Cuticular cycle and molting hormone levels during the metamorphosis of Tenebrio molitor (Insecta Coleoptera)

The cuticular cycle of Tenebrio molitor (apolysis, synthesis of outer and inner epicuticle, fibrous cuticle deposition) was studied during the last larval and pupal stages by electron microscopy. Concurrently, molting hormone (MH) titers in the hemolymph were determined by a radioimmunoassay method. It appears, both in larvae and in pupae, that the MH peak coincides with the initiation of pre-ecdysial cuticle deposition (i.e., outer epicuticle synthesis). Thus MH is involved in the induction of cuticular synthesis; however, its role in inducing larval-pupal apolysis is questionable. We note that this peculiar apolysis occurs long before MH release.     

Changes in the morphogenetic response of Tenebrio molitor pupae to juvenile hormone in relation to age

Morphogenetic effect of juvenile hormone (JH) and its analogues, dodecyl methyl ether, ethyl trimethyl dodecadienoate and methylenedioxyphenoxy-6-epoxy-3-ethyl-7-methyl-2-nonene, on carefully timed Tenebrio pupae was determined. These results show that the response of pupal epidermal cells to JH varied with age during the first 48 hr after larval-pupal ecdysis. The pupae showed low morphogenetic response soon after pupal ecdysis but their response increased gradually until 18 hr. The response to JH decreased in pupae older than about 32 hr; and 48 hr old pupae were unresponsive to low doses of JH employed in this study. Age-related differences in the pattern of response of the individual body regions to JH were also observed.The synergistic effect of 1 μg of ecdysterone with these JH compounds was also tested in relation to the age of Tenebrio pupa. The results show that the synergistic effect of ecdysterone was generally limited to >18 hr old pupae. This suggests that the physiological basis of the synergistic effect of ecdysterone may be the latter's ability to synchronize epidermal cells.The significance of these observations in the analysis of time of action of juvenile hormone is discussed.     

Composition,synthetic and cytolytic activities of Galleria mellonella silk glands during the last-larval instar under the action of juvenile hormone

Within the first 48 hr of the last-larval instar of Galleria mellonella the silk glands grow but silk production is restrained. This ‘preparatory phase’ of the glands is probably maintained by juvenile hormone. Silk production and accumulation are stimulated in the ‘accumulation phase’ between 60 and 132 hr by unknown factors in the absence of juvenile hormone. The rate of RNA synthesis culminates at 84 hr but the RNA content increases until the end of cocoon spinning at 144 hr. In the following ‘regression phase’ (144–160 hr), when the glands exhibit high activities of acid and alkaline DN-ases and of acid phosphatase, the RNA and protein contents rapidly decrease, but that of DNA remains high. This phase is typical of moulting insects, is independent of juvenile hormone, and seems to be caused either by an increase in ecdysteroids or by lack of nutrients. The following ‘degeneration phase’ occurs when the surge of ecdysteroids terminates the larval-pupal transformation. Disintegration of silk glands by autolysis and phagocytosis is completed after pupal ecdysis (180 hr). Treatment of larvae with a juvenoid (ZR 512) at 48 or 132 hr in the last instar dramatically alter the composition, synthetic and cytolytic activities of silk glands. At the next ecdysis the glands attain a state very similar to that of the preparatory phase. They are capable of intensive silk production and completion of developmental cycle when the supernumerary larvae prepare for pupation. The results indicate that juvenile hormone can reverse the development of the silk glands.     

The onset of metamorphosis in Tenebrio molitor L.: Effects of a juvenile hormone analogue and of 20-hydroxyecdysone

Sensitivity to juvenile hormone and to 20-hydroxyecdysone has been investigated during the last-larval stages of Tenebrio molitor. Topical applications of a juvenile hormone analogue (K-421d) showed that the sensitive period, occurring before apolysis, is relatively short (less than 4 days in a 3-week instar) and divided into two phases. Treatment during the first and longest phase induced a delay in development and then an increase in larval moult percentage. Treatment during the second phase induced several abnormal moults (prothetelic larvae and larval-pupal intermediates).Injections of massive doses of 20-hydroxyecdysone (10 μg per animal) also evidenced a period of disturbance of the morphogenetic programme, beginning before pupal apolysis but continuing several days after.Comparison of the sensitive periods to both hormones suggests that a very important and rapid step of the larval-pupal programme change is controlled hormonally just before pupal apolysis.     

Time studies of ecdysone-action on in vitro apolysis of Chilo suppressalis integument

The relationship between induction of in vitro apolysis and the duration of hormone treatment, and the effects of metabolic inhibitors on the ecdysone-induced apolysis were investigated in the cultured integument taken from the rice stem borer larva, Chilo suppressalis. When fragments of integument were subjected to 0.3 μg/ml β-ecdysone for more than 5 hr and then transferred to hormone free medium, they were induced to apolyse one day after treatment. If the fragments of integument were treated with hormone for 1 to 4 hr at first and then treated with hormone for 2 to 5 hr again after a 5 day interval in hormone free medium, almost all the fragments were induced to apolyse one day after treatment. This result suggests that the action of β-ecdysone on the cultured integument is accumulative. If the fragments of integument were cultured in the medium containing actinomycin-D and then transferred to medium containing β-ecdysone, a strong inhibitory effect on the apolysis of the integument was observed. Similarly, an inhibitory effect appeared when fragments of integument were treated first with hormone and then with puromycin. These results show that the m-RNA synthesis necessary for apolysis was completed within 6 hr after hormone treatment. However, the protein synthesis required for apolysis was not. The relationship of the results obtained from these in vitro experiments to the mode of action of ecdysone is discussed.     

Effects of NeemAzal on marker enzymes and hemocyte phagocytic activity of larvae and pupae of the vector mosquito Aedes aegypti

Many of the neem based botanical biocides are currently studied to a greater extent because of the possibility of their use in eco-friendly control of pests and vectors. However, no report was available to assess the impact of neem based formulation, NeemAzal on marker enzymes and hemocyte mediated cellular immune responses of important vector mosquito A. aegypti. The NeemAzal found to exert larvicidal and pupicidal activities against A. aegypti developmental stages. The pupae appear to be more susceptible to the treatment. Further, a significant increase in the level of total protein (31%), α-carboxylesterase (121%), β-carboxylesterase (46%), acid phosphatase (62%) and alkaline phosphatase (37%) was observed in larvae upon exposure to NeemAzal. Moreover, treated pupae showed increased level of acetylcholinesterase (116%) and acid phosphatase (43%) while α-carboxylesterase (34%), β-carboxylesterase (12%) levels were simultaneously decreased, and no significant changes in alkaline phosphatase were noticed. Qualitative analysis also revealed that the exposure considerably modulated the larval β-carboxylesterase isoenzyme profile whereas little changes were noticed on phosphatases. On the other hand hemocyte viability of larvae (18%) and pupae (16%) as well as phagocytic ability of larval (48%) and pupal hemocytes (44%) against yeast target was significantly reduced upon NeemAzal exposure. We demonstrated for the first time that the NeemAzal differentially affected the marker enzymes and created immuno-suppressive state by reducing the phagocytic ability of hemocytes of larvae and pupae of A. aegypti.     

Glycogen phosphorylase activity in pharate adults of the stable fly and the effects of a juvenile hormone analogue

Glycogen phosphorylase was assayed in homogenates of pharate adults of the stable fly, Stomoxys calcitrans, by the release of inorganic phosphorous (Pi) from glucose-1-phosphate (G-1-P) in the presence of glycogen. Activity was determined as active and total phosphorylase present by the absence or presence of adenosine-5-monophosphate (AMP) in homogenates. Homogenates of the pharate adults (that were treated topically immediately after larval-pupal apolysis with a synthetic insect juvenile hormone analogue, (E)-4-[(6,7-epoxy-3-ethyl-7-methyl-2-nonenyl)oxy]-1,2-(methylenedioxy)benzene) displayed no inhibition or enhancement of phosphorylase activity when compared to untreated pharate adults.     

Control of juvenile hormone esterase activity in Galleria mellonella larvae

The juvenile hormone esterase (JHE) activity in Galleria mellonella larvae was measured after exposure to different experimental conditions that affect larval-pupal transformation. The data show that stimulation of production of JHE is closely coupled with the developmental signals that intiate larval-pupal metamorphosis. Injury, which delays pupation, delays the appearance of JHE activity if the larvae are injured within 48 hr after the last larval moult. Chilling of day-0 larvae induces a supernumerary larval moult and inhibits the appearance of JHE. However, JHE activity increases in chilled larvae when their commitment for an extra larval moult is reversed by starvation. Starvation is effective in reversing the commitment for an extra larval moult if commenced within 48 hr after chilling, thereby suggesting a critical period for that commitment. These data suggest that the stimulus for JHE synthesis and/or release occurs approximately within 48 hr after the last larval ecdysis. A series of studies involving implantation of brain, suboesophageal ganglion and fat body into chilled, as well as chilled and ligated larvae suggest that a factor from the brain is involved in stimulation or production of JHE in Galleria larvae.JH, which suppresses JHE activity in day-3, -5 and early day-6 Galleria larvae, stimulates the production of JHE in late day-6 larvae, suggesting that reprogramming in larval fat body may occur on day 6 of the last larval stadium.     

Study of catheptic and acid phosphatase activities during development and metamorphosis ofDrosophila melanogaster

Summary Developmental stages ofDrosophila melanogaster cultured at 22 ± 1° C were collected and tested for catheptic activity and acid phosphatase activity.It was found that catheptic activity was absent in the egg as well as in the first and second larval instars. The activity first appears in the third instar larva and reaches its peak 24 to 48 hr after puparium formation. It then decreases, at first gradually and at pupal stage 9 (120 to 144 hr after puparium formation) abruptly, reaching zero level just before the emergence of the imago.The pattern of acid phosphatase activity in different developmental stages was found to be broadly similar to that of catheptic activity in the larval and pupal stages. However, the acid phosphatase activity was found to be exceptionally high in the egg in contrast to the catheptic activity.The authors are grateful to Prof. Dr. R. Weber, Zoological Institute of the University of Bern, for constructive criticism of this paper.     

The origin of plasma alkaline phosphatase activity in mice and rats

Swiss albino mice displayed the highest activity of alkaline phosphatase at 4-6 weeks with a precipitous decline by 18 weeks of age to a value seen in the mature animal. Circulating activity of alkaline phosphatase was significantly higher in the rat than the mouse in the fed state. With fasting, enzyme activity declined in the rat yet increased in the mouse. The net result was significantly higher plasma alkaline phosphatase activity in the mouse than the rat after the 48 hr fast. L-Phenylalanine inhibition of plasma alkaline phosphatase was greater in plasma from the rat than the mouse in the fed state. Yet in the fed condition, L-homoarginine and L-p-bromotetramisole inhibited alkaline phosphatase activity in plasma from mice to a greater extent than in rats. Heat inactivation as well as urea denaturation of alkaline phosphatase was significantly faster with plasma of the mouse than the rat in the fed state. Thus, it appears that the alkaline phosphatase isoenzyme of skeletal origin contributes a greater proportion of the circulating activity in the fed Swiss albino mouse than occurs in the Sprague-Dawley rat in which the intestinal isoenzyme plays a greater role in the fed condition.     

Étude du contrôle neuro-endocrine du fonctionnement du corpus allatum chez les larves du quatrième stade de Rhodnius prolixus

This paper concerns the effects of electrocauterization of the pars intercerebralis (P.I.) in the penultimate instar (fourth) of Rhodnius prolixus performed at various intervals following a blood meal. The following ecdysone-induced apolysis is strongly adultoid in the larvae whose P.I. was destroyed within 20 hr after a meal. The same operation at 24 hr, 48 hr, and 7 day intervals accompanied with ecdysone injections does not produce such effects since the resulting cuticle synthesis is normal (larval). These experiments demonstrate that the protocerebrum has an allatotropic action during the 20 hr following the blood meal. The stimulation of the corpus allatum is effected by a humoral pathway as shown by experiments of disconnecting C.A., cauterization of the P.I., and the application of juvenile hormone. Allatotropic and prothoractotropic effects of the brain cannot be induced by implanting entire brains or only P.I. The results of parabiosis between a fed allatectomized insect and a parsectomized one just after a meal do not allow us to evaluate the allatotropic effects as the apolysis does not occur in such pairs.These results have permitted us to discuss the secretory activity of the brain and its interactions with other endocrine glands.     

Alkaline and acid phosphatase in the intestines of rats infected with a virulent strain of Entamoeba histolytica and treated with emetine and Flagyl

The distribution patterns of alkaline phosphatase (EC 3.1.3.1) and acid phosphatase (EC 3.1.3.2) in the intestine of rats inoculated intracaecally with a virulent strain of Entamoeba histolytica and treated with emetine hydrochloride and metronidazole (Flagyl) were studied. The caecum and the large intestine showed a highly significant increase in alkaline phosphatase activity after amoebic inoculation, and the enhanced activity was lowered by emetine and Flagyl treatment. There was no significant increase in acid phosphatase activity either in the caecum and the large intestine or in the small intestine (ileocaecal end). Intracaecal inoculation of bacterial associates alone from E. histolytica cultures did not produce any significant change in the level of these enzymes in the intestine.     

Ecdysteroid titres during postembryonic development of Sarcophaga bullata (Sarcophagidae: Diptera)

The level of ecdysteroids in Sarcophaga bullata was determined by radioimmunoassay (RIA) from the time of larviposition (0 hr) until adult eclosion. Five distinct peaks of ecdysteroid activity were recorded. The first two, which occurred midway through the duration of the stadia (14 and 30 hr, respectively), resulted in larval/larval moults (24 and 44 hr). The third peak of ecdysteroid activity commenced at 131 hr and was associated with formation of the white prepuparium. The fourth peak was sustained over a long time period (from 79 hr post pupariation to 120 hr) and resulted in pupal/adult apolysis and the definition of the adult form. The last elevation of the ecdysteroid titre at approx. 160 hr post pupariation) was associated with the synthesis and secretion of adult cuticle.     

Quantification of acetylcholinesterase,acid and alkaline phosphatases in the central nervous system of Schizodactylus monstrosus, D. (schizodactylidae:orthoptera). Effect of topical application of the pyrethrum

The newly emerged adult male and female Schizodactylus monstrosus D. were treated with an insecticide, pyrethrum or with petroleum ether in the case of controls. At selected intervals of 6 h, 12 h and 18 h after treatment, the brain and ventral nerve cord and ganglia were homogenized to estimate the activity of acetyl-cholinesterase, and acid and alkaline phosphatase. The resultant activities of these three enzymes showed that: acetylcholinesterase decreased rapidly, and acid and alkaline phosphatase increased significantly after pyrethrum treatment in both brain and ventral nerve cord with ganglia compared with the controls. The activities of alkaline phosphatase showed a marked fluctuation at different post-treatment periods in both the tissues. The results have been discussed in relation to the impact caused by the treated insecticide and its metabolism.     

Effect of alteration of endocrine mechanism on the development of nuclear polyhedrosis in the isolated pupal abdomens of the silkworm, Bombyx mori

Cyasterone injection into isolated pupal abdomens of Bombyx mori in an arrested state of development results in a hastened death of the abdomens from NPV. The time taken by the isolated abdomens to die was shortened even when cyasterone injection was delayed until 72 hr after NPV inoculation. When abdomens of pupae inoculated with NPV were isolated at different times after larval-pupal ecdysis, those prepared after the critical period of molting hormone secretion died sooner than those prepared before the critical period. These results imply that activated metabolism plays an active role in NPV development, possibly due to the increased rate of synthesis of viral constituents.     

Tyrosine and phenylalanine concentrations in haemolymph and tissues of the American cockroach, Periplaneta americana, during metamorphosis

Phenylalanine and tyrosine concentrations were measured in the haemolymph, fat body, and abdominal integument of the American cockroach, Periplaneta americana, during the pre- and post-ecdysial periods of cuticle formation and sclerotization.Gas-liquid chromatography of trimethylsilyl derivatives of phenylalanine, tyrosine, and their metabolites provided a very sensitive and rapid method for determining those amino acids in small haemolymph and tissue samples.Haemolymph tyrosine increased in two stages: initially near apolysis and 16 to 25 hr pre-ecdysis, reaching its highest concentration at ecdysis (3·5 μg tyrosine/mg haemolymph). During that time, total haemolymph tyrosine increased by approximately 700 μg/insect. Fat body and abdominal integument began to accumulate tyrosine near apolysis. Fat body tyrosine peaked between ecdysis and 3·3 hr post-ecdysis whereas abdominal integument tyrosine peaked at ecdysis. Maximum concentrations were 6·0 μg and 4·1 μg tyrosine/mg wet wt. of tissue, respectively. Between ecdysis and 24 hr post-ecdysis, the period of maximum sclerotization, total tyrosine in haemolymph and fat body decreased by approximately 600 μg and 420 μg/insect, respectively. Phenylalanine concentrations did not change significantly in the haemolymph, fat body, or abdominal integument during the pre- and post-ecdysial periods.The cockroach apparently does not store free phenylalanine or tyrosine in the fat body during larval development as compared to tyrosine storage in some Diptera. The rapid increase of haemolymph, fat body, and integument tyrosine just prior to ecdysis suggests another form of storage for this important amino acid.     

Kinetic properties, and location of nonspecific phosphomonoesterases in subcellular fractions of fasciola hepatica

Optimal activity was recorded at pH 4.5–5 and pH 9.0–9.5 and specific activity was seen to be 0.013 μmoles of p-nitrophenyl phosphate/min/mg protein at 37 C at pH 4.5 and 0.00169 μmoles at pH 9.0. The ratio of acid to alkaline phosphatase was 7.7:1.0. The K_m for acid phosphatase (EC 3.1.3.2) was 0.5 mM with a V_max of 0.0128 units/mg protein and 0.2mM for alkaline phosphatase (EC 3.1.3.1) with a V_max of 0.00175 units/mg protein. Acid phosphatase activity was optimal at 60 C and alkaline at 37 C. Linearity of enzyme activity was observed with time after the first 15 min of incubation and with homogenate concentration. KCN at 20 mM inhibited 82% of activity at pH 9.0 but also 91.5% activity at pH 4.5. NaF at 10^?2M inhibited 92% of activity at pH 4.5 but had no effect at pH 9.0. The two flukicides rafoxanide and nitroxynil at 20mM had little effect on activity at pH 9.0 and pH 4.5. Enzyme activity at pH 4.5 was found to be greatest in the microsomal fraction with high activity in the lysosomal and soluble fractions. Histochemically, alkaline phosphatase was restricted to the excretory system, vitellaria, and uterus while acid phosphatase was found in the integument and gastrodermis.     

Effect of Bacteria and Amoebae on Rhizosphere Phosphatase Activity

The contributions of various components of soil microflora and microfauna to rhizosphere phosphatase activity were determined with hydroponic cultures. Three treatments were employed: (i) plants alone (Bouteloua gracilis (H.B.K.) Lag. ex Steud.) (ii) plants plus bacteria (Pseudomonas sp.), and (iii) plants plus bacteria plus amoebae (Acanthamoeba sp.). No alkaline phosphatase was detected, but an appreciable amount of acid phosphatase activity (120 to 500 nmol of p-nitrophenylphosphate hydrolyzed per h per plant) was found in the root culture solutions. The presence of bacteria or bacteria and amoebae increased the amount of acid phosphatase in solution, and properties of additional activity were identical to properties of plant acid phosphatase. The presence of bacteria or bacteria and amoebae increased both solution and root phosphatase activities at most initial phosphate concentrations.     

Changes in the activity of dopa quinone imine conversion factor during the development of Bombyx mori

The activity of DOPA quinone imine conversion factor (QICF) in tissues at different developmental stages of the silkworm, Bombyx mori, was determined. QICF activity was detected in all developmental stages from egg to pupa although the activities, other than in fifth instar larvae, were quite low. Activity in whole larvae peaked one day before the onset of larval-pupal development and declined to a low level shortly before ecdysis. In whole pupae, maximal QICF activity was obtained 1 h after pupation. The activity in larval cuticles was elevated on the last day of the fourth instar and again between days 4 and 8 of the fifth instar, decreasing to very low levels before pupal ecdysis. QICF was detectable in pupal cuticles with most of the pupal activity found in homogenates of mid and hind guts. A major part of the total larval QICF activity was found in hemolymph. Activity in hemolymph varied in a different manner from that in cuticles, with markedly raised levels immediately before pupal ecdysis when the cuticular activity had declined. It is postulated that QICF in cuticles plays some role in wound healing and/or sclerotizatio,, while QICF in hemolymph participates in melanization in the humoral immune system.