigen) was recovered from mid‐larval to pupal stages with the highest parasitism rate (5.5%) occurring in third to fourth instar larvae. The host stages for developing A. fuscicollis completely overlap with those of D. delecta, and with those of H. brunicornis to some degree. A statistically significant negative correlation exists between A. fuscicollis and these dominant parasitoids, indicating competitive interaction within the host.     

Morphogenesis and cellular proliferation pattern in the developing antennal lobe of Drosophila melanogaster

The adult antennal lobe of Drosophila melanogaster emerges from a precursor, the larval antennal lobe. Pulse and pulse-chase labelling of dividing cells in larvae and pupae with bromodeoxyuridine confirmed previous data that some of the interneurons of the adult antennal lobe derive from a lateral neuroblast which starts to divide early in the first larval instar. However, the majority of these interneurons originate from neuroblasts that initiate mitosis at later stages, with a peak of about 10–12 pairs of dividing neuroblasts in the late third larval instar. No clustering of adult antennal lobe neurons according to their birthdates was observed. In contrast to neurons, terminal divisions of glia in the antennal lobe reach their maximum only 12 h after puparium formation.     

Monarda fistulosa hydrolate as antimicrobial agent in artificial media for the in vitro rearing of the tachinid parasitoid Exorista larvarum

Exorista larvarum (L.) (Diptera: Tachinidae), a larval parasitoid of Lepidoptera, can be reared from egg to fecund adult on artificial media composed of crude components. The standard in vitro culture is performed in 24‐well plastic rearing plates. Exorista larvarum eggs, removed from superparasitized larvae of Galleria mellonella (L.) (Lepidoptera: Pyralidae), are individually placed in the wells, each containing a cotton swab soaked in liquid medium. The plates are then sealed until parasitoid puparium formation. To avoid contamination by microorganisms, the artificial medium is routinely supplemented with 0.01% solution of gentamicin. Experiments were carried out to assess whether this broad‐spectrum antibiotic may be replaced with hydrolate of Monarda fistulosa L. (Lamiaceae), which was selected due to its high in vitro activity against pathogenic microorganisms for humans and plants. The hydrolate was either supplemented to the artificial medium (0.5% wt/wt) (first experiment) or placed in an empty well (200 μl) of the rearing plate, to be supplied as saturated air due to evaporation (second experiment). In both experiments, a standard medium with gentamicin and an antimicrobial‐free medium were maintained as positive and negative controls, respectively. In the first experiment, in the hydrolate‐supplemented medium fewer E. larvarum completed egg‐to‐adult development than in the standard medium, but significantly more parasitoids developed from egg to adult compared to the antimicrobial‐free medium. No significant difference was found between the numbers of eggs laid by the females obtained from the standard medium vs. those from the hydrolate‐supplemented medium. In the second experiment, the hydrolate‐saturated air significantly decreased E. larvarum egg hatching, puparium formation, and female fecundity compared to the standard medium. In perspective, M. fistulosa hydrolate supplemented to the artificial media for E. larvarum may be considered as a promising candidate to replace the gentamicin solution, as suggested also by the microbiological analyses of the media, performed at various growth stages of the parasitoid in a separate trial. Conversely, the hydrolate‐saturated air treatment was deemed unsuitable.     

Comparative characteristics of the puffing pattern and the endocrine system in an oregon laboratory stock and in l(2)gl Drosophila melanogaster mutants differing in the time of their death

This study shows that homozygotes for different alleles of the lethal mutant, l(2)gl, differing in the time of death also vary in the state of their endocrine system and the puffing patterns of their salivary gland chromosomes. Homozygotes which die at the larval stage have underdeveloped prothoracic glands and normal corpora allata (CA); in those dying at the prepupal stage both the prothoracic glands and the CA are equally underdeveloped. — All the early third instar larval puffs (96–110 h., PS 1–2) develop in homozygotes; however, the reduction of some early larval puffs, normally occurring before pupariation or at puparium formation, is delayed. Some puffs are more developed than normal. — The differences in puffing patterns chiefly concerned puffs which normally appear 4–5 h before puparium formation and at puparium formation. In homozygotes lethal as larvae some of the puffs normally active at this time did not develop. However, along with some of the late larval puffs, there appeared many puffs characteristic of prepupae. — In homozygotes lethal as prepupae only the time and sequence of puff appearance was altered. Many late larval puffs were active in prepupae rather than in larvae, whereas some of the puffs, normally appearing in prepupae, were active in the larval stage.Accordingly, we propose to distinguish two groups of puff loci. 1) Hormone dependent puffs: These do not develop in larval lethals and are active only after puparium formation in pupariated lethals. 2) Autonomous puffs: Their appearance depends more on the time of development, than on hormonal background. It is suggested that the induction of hormone dependent puffs and of puparium formation is possible at low ecdysone levels, provided that the juvenile hormone level is also low.     

Biochemical and ontogenetic aspects of glycoprotein synthesis in Drosophila virilis salivary glands

After SDS-polyacrylamide gel electrophoresis two glycosylated glue proteins are found in the salivary glands of Drosophila virilis late third instar larvae. Synthesis of larval glue protein 1 occurs in three successive steps: at first a precursor protein with a molecular weight of about 138,000 daltons is formed. This is modified by two subsequent steps of glycosylation, the first one involving hexosamine, the second one hexoses. Studies with tunicamycin and β-hydroxynorvaline suggest that glycosylation occurs at threonine residues. Larval glue protein 2 has a molecular weight of approximately 15,000 daltons and is weakly glycosylated. The synthesis of glue proteins is stage specific. It starts at about 120 hr after oviposition and attains its maximal rate about 20 hr later. At this time the larvae leave the food. Between ecdysone release and puparium formation (146–151 hr) larval glue protein synthesis is terminated. Throughout the prepupal stage a different set of glycoproteins is synthesized. Thus, the larval-prepupal transition is accompanied by the reprogramming of glycoprotein synthesis in salivary glands. The secretion products formed during the two developmental stages seem to possess different biological functions.