Possible role in uptake of water vapour by ixodid tick salivary glands

The mouth is confirmed as the site of water vapor uptake in the lone star tick, Amblyomma americanum. It was shown that the level of chloride (³⁶Cl) increased in the mouthparts of desiccated ticks. The highest levels of ³⁶Cl were found in the mouthparts, salivary glands, and gut tissue during rehydration. It is suggested that ions are secreted by the salivary glands into the mouth where water is picked up hygroscopically by the secretion. It is further suggested that the water and ions are then swallowed and absorbed from the lumen of the gut.     

Control of fluid secretion by isolated salivary glands of the lone star tick.

Adrenaline, noradrenaline, cyclic-AMP, and theophylline stimulated salivary fluid secretion in glands isolated from rapidly engorging females of the lone star tick, Amblyomma americanum. Cyclic-AMP and theophylline effectively initiated secretion only after the glands were pre-incubated and pre-stimulated to secrete with adrenaline. Pilocarpine nitrate, 5-hydroxytryptamine (5-HT), and glutamate did not stimulate glandular secretion, 2,4-Dinitrophenol (DNP) slowed adrenaline's ability to stimulate secretion. Results obtained after additions of harmaline and ouabain point to the possibility of a (Na⁺+K⁺)-ATPase system for helping to move fluid across the glandular epithelium. The significance of these findings to the mechanisms and control of tick salivary glandular function is discussed.     

Juvenile hormone-induced vitellogenesis in Apterous4, a non-vitellogenic mutant in Drosophila melanogaster

D. melanogaster females homozygous for the ap⁴ mutant synthesize yolk protein and circulate this protein in the haemolymph at concentrations not different from concentrations found in normal females. However, ap⁴ females deposit little or no yolk protein into developing oöcytes. Topical application of a juvenile hormone analogue (JHA), ZR-515, stimulated sequestration of yolk protein by developing oöcytes of ap⁴ females. JHA had no detectable effects on haemolymph concentrations of yolk protein in either normal or ap⁴ females nor on the protein profiles obtained from electrophoresis of haemolymph samples.     

Effects of farnesyl methyl ether on the haemolymph and ovarian proteins of the tent caterpillar, Malacosoma americanum

The mode of antigonadotropic action of farnesyl methyl ether (FME) on Malacosoma americanum was investigated by studying the haemolymph and ovarial proteins during vitellogenesis. Low doses of FME which permitted apparently normal adult development but inhibited ovarian development were used.As indicated by the incorporation of ¹⁴C-glycine, FME treatment had no effect on the protein biosynthetic activity by the fat body. However, it resulted in significant accumulation of several haemolymph proteins apparently caused by their reduced uptake by the ovarioles.Radioassay of individual proteins revealed that all the haemolymph proteins had incorporated ¹⁴C-glycine. However, proteins G and H, and to some extent protein B of the treated females, showed significantly higher incorporation of the labelled glycine. It is concluded that the antigonadotropic action of the JH mimic was due to reduced incorporation of various sex-specific and non-specific haemolymph proteins into the ovarioles.     

Gene-controlled incorporation of haemolymph protein into the ovaries of Bombyx mori

The haemolymph protein concentration in Bombyx mori decreases normally by about one-fourth during pharate adult development. In females homozygous for the small egg gene, the concentration of haemolymph protein remained constant throughout the pupal and pharate adult stages. The sm gene does not influence the synthesis of vitellogenic female protein of pupal and pharate adult haemolymph (FP). Normal ovaries transferred to the haemocoele of sm females undergo normal vitellogenesis. In the absence of normal alleles of sm, the ovaries encounter difficulties in the incorporation of FP into their oöcytes from pharate adult haemolymph. These results suggest that an active translocation mechanism is involved in the transfer of haemolymph protein into the ovaries.     

Biochemical characterization of α- and β-glucosidases in alimentary canal, salivary glands and haemolymph of the rice green caterpillar, Naranga aenescens M. (Lepidoptera: Noctuidae)

The biochemical properties of ??- and ??-glucosidase in salivary glands, alimentary canal and haemolymph of Naranga aenescens larvae, one of the most damaging pests of the rice crop in Iran, were investigated. The specific activity of ??-glucosidases were 3.88, 2.74 and 1.58 ??mol/min per mg protein in the alimentary canal, salivary glands and haemolymph of last instar larvae, respectively. The specific activity of ??-glucosidases were 1.27, 0.077 and 0.414 ??mol/min per mg protein in the alimentary canal, salivary glands and haemolymph of last instar larvae, respectively. The optimal pH for ??-glucosidases were 6.0, 6.0?C8.0 and 6.0 and the maximum activity for ??-glucosidases were obtained at pH 6.0, 5.0?C7.0 and 5.0 in alimentary canal, salivary glands and haemolymph, respectively. The optimum temperatures for ??-glucosidases were determined at 55°C in alimentary canal, 35?C45°C in salivary glands and 55°C in haemolymph, whereas the ??-glucosidases reached their optimum at 45°C in all three tissues. Effect of metal ions on the activity of ??- and ??-glucosidases showed that K⁺ (20 mM) and Mg²⁺ (10 and 20 mM) increased N. aenescens ??- and ??-glucosidases activities from salivary glands, while Ca²⁺ increased ??- and ??-glucosidases activities in haemolymph. In the presence of Fe²⁺, Mn²⁺, Hg⁺ and Zn²⁺ (10, 20 mM) and Hg²⁺ (20 mM), these enzymes from all tissues were completely inactivated. K _m values were estimated for the ??-glucosidases as 3.96, 0.547 and 3.084 mM and for ??-glucosidases as 1.93, 1.014 and 1.93 mM in the alimentary canal, salivary gland and haemolymph, respectively. The zymogram analyses of N. aenescens crude extracts indicated the presence of at least two isoforms for ??-glucosidase and one isoform for ??-glucosidase.     

Re-evaluation of the role of corpora cardiaca in calling and oviposition behaviour of giant silk moths

Re-investigation of the role of the corpora cardiaca in the reproductive behaviour of the giant silkmoths, Hyalophora cecropia and Antheraea polyphemus, showed that this pair of glands plays no essential role, either in “calling” behaviour by virgin females or in increased oviposition due to mating. Removal of corpora cardiaca-corpora allata complexes, either from diapausing pupae or from freshly eclosed adult females, had no effect on the calling behaviour or on its timing in either species. Moreover after mating, these operated females laid eggs in the typical mated oviposition pattern. Furthermore, females in which there was only a nervous connection between the brain and the abdomen but no haemolymph circulation called normally and oviposited after mating.Although the corpora cardiaca were not essential for calling behaviour, hormogenates of corpora cardiaca-corpora allata complexes and blood from calling or ovipositing females induced a typical “calling” response in 30–60% of the isolated virgin H. cecropia abdomens tested. This activity was not species-specific as it was also found in Manduca sexta, but the restriction of major activity to corpora cardiaca extracts and haemolymph suggested that a neurosecretory factor may modulate the normal neural control of calling behaviour.     

Transfer and fate of male secretions deposited in the spermatophore of females of Acanthoscelides obtectus Say (Coleoptera Bruchidae)

During mating, males of Acanthoscelides obtectus deposit a spermatophore in the female genital tract. Spermatophore structure subsequently undergoes considerable modification, especially the central portion, which becomes vacuolated. Two methods were used to show that certain male secretions could thus pass into female haemolymph. When young males were injected with [¹⁴C]-arginine or [¹⁴C]-histidine, the accessory glands actively incorporated the isotope and the resulting spermatophores were radioactive. After mating, spermatophore radioactivity declined and then appeared in the haemolymph of females and in the oöcytes after a delay. Immunoelectrophoresis also showed that antigens appeared in the haemolymph of females after mating which reacted against male-gland antiserum. This technique, however, did not enable us to detect the presence of male antigens in the oöcytes formed after mating. The fate of some male secretions in the female and their physiological importance in the control of the female reproductive function were analysed in the present work.     

Metabolism of U14C leucine and U14C valine by adult female Glossina morsitans during pregnancy

After U¹⁴C leucine or U¹⁴C valine injections into haemolymph of adult female Glossina morsitans during late pregnancy, radioactivity was detected in the post-parturient female and its larval offspring in the injected material, lipids, and a range of non-essential amino acids. The level of radioactivity recorded from the third instar larva was higher than that remaining in the injected adult, and the activity was higher in amino acids than in the lipid fraction. Radiometric analysis of oöcyte and intra-uterine progeny 24 hr after haemocoelic administration to females of labelled leucine or valine revealed a pattern of radioactivity coincident with growth characteristics of these young stages. Rate of leucine uptake by the in utero third instar larva was slightly higher than that of valine, and this instar continues feeding even only a few hours before parturition. For both labelled materials, expired carbon dioxide and excreta from remales in early pregnancy showed significantly higher radioactivity than those in late pregnancy. Uric acid is the main nigrogenous waste of leucine and valine metabolism, though small amounts of these amino acids are also lost during excretion, with valine elimination being higher than leucine.     

Adipokinetic hormone-induced lipid mobilization and lipophorin interconversions in fifth larval instar locusts

The response of fifth larval instar locusts to injected adipokinetic hormone (AKH) is only poor, as is reflected in both a very moderate elevation of the haemolymph lipid concentration and the slight occurrence of the haemolymph lipophorin interconversions characteristic for adult locusts, resulting in formation of only small quantities of the low density lipophorin (A⁺). However, an additional lipophorin fraction (A′) is induced, which is intermediate in density and size between high and low density lipophorin and which is not identified in adult haemolymph. As in adults, larval A⁺ formation includes association of the resting high density lipophorin with a non-lipid containing protein (C₂), the haemolymph concentration of which is only one-fifth relative to adults. However, the larval haemolymph protein composition is not the primary cause of the incomplete adipokinetic response, as elevation of the concentration of protein C₂ by injection of isolated adult C₂, whether or not in combination with adult high density lipophorin, did not increase lipophorin conversions nor haemolymph lipid elevation.In vitro incubation of larval fat bodies in adult haemolymph showed that competency to both the AKH-induced lipid release and the haemolymph lipophorin conversions of the larval fat body are reduced compared to equal amounts of adult tissue. Reciprocal incubation of adult fat body in larval haemolymph resulted in only a very moderate adipokinetic response, demonstrating that larval haemolymph protein composition is restrictive for full development of hormone action.Both immunoblotting experiments and enzyme-linked immunosorbent assays (ELISA), using monoclonal antibodies specific for the adult lipophorin apoproteins, indicated that the larval lipophorins closely resemble the adult forms. Apparently the structure of locust lipophorins is remarkably constant throughout development despite changes in metabolic functions.     

Protein metabolism by the salivary glands and other organs of the larva of the blowfly, Calliphora erythrocephala

Protein metabolism in salivary glands, gut, haemolymph, and fat body during the last larval instar of the blowfly, Calliphora erythrocephala, has been investigated. In salivary glands, protein release, protein synthesis, amylase, and pepsin-like protease activity were maximal in 6 day larvae, this being at a time when the larvae had finished feeding. All these functions declined in glands from the rounded-off white puparial stage (R.O.) while acid phosphatase activity rose throughout the third instar to a maximum at the R.O. stage, Glands from 6 and 7 day larvae released protein which on disk gel electrophoresis separated into four minor bands and two major bands one of the latter possessing protease activity.In the gut, pepsin-like protease activity was maximal in 4 day larvae after which it fell rapidly thus following the feeding pattern of the larva in contrast to that in the salivary glands which did not.In vitro experiments showed that protease was released from 6 day glands through the basal membrane of the cells and not via the duct. A pepsin-like protease was also found in the haemolymph and fat body, the activity in the fat body rising rapidly during the latter part of the third instar, a rise which is attributed to the fat body sequestering protease from the haemolymph. Acid phosphatase activity in the fat body was maximal in 5 day larvae indicating that this enzyme was synthesized early in the third instar. It was shown that fat body sequestered ¹⁴C-labelled protein synthesized by and released from the salivary glands, most of the ¹⁴C activity being associated with a 600 g precipitable, acid-phosphatase rich fraction.It is proposed that in late third instar larvae the salivary glands function as glands of internal secretion, releasing protease into the haemolymph, which is then sequestered by the fat body (and perhaps other tissues) and is subsequently used in the lysis of the tissues at the time of metamorphosis.     

Vitellogenin fluctuations in haemolymph and fat body and dynamics of uptake into oöcytes during the reproductive cycle of Diploptera punctata

Haemolymph and fat body soluble protein titres have been examined during the reproductive cycle of Diploptera punctata, with particular emphasis on the occurrence of vitellogenin and its uptake into the developing oöcytes. Vitellogenin was first detected in the haemolymph of mated females 2 days after adult eclosion at about the same time that vitellin deposition in basal oöcytes began. Peak haemolymph titres of vitellogenin occurred on day 6, correlated with the completion of yolk uptake. Thereafter vitellogenin levels declined and were generally undetectable throughout most of gestation, rising again shortly before parturition in association with the second gonotrophic cycle. Total haemolymph protein levels were not correlated with vitellogenesis.Soluble fat body vitellogenin titres of mated females remained low during the first oöcyte growth period but then rose several-fold at its completion and remained high throughout pregnancy and the second gonotrophic cycle. Total fat body soluble proteins decline after adult eclosion in association with oöcyte growth.Vitellin accumulation in basal oöcytes was related linearly to increase in volume until the onset of chorion formation. Thus no post-vitellogenic growth period was detected.     

On the relative importance of juvenile hormone and vitellogenin for oöcyte growth in the cockroach Nauphoeta cinerea

The corpora allata of castrated females of Nauphoeta grow only very slightly and do not reach a volume greater than that of the glands of normal females during gestation. These small corpora allata are, however, active and are responsible for the synthesis of vitellogenin (female specific protein) in large amounts. Besides vitellogenin the other haemolymph proteins are also synthesized and accumulated in the haemolymph in much higher concentrations than in normal females. Implanted oöcytes grow in castrated as well as in normal females at about the same rate until the tenth day of the oöcyte maturation period. Thereafter they only grow in castrated females. If castrated and normal females are decapitated, their protein content decreases. At the same time the growth stimulating capacity of their haemolymph decreases at a much faster rate. If oöcytes are implanted in castrated and decapitated females after 4 days they cannot grow any more although the vitellogenin titre of the haemolymph is still much higher than it is at any time in normal females. It can be concluded that vitellogenin alone cannot induce oöcyte growth and that juvenile hormone is necessary as well for vitellogenin synthesis as for its incorporation into the oöcytes. However, in insects rich in vitellogenin juvenile hormone leads to a more rapid oöcyte growth than in insects containing only small amounts of this protein.     

The vitellogenin of the ring-legged earwig,Euborellia annulipes (Lucas) (Insecta: Dermaptera)

Using Polyacrylamide gel electrophoresis, labelling procedures and immunological methods, an extraovarian synthesis of vitellogenin has been demonstrated in Euborellia annulipes. Electropherograms of the haemolymph of 1-wk-old mated females show an extra protein fraction on the second day after mating; the concentration of this fraction in the haemolymph falls from the fifth day after mating. The fatbody incorporates [³H]leucine into the proteins synthesised by it during the period of oocyte formation. The proteins extracted from the fatbody and ovary of females of the first reproductive cycle show a high level of radioactivity 3 days after mating, suggesting a simultaneous release of proteins from the fatbody and uptake by the oocytes. On the other hand, TCA-precipitable fractions of the ovary, obtained from females on the pre-ovipositional day, record a high [³H]activity but similar fractions from the fatbody only show minimum radioactivity. Antibodies prepared against the antigens obtained from the crude yolk extract reacted with the proteins from the haemolymph and also with the proteins from fatbody extracts of females of the first reproductive cycle.     

Radial transport of ions in roots

Measurements were made of the relative amounts of ⁸⁶Rb, ³⁶Cl, and ³²P accumulated in the cortex and stele of intact roots of corn (Zea mays), either detached or attached to their shoots. Both 4- and 7-day-old roots accumulated as much or more ⁸⁶Rb in the stele as in the cortex. In experiments with ³⁶Cl, cortex and stele accumulated the same amount, except for 4-day-old and 7-day-old attached roots, in which the cortex contained more ³⁶Cl than the stele after 23 hr. An additional study of ³²P uptake showed greater accumulation in the cortex than the stele for a short period of time, but as much in the stele as in the cortex after 8 to 24 hr. Transport of ⁸⁶Rb, ³⁶Cl, and ³²P into the xylem exudate increased with increasing accumulation of these ions in stele and cortex of the root. These experiments show no consistent difference between cortex and stele of intact corn roots with respect to their ability to accumulate several kinds of ions.     

Juvenile hormone induced ovarian uptake of a female-specific blood protein in Oncopeltus fasciatus

An adult female-specific blood protein was demonstrated in Oncopeltus by gel electrophoresis. This protein is the major band in soluble yolk fractions. It is also present at substantial concentrations in the haemolymph of starved and diapausing adult females. Thus, the failure of the ovaries to form yolk under these conditions is characterized by an inability to remove vitellogenin from the blood. Application of a juvenile hormone analog (JHA) restored protein yolk deposition in starved and diapausing adult females. Whereas other blood proteins decreased no more than two-fold upon JHA treatment, the vitellogenin concentration decreased 20-fold in starved females. The vitellogenin concentration in the blood of diapausing females was not significantly affected by JHA, apparently because synthesis kept pace with ovarian uptake in this case.     

Vitellogenesis by the American cockroach: Haemolymph and follicle protein patterns during vitellogenin synthesis

Radioactive ¹⁴C-leucine is removed from the blood within 4 hr of injection during the first 2 days of the vitellogenic cycle. Injections during the 3rd to 6th day result in leucine retention and a rise in labelled protein.Label appears in the follicle by day 3 with most of the protein being incorporated during day 5. Comparison of haemolymph and follicle proteins suggests that fat body synthesis, subsequent haemolymph transport and follicle uptake all occur primarily on days 4 and 5 of the cycle.In vivo follicle incubations reveal ¹⁴C-leucine uptake during the last 4 days of the cycle. During days 4, 5, and 6, leucine is incorporated into protein by the follicle. Injections of ¹⁴C-haemolymph proteins into 6 day females result in the incorporation of label into the terminal oöcytes.     

Änderungen im muster der Haemolymphproteine von adulten Königinnen der Hummelart Bombus terrestris

The soluble proteins of haemolymph during the life cycle from adult bumblebee queens (Bombus terrestris) were separated by disk electrophoresis on polyacrylamide gels. Twenty-three fractions stainable with amido black were detected. Every phase of the bee's adult life is characterized by a specific pattern of haemolymph proteins.Newly emerged queens have a low haemolymph protein concentration which increases in the first 5 days to a maximum. The high concentration is probably connected with the synthesis of hibernation reserves. Before the beginning of hibernation the concentration of some protein fractions seems to decrease; the concentration of these fractions is low also after hibernation.During the spring the first oöcytes begin to grow and the activity of corpora allata, hypopharyngeal glands, and wax glands reaches a maximum at the time of starting nests. A large increase in the concentration of haemolymph proteins is correlated with the activity of these glands. This high concentration does not change during the whole egg-laying period; however, the concentration decreases to a minimum in old queens with degenerating ovaries.In the protein pattern of ovary homogenate we detected three fractions with an RF identical to haemolymph fractions. Investigations on queens parasitized with the nematode Sphaerularia bombi confirmed that these fractions are yolk material (vitellogenin) taken up by ovaries. In parasitized queens oöcytes do not grow and the fractions are of a much lower concentration than in nonparasitized queens with maturing eggs. Therefore it appears that the parasite injures primarily the corpora allata known to stimulate the synthesis of yolk protein.     

The occurrence of vitellogenin in workers and queens of Apis mellifica and the possibility of its transmission to the queen

Immuno-diffusion tests show that worker and queen haemolymph contains a protein fraction which does not occur in the haemolymph of drones. Its immunological and electrophoretical properties are identical with those of the main soluble fraction in the ovaries of queens in oviposition. It therefore is a vitellogenin.The titre of this vitellogenin in the haemolymph of 0- to 28-day-old workers was determined by rocket-immunoelectrophoresis. It attains a maximum on day 12. Its changes seem to be positively correlated with the volume of the corpora allata during the first 12 days of adult life.The hypopharyngeal, mandibular, and salivary glands and the content of the honey stomach of workers were immunologically examined. Vitellogenin could not be found in these organs nor in worker or royal jelly. It is also absent from the digestive tract of queens.¹⁴C-labelled amino acids were injected into 5-day-old workers. Later the uptake of radioactive proteins by the queen was examined. Autoradiography of immuno-diffusion plates showed that within 72 hr active material passed from the injected workers into the eggs laid by the queen. The soluble proteins were extracted from the ovaries and the thorax of the queens and their radioactivity determined. The ratio of ovary to thorax radioactivity of queens directly injected was significantly different from that of queens kept with injected workers.Several proteins of the homogenized hypopharyngeal glands of workers showed precipitation reactions with the antiserum against homogenate of queen ovaries. This together with the results of the tracer experiments indicates that the proteins of the worker hypopharyngeal glands may be precursors of queen yolk components.     

Possible involvement of ecdysteroids in photoperiodically induced suppresion of ovarian development in a Japanese strain of the migratory locust,Locusta migratoria

In a Japanese population of Locusta migratoria, adult females become reproductively inactive under crowding and long days (LD) and reproductively active under crowding and short days (SD). The identity and titre of ecdysteroids in the haemolymph and ovaries from adult females reared under SD and LD were investigated by RIA/HPLC. The effects of exogenous juvenile hormone (JH) III treatments on the termination of such reproductive arrest and ecdysteroid contents in LD females were also examined. In general, ecdysteroid titres in both haemolymph and ovaries were significantly higher in reproductively active SD females than in reproductively inactive LD females. A clear difference was also observed in oocyte growth between SD and LD individuals. JH III applications (three consecutive topical applications, 150 μg per insect per day from day 3) stimulated ovarian development in LD females and significantly increased the haemolymph and ovarian ecdysteroids to a level comparable to that of reproductively active SD adult females.