Haemolymphal activation of protyrosinase and the site of synthesis of haemolymph protyrosinase in larvae of the fleshfly, Sarcophaga barbata

When whole blood from 5 day third instar larvae of the fleshfly, Sarcophaga barbata was incubated under nitrogen at 25°C for 16 hr in the presence of salivary glands there was an increase in its protyrosinase content, which amounted to 53% of that which occurs in vivo over the same period. The protyrosinase in ammonium sulphate fractions of haemolymph that were allowed to stand at 4°C for 24 hr following the incubation at 25°C was found to have autoactivated. Analysis of all these fractions revealed the presence of a protyrosinase activator in the 30% saturated ammonium sulphate fraction. When proenzyme and haemolymph activator were mixed there followed a lag period before the rapid phase of activation, the duration of the lag being dependent upon the concentration of both proenzyme and activator. The final activity attained was dependent upon the concentration of proenzyme, but was independent of the activator concentration and was comparable to that obtained using the cuticle activator. The level of activator in the haemolymph increased as larvae aged from 4 to 7 days.The effect of several compounds on the catecholase activity of the activated haemolymph protyrosinase and on the cuticle enzyme is reported and the significance of haemolymphal activation of protyrosinase is discussed.     

Bacteria-induced haemolymph proteins of Manduca sexta pupae and larvae

The haemolymph of Manduca sexta larvae and pupae has been analyzed for proteins potentially associated with the bacterial defence response of the insect. Five proteins, M13, M18, M20, M23, and M24 in pupae, and M4, M11, M13, M18 and M23 in larvae, are induced by the injection of bacteria into the haemolymph. Proteins M4 and M11 are always present at high levels in uninjected pupae. Proteins M20 and M24 could not be induced in larvae. These proteins, as well as those not showing a response to bacterial challenge or injury, were also analyzed for presence of disulphide bonds and carbohydrate moieties, and their apparent molecular weights determined.     

Clock genes period and timeless are rhythmically expressed in brains of newly hatched, photosensitive larvae of the fly, Sarcophaga crassipalpis

While roles of the clock genes period (per) and timeless (tim) are relatively well understood in relation to circadian clocks, their potential roles in insect photoperiodism remain enigmatic. In this study, the expression of per and tim genes under two contrasting photoperiods is described in the central nervous system of photoperiodically sensitive, newly hatched first instar larvae of the flesh fly, Sarcophaga crassipalpis. Using qPCR, diel oscillations were observed in the mRNA levels of both genes under long-day (15 h light:9 h dark, promotes direct development) and short-day conditions (11 h light:13 h dark, induces pupal diapause). Peak per and tim mRNA oscillations were closely associated with the light/dark transition. The conspicuous difference between the two photoperiodic conditions was that the sharp increase in per and tim mRNA abundance occurred during the light phase under long days but during the dark phase under short days. The diel oscillations were, at least in part, driven by an endogenous component, as demonstrated by transferring larvae to continuous darkness. The cells displaying Tim- and Per-like immunoreactivities (Tim- and Per-LIRs) were localized using anti-Drosophila-Per and anti-Chymomyza-Tim antibodies. Per-LIR and Tim-LIR co-localized in three groups of cells in each brain hemisphere. Two other groups, one in the brain hemispheres and the other in the fused ventral nerve ganglion, expressed only the Per-LIR.     

Isothiocyanates in myrosinase-treated seed extracts of Moringa peregrina

Seeds of Moringa peregrina (Moringaceae), on treatment with myrosinase, produce 2-propyl, 2-butyl and 2-methylpropyl isothiocyanate in addition to 5,5-dimethyl-oxazolidine-2-thione, all new to the family but known as natural derivatives from other sources. 4-(4′-O-Acetyl-α(-l-rhamnosyloxy)benzyl isothiocyanate, not previously described, together with substantial quantities of its non-acetylated counterpart, formerly recognized as a component in hydrolysed seeds of M. oleifera, constitute the additional mustard oils observed in seeds of M. peregrine.     

Factors that influence the post-emergence growth in Sarcophaga falculata

The post-emergence growth (deposition of endocuticle and growth of skeletal muscles) in Sarcophaga falculata is induced by a blood-borne factor secreted from the head of the pharate adult. The secretion begins 24 to 28 hr before eclosion (at 28°C). A second blood-borne factor, appearing in the haemolymph of the flies a few hours after tanning, suppresses the post-emergence growth in flies, the heads of which have been ligatured at the time of eclosion.     

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.     

Tyrosinase activity in the larva of the fleshfly, Sarcophaga barbarta

When plasma from third instar larvae of the fleshfly, Sarcophaga barbarta, was diluted tenfold with distilled water, lipoproteins precipitated out. After centrifuging, the water supernatant was rendered 30, 50, and 65% to ammonium sulphate, and it was found that the 50% fraction contained 95% of the tyrosinase activity in all the fractions, the enzyme being present in its inactive form or proenzyme. The proenzyme was activated by mixing it with activator isolated from the larval cuticle. After addition of activator there followed a lag period before the rapid phase of activation, the duration of the lag being dependent upon the concentration of both proenzyme and activator. The final activity attained was dependent upon the concentration of proenzyme but was independent of the activator concentration.The level of proenzyme in the plasma rose steadily throughout the third larval instar reaching a maximum in 7 day larvae, formation of the puparium commencing about 24 hr later, the rounded-off white stage (r.o.). At the r.o. and golden-brown stage (1 hr later) the level was still maximal, but 12 hr later at the dark-brown puparial stage no proenzyme was isolatable from the plasma, all the enzyme at this stage behaving as active enzyme.The vast majority (95%) of the proenzyme isolated from plasma in the larval stages and at the r.o. white stage was present in the 50% ammonium sulphate fraction, whereas 1 hr later at the golden-brown stage only 33% of the proenzyme was found in the 50% fraction, 62% now being found in the 65% fraction. At the dark-brown puparial stage 12 hr later, not only was there a further redistribution, but all the enzyme behaved as active enzyme. It is suggested that these changes in the distribution and behaviour of the proenzyme indicate that, in vivo, activation of the enzyme in the blood has taken place over the period r.o. white to the golden-brown to dark-brown puparial stage.     

Freeze-tolerance adaptations,including haemolymph protein and lipoprotein nucleators,in the larvae of the cranefly Tipula trivittata

The terrestrial overwintering larvae of the cranefly Tipula trivittata were freeze tolerant (able to survive the freezing of their extracellular body fluids) throughout the winter and spring of 1982–1983 until they pupated in mid-May. The larvae were most cold tolerant (24 h lower lethal temperatures of ?25 to ?30°C) in late January and early February. Sorbitol, at a maximal concentration of ～0.4 M, was the only polyol determined to be present at high levels and sorbitol accounted for most of the seasonal fluctuation in osmotic concentration. Haemolymph inorganic ion (Na⁺, K⁺, Ca²⁺, Mg²⁺, Cl^?) concentrations did not vary seasonally.The supercooling points of the larvae remained constant at ?6 to ?7°C over the study period because of the presence of haemolymph ice nucleating factors. These ice nucleating factors consist not only of haemolymph proteins, as had been demonstrated previously in other insect species, but also lipoproteins.     

Metabolic reserves associated with pupal diapause in the flesh fly, Sarcophaga crassipalpis

Larvae of Sarcophaga crassipalpis destined for pupal diapause (light:dark 12:12, 20°C) contain nearly twice as much lipid and twice the haemolymph protein concentration as larvae that will not enter diapause (light:dark 15:9, 20°C). This conspicuous difference in metabolic reserves provides the earliest indication of the developmental fate of the larva. Lipid reserves are utilized rapidly during the first half of diapause and then remain stable until adult eclosion. In contrast, residual dry weight changes very little early in diapause but drops sharply late in diapause, thus implying a transition from lipid utilization to protein or carbohydrate utilization in mid-diapause. We suggest that this metabolic transition marks the end of the “fixed latency period”: pupae readily respond to environmental or hormonal stimulation after this point. Diapause-destined larvae did not accumulate more glycogen than nondiapause-destined larvae, but an 80% decrease in glycogen at the onset of diapause and its elevation at the end of diapause suggests the utilization of glycerol or related compounds as cryoprotectants during diapause. Profiles of water content are very similar in short-day and long-day flies, thus suggesting that dehydration is not a mechanism exploited by the flesh fly to achieve cold hardiness. Adult flies that have experienced pupal diapause emerge from the puparium with lipid, glycogen, and water content nearly identical to flies that have not experienced diapause, but the residual dry weight is much lower. The severe depletion of protein may account for the reduced fecundity of flies that have experienced diapause.     

Active Na+ uptake in the isolated midgut of larval Sarcophaga bullata

The isolated midgut of larval Sarcophaga bullata actively accumulates Na⁺ from the gut lumen into the haemolymph. The active transport of Na⁺ out of the gut lumen is responsible for the transepithelial potential difference measured across the midgut epithelium, such that the midgut lumen is negative in respect to the haemolymph side. Both the net movement of Na⁺ out of the midgut lumen and the transepithelial potential are inhibited by CN^? and, in addition, the potential in blocked by ouabain.     

Female specific proteins in Triatoma infestans haemolymph

The presence of female specific proteins in triatoma infestans haemolymph, as well as the relationship between the female specific proteins and egg proteins, were analysed. At the same time, the presence of specific female proteins in different instars was studied. Cellulose acetate electrophoresis, polyacrylamide gel electrophoresis, and immunochemical methods were used.No differences between immature female and male haemolymph were established. Female haemolymph obtained from insects with ovary development revealed quantitative differences, with cellulose acetate, with respect to the control males. With polyacrylamide gel electrophoresis, one component that is not detected in control males was detected in mature female haemolymph. With immunochemical assays, at least two antigenic components that were not observed in male haemolymph were detected.Egg extract showed, with cellulose acetate, two bands with a mobility similar to that of the proteins increased in the haemolymph of the mature female; with polyacrylamide gel, two major bands with a mobility similar to that of the specific female haemolymph protein were detected. Egg extract contains at least two components demonstrated by double-diffusion assays and three components by immunoelectrophoresis, with immunological identity to specific mature female haemolymph proteins.The extract obtained from recently hatched insects revealed two components with immunological identity to specific female proteins. Haemolymph from first, second, third, fourth and fifth instars do not appear to contain any femalespecific haemolymph protein.     

Interaction of Xenorhabdus nematophilus subsp. nematophilus with the haemolymph of Galleria mellonella

The lethal dose (LD)₅₀ values and probit-mortality regression slopes of the primary and secondary forms of Xenorhabdus nematophilus subsp. nematophilus for Galleria mellonella were equal. The two bacterial forms grew at equal rates in larval serum-supplemented media. The secondary form grew less well in larval serum-supplemented media than in synthetic larval serum.The secondary bacteria adhered to the haemocytes to a greater extent than did the primary bacteria. Both types of bacteria did not produce metabolites suppressing the ability of the haemocytes to respond to Bacillus cerues.Differences were observed in the rate of clearance of the primary and secondary bacteria from and their subsequent re-entrance into the haemolymph in vivo. This appeared to be independent of bacterial metabolism. Evidence is presented showing multiplication of the primary bacteria during their association with the haemocytes.The total haemocyte counts increased during bacterial infection. All the haemocytes were killed. The rate and pattern of change of the total haemocyte counts was influenced by the form of bacteria and independent of bacterial metabolism.     

Nonadecan-7-ol-2-one an aliphatic ketol from Diospyros peregrina

A new aliphatic ketol isolated from the stems of Diospyros peregrina was characterized as nonadecan-7-ol-2-one by analyses MW determination chemical degradation and spectroscopic data.     

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.     

The circadian eclosion rhythm in Sarcophaga argyrostoma: A limit cycle representation of the pacemaker

We examine the effects of light upon the phase and coherence of the Sarcophaga eclosion rhythm and show that the limit cycle representation developed previously for the mosquito Culex pipiens quinquefasciatus can be extended, with very slight modification, to Sarcophaga.     

Control of extracardiac haemolymph pressure pulses in Tenebrio molitor

The pupae of Tenebrio exhibit periodic pulsations in the haemolymph pressure which are independent of the heartbeat. Tensometric records of the extracardiac pulses show certain specific modifications caused by changes in either the internal or external environment. Chronological changes in the pulse pattern were associated with adult morphogenesis and ecdysis. The abdominal pump controlling extracardiac pressure pulsations is independent of the brain or of any other cephalic part of the nervous system. The nerve impulses controlling the pump arise only in the mesothoracic ganglion. They are carried by the connectives to certain abdominal ganglia from which they are further transmitted to the contracting intersegmental abdominal muscles. The extracardiac pulses in haemolymph pressure aid in the maintenance of water balance and respiration and assist with the circulation of haemolymph through the appendages.     

Growth of Musca domestica (Diptera: Muscidae) and Sarcophaga dux (Diptera: Sarcophagidae) larvae in poultry and livestock manures: Implication for animal waste management

Natural diets commonly exploited by the flies are animal manures including production from the poultry and livestock facilities. The larvae of the common filth flies such as Musca domestica and Sarcophaga dux are known as voracious feeders and may thus be used to convert manures into non-polluted residue. This study was conducted to observe the impact on flies' growth rate and capability of the larvae to process animal manures using chicken, goat and cow manures. One hundred newly hatched larvae of M. domestica and S. dux were introduced separately into 150?g manures under laboratory conditions. The initial wet mass and larvae length were recorded while mortality rate and dry mass were measured after the larvae were placed into the manures. The results showed that the manure types give significant effects (p?<?.05) on the growth of M. domestica and S. dux larvae. Cow manures and chicken manures contributed the highest growth for M. domestica and S. dux respectively. This result confirmed by the mean increment in wet mass and larvae length. In contrast, M. domestica greatly reduced 59.9?±?4% chicken manures while 25.0?±?1.8% goat manures reduced by S. dux. The potential of M. domestica and S. dux larvae to reduce animal waste products were further discussed in this study.