Haemolymph ecdysteroid in the housefly,Musca domestica,during oögenesis and its relationship with vitellogenin levels

Ecdysteroid titre in the haemolymph of the housefly, Musca domestica, cycled during oögenesis and peaked at ～50 pg/μl during stages 5, 6 and 7. Levels of 10–20 pg/μl were found in houseflies with pre- and post-vitellogenic ovaries. Removal of the corpus allatum and corpus cardiacum complex resulted in low ecdysteroid levels (10 pg/μl). Ovariectomized flies also had lower ecdysteroid levels than the controls at 2 days (5 pg/μl) after emergence but not at 6 days (22 pg/μl). It is possible that the ecdysteroid peak that occurred during stages 5, 6 and 7 was produced by the ovaries because ovaries secreted and synthesized ecdysteroid in vitro. Endogenous haemolymph ecdysteroid levels had a linear correlation with the amount of vitellogenin that held for hormone concentrations of 5–43 pg/μl. Furthermore, the injection of 20-hydroxyecdysone at doses of 10 ng?1.0 μg/fly increased the amount of vitellogenin from 6 h to 12 h after injection; by 24 h, the vitellogenin returned to control levels. When 20-hydroxyecdysone was injected into ovariectomized flies, it was rapidly degraded and 96% was cleared from the haemolymph within 1 h.     

Vitellogenin concentrations in the haemolymph and ovaries of Ixodes scapularis ticks during vitellogenesis

The vitellogenin and vitellin concentrations in the haemolymph and ovaries of Ixodes scapularis females were determined using a double sandwich enzyme-linked immunosorbent assay. The level of vitellogenin in the haemolymph began to increase just prior to tick detachment from the host and continued to increase until 2 days after detachment. There was a slight decrease in the vitellogenin level 4 days after detachment, but a second peak was observed approximately 5 days after oviposition. Subsequent to oviposition, the vitellogenin levels in the haemolymph quickly decreased. The concentration of vitellogenin in the haemolymph ranged from 1.55 to 11.48 g l^-1 during the period after dropping from the host through oviposition. The concentration of vitellin in the ovaries began to increase as the female began rapid engorgement (0.03 mg per female) and declined after oviposition (0.1 mg per female).     

Quantitative studies on the release of locust adipokinetic hormone

Fractionation of methanolic extracts of haemolymph on Sephadex LH-20 made possible the measurement of the titre of adipokinetic hormone in the haemolymph of locusts. Experimentally produced high concentrations of haemolymph carbohydrate caused a delay in the mobilization of lipid during flight, and very low titres of the hormone were present in the haemolymph of locusts injected with trehalose immediately before a 25 min flight. In these locusts flight speed was higher than saline-injected controls. Although delayed lipid mobilization during flight was also seen in locusts injected with sucrose, sucrose is not utilized for flight metabolism and flight speed was not increased by the injection. Tentative estimates of the release rate (c. 1000pg/20min flight) and half life (c. 20 min) of adipokinetic hormone during flight are made. The results described suggest that during flight the rate at which trehalose disappears from the haemolymph does not play a major role in the initiation of the release of adipokinetic hormone.     

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.     

Effects of ablation of the pars intercerebralis on ecdysteroid quantities and yolk protein expression in the blowfly Protophormia terraenovae

Quantities of ecdysteroid are compared in the haemolymph and ovaries of the blowfly Protophormia terraenovae Robineau‐Desvoidy (Diptera: Calliphoridae) under reproductive (LD 18 : 6 h at 25 °C) and diapause (LD 12 : 12 h at 20 °C) conditions. The effects of ablation of the pars intercerebralis or ovaries on ecdysteroid quantities and of ablation of the pars intercerebralis on yolk protein expression are examined. Under reproductive conditions, the levels of ecdysteroid in vitellogenic females are high, although the levels in previtellogenic females and females with mature ovaries are low. Under diapause conditions, there are low quantities of ecdysteroid in both the haemolymph and ovaries. Ecdysteroid titres in the haemolymph are not significantly affected by the removal of the ovaries, suggesting that tissues other than the ovaries are also involved in the production of ecdysteroids. Reproductive females in which the pars intercerebralis of the brain is experimentally ablated have ecdysteroid levels that are not significantly different from sham‐operated or intact females. However, yolk protein expression in the fat body is suppressed after removal of the pars intercerebralis. These results suggest that the suppression of ecdysteroid levels in the haemolymph and ovaries is associated with reproductive diapause, and that the pars intercerebralis could play a role in yolk protein synthesis without mediating ecdysteroid production.     

Ecdysteroids in females and eggs of the Ixodid tick Amblyomma hebraeum

Summary

A mated Amblyomma hebraeum female will engorge on a host for about 8 days before detaching and beginning the maturation of its single egg batch which is laid during a period of about 30 days. The feeding period is characterized by an important synthesis of endocuticular material occurring before the rapid feeding phase. This latter phase, correlated with an enormous weight uptake, shows an increase of ecdysteroid levels measured in the whole animal by RIA. However, the hemolymphatic levels of ecdysteroids remain very low (12 pg 20-hydroxyecdysone equivalent (20-OH-E eq.) per μ1. Within 4 days after detachment, the salivary glands degenerate. Ecdysteroid levels in the whole animal continue to increase, reaching high values (about 500 ng 20-OH-E eq./tick) at the moment of oviposition which begins 10–14 days after dropping. During the same period, hemolymphatic ecdysteroid levels increase, rising to a peak (600 pg 20-OH-E eq./μ1) 1 day prior to the beginning of oviposition. Then, the levels decrease and stabilize around 250 pg 20-OH-E eq./μl during egg-laying. Freshly laid eggs contain large amounts of ecdysteroids (2744 pg 20-OH-E eq./mg).

20-Hydroxyecdysone and ecdysone have been found to be the major free ecdysteroids in hemolymph, ovaries and eggs (verified by the HPLC-RIA technique and GC-MF of silylated HPLC fractions). Helix juice (or esterase) labile ecdysteroid conjugates do not seem to be present to any noticeable extent in hemolymph, ovaries and eggs.     

Radioimmunological Quantification of C21, C19 and C18 Steroids in Haemolymph of the Insect Locusta migratoria

Summary

Titers of pregnenolone, progesterone, testosterone, 5α-dihydrotestosterone, estrone and estradiol were measured by radioimmunoassay in purified haemolymph extracts of larval and adult male and female Locusta migratoria. They varied between following values (in pg/ml): pregnenolone: 467–757; progesterone: 37–119; testosterone: 11–54; 5α-dihydrotestosterone: 13–41; estrone: 25–1392; estradiol: 12–26. Titers of pregnenolone progesterone, testosterone, 5α-dihydrotestosterone and estradiol did not substantially fluctuate among the developmental stages we examined. Peaks of estrone were found in males and females in the middle of the fifth larval instar and in 3 week old adult males. The titers of most of the above six steroids are about 5 to 10 times lower than the concentrations found in purified extracts of several tissues of this insect.     

Ecdysteroid-dependent protein synthesis in caste-specific development of the larval honey bee ovary

In the honey bee, Apis mellifera, the fifth larval instar is a critical period for caste differentiation. During this premetamorphic phase the hormonal milieu shows pronounced caste differences and several organs, particularly the ovaries, enter different developmental pathways leading to highly fertile queens and nearly sterile workers. Developmental profiles of total protein synthesis in larval ovaries showed marked caste differences starting with the early fifth instar. By two-dimensional electrophoresis, caste-specific patterns could be detected in the synthesis of a 29 kDa/pI 4.6 and two 24 kDa/pI 5.2–5.5. proteins (pI=isoelectric point). A marked decrease in the expression of these proteins was found to coincide with caste-specific differences in the haemolymph ecdysteroid titer. In vitro exposure of larval worker ovaries to physiological (10^–7 m) concentrations of synthetic makisterone A elicited an identical response. Juvenile hormone did not affect protein synthesis patterns in larval ovaries, and also did not inhibit or reverse the ecdysteroid-induced effects. Heat shock experiments revealed that the 29 kDa/pI 4.6 ecdysteroid-regulated protein belongs to the class of small heat shock proteins.     

Effect of JH and PII on the protein content of haemolymph and ovaries of Spilostethus pandurus

• 1.1. An SDS-PAGE study of the qualitative and quantitative differences in protein bands from haemolymph and ovaries of Spilostethus pandurus females treated with JH or chemically allatectomized with precocene II, has been done.
• 2.2. The SDS-PAGE study of haemolymph revealed the occurrence of three female-specific proteins. In the ovary appeared three protein fractions (A, Band C) with mol. wts similar to those from haemolymph.
• 3.3. The three female-specific proteins from the haemolymph, and the ovary bands B,C and D were absent in the samples from PII-treated females.
• 4.4. JH accelerates ovary growth and the relative amounts of bands B, C and D were in relation to the physiological stage of the considered ovaries.

     

Endogenous ecdysteroid levels and rates of ecdysone acylation by intact ovaries in vitro in relation to ovarian development in adult female house crickets,Acheta domesticus

Ecdysteroid titres have been determined in adult female house crickets (Acheta domesticus) in relation to reproductive maturation. Ecdysteroid levels in newly emerged adult females are low except in the gut and carcass, which probably reflect the remnants of the preecdysial ecdysteroid peak. Ecdysteroid levels in all compartments increase markedly once ovarian weight surpasses 10 mg. Apolar ecdysteroid conjugates (ecdysone 22-fatty acyl esters) predominate in ovarian tissue throughout ovarian maturation, but low levels of free ecdysteroid and polar conjugated ecdysteroids are also present. During this period, two peaks of ecdysteroids (mainly free and apolar conjugated ecdysteroids) are observed in the haemolymph, gut, and carcass compartments. The peaks in the haemolymph occur when the ovarian mass reaches 30 and 100 mg. The gut and carcass may be acting as sinks or sites of metabolism for the hormone released from the ovaries. The rate of ecdysone acylation by ovaries was found to be developmentally regulated, increasing from low levels in the immature ovaries of newly emerged females as the ovaries increase in size. A semiquantitative assay has been developed to identify compounds which inhibit the conversion of [³H] ecdysone into 22-fatty acyl [³H] ecdysone by ovaries in vitro. A number of ecdysteroids possessing a free hydroxyl group at C-22 as well as the side-chain stereochemistry of ecdysone effectively inhibit this conversion, probably by acting as competitive substrates. In the cases of 20-hydroxyecdysone and ponasterone A, it was clearly demonstrated that these compounds are converted to a mixture of C-22 fatty acyl esters. Several other compounds which have been sugested to affect ecdysteroid metabolism/mode of action in other systems were also tested for their effects on the acyltransferase activity of ovaries in vitro. Arch. Insect Biochem. Physiol. 35:279-299, 1997.© 1997 Wiley-Liss, Inc.     

PRODUCTION OF RECOMBINANT OVINE INTERFERON TAU USING A BOMBYX MORI NUCLEAR POLYHEDROSIS BACULOVIRUS EXPRESSION SYSTEM

ABSTRACT

Ovine interferon tau (oIFN-τ) is an embryonic protein of critical importance in the establishment of pregnancy in the sheep. We have produced recombinant (r) oIFN-τ using a baculovirus expression system and demonstrated the biological activity of the protein produced. Bombyx mori larvae were infected with B. mori nuclear polyhedrosis virus (BmNPV), modified by inserting a cDNA coding for oIFN-τ downstream of the strong polyhedron promoter. Following infection, antiviral activity of the haemolymph rose to a maximum of 3.6?×?10⁸?u/mL (equivalent to 3?mg roIFN-τ/mL) by day 5, when haemolymph was collected and stored frozen. Control haemolymph, collected from uninfected insects at an equivalent time, contained no antiviral activity. The roIFN-τ was partially purified by gel filtration column chromatography and the presence of roIFN-τ confirmed by western blotting. The biological activity of the partially purified roIFN-τ was tested in ewes. Treatment with roIFN-τ caused a significant delay in luteolysis confirming biological potency. The results demonstrate that this system can be successfully used to produce large quantities of roIFN-τ.     

Aerial and Aquatic Respiration in the River Crab Potamonautes Warreni Calman with Notes on Gill Structure

Summary

The oxygen consumption rate (?O₂) for Potamonauteus warreni Calman (= Potamon warreni (Calman) kept in 25 °C water was 34,4 μmol 1^?1 O₂ kg^?1 and after 72 hours in 98% R.H. air the rate was 31,9 μmol 1^?1 O₂ kg^?1 min^?1. The ?O₂ values for each of the two groups are not significantly different (P > 0,05). The partial oxygen tension of pre-branchial (v = venous) haemolymph (P_vCO₂) is 15,3 mm Hg in water and 13,0 mm Hg in air); partial carbon dioxide tension of pre-branchial (v) haemolymph (P_vCO₂) is 13,2 mm Hg in water and 13,0 mm Hg in air); the total carbon dioxide concentration in pre-branchial (v) haemolymph (C_vCO₂) tot. is 12,3 mmol 1^?1 in air and 13,9 mmol 1^?1 in water) are not significantly different for the two groups (P > 0,05). The haemolymph pH and the lactate concentration for crabs in water was found to be 7,51 and 0,38 mmol 1^?1 respectively. No significant differences were found in pre-branchial haemolymph oxygen tension, carbon dioxide tension, total carbon dioxide content, haemolymph pH, lactate level, chloride concentration, P₅₀ and haemocyanin-oxygen cooperativity in control crabs kept in water, and experimental crabs held in air for 72 hours. The chloride concentration, (327,0 mmol 1^?1) for crabs kept in water does not differ from that of crabs held in air for 72 hours but is at least 15% higher than the sodium concentration (255 mmol 1^?1) for crabs kept in water. The gill surface area is 520 mm² g^?1 wet body mass; on average 9,2 gill platelets (lamellae) can be found on a gill length of one millimetre. Each lamella is spaced 60–70 μm apart, each with a thickness of 30–40 μm. It is concluded that P. warreni may be described as a truly amphibious fresh-water crab.     

Experimental infection withRickettsiella phytoseiuli in adult femaleDermacentor reticulatus (Ixodidae): an electron microscopy study

Rickettsiella phytoseiuli naturally occurring inPhytoseiulus persimilis mites was cultivated in adult femaleDermacentor reticulatus ticks. It demonstrates all six known developmental stages: dense, intermediate, bacterial, giant, crystal-forming and small dark particles. These stages of rickettsiae were found in salivary glands, Malpighian tubules, synganglion, ovaries, tracheal complex, haemolymph, fat body and alimentary tract.Rickettsiella phytoseiuli did not infect the Gené's organ. It multiplied in female ticks in a manner similar to that in the typical host mite,P. persimilis.     

Metabolomic analyses of the haemolymph of the Asian citrus psyllid Diaphorina citri,the vector of huanglongbing

The Asian citrus psyllid Diaphorina citri Kuwayama is currently threatening the citrus industry by transmitting the causative agent Candidatus Liberibacter asiaticus (CLas) of huanglongbing. Multiplication of CLas in haemolymph of D. citri indicates that it contains the necessary nutrients for CLas. Although many studies examine D. citri, the haemolymph composition of this dangerous pest remains to be investigated. In the present study, the haemolymph of D. citri is collected using a nanolitre syringe after the removal of one of its forelegs. The haemolymph is either derivatized with methyl chloroformate (MCF) or trimethylsilyl (TMS) derivatizing reagent and analyzed with gas chromatography–mass spectrometry. Nineteen amino acids, two organic acids and seven fatty acids are detected in the haemolymph after MCF derivatization. More metabolites are detected after TMS derivatization. Sugars are the most abundant metabolites in the haemolymph. Glucose and fructose are the main monosaccharides. Trehalose and sucrose are the major disaccharides. Furthermore, three inositol isomers (myo‐inositol, scyllo‐inositol and chiro‐inositol) are detected in the haemolymph. Organic acids are found in low amounts, whereas phosphoric acid is found at a higher concentration. Twenty‐four nucleotides and sugar nucleotides, including ATP, ADP and AMP, are detected using high‐performance anion‐exchange chromatography. Adenine nucleotides are the most abundant nucleotides followed by uridine and guanosine. The adenylate energy charge for the haemolymph is 0.77. Our results show that many metabolites found in the citrus phloem sap are also found in the haemolymph of D. citri.     

Haemolymph composition in the larva of the dragonfly,Uropetala carovei

Analysis of the haemolymph composition of the semi-terrestrial larva of the dragonfly, Uropetala carovei, yielded the following data. The osmotic pressure of the haemolymph is equivalent to 174.0 mM/1 solution of sodium chloride, the pH 8.2, sodium ion concentration 152.0 mM/1, potassium 4.6 mM/1, calcium 3.8 mM/1, magnesium 3.9 mM/1, bicarbonate 27.6 mM/1, and chloride 121.0 mM/1; total ninhydrin-positive substances 58.7 mM/1 (of which the major components are alanine, glycine, glutamic acid, and leucine); total carbohydrates 8.9 ± 0.4 μg/μl haemolymph (trehalose 7.88 μg/μl and glucose 0.85 μg/μl). Twelve protein bands were found, one of which showed alkaline phosphatase activity, three, acid phosphatase, and four, esterase activity. The significance of these results is discussed.     

Morphological study of the reproductive system and ovarian growth of the univoltine bamboo borer,Omphisa fuscidentalis (Hampson, 1896) (Lepidoptera: Crambidae)

ABSTRACT

The female reproductive system and protein deposition in the ovaries during development have never been examined in the bamboo borer Omphisa fuscidentalis Hampson. The aim of this study was thus to study the morphology of the female reproductive system of each stage of development. The female reproductive system of the borer consists of a pair of ovary, oviduct and accessory glands. Each ovary is composed of four polytrophic ovarioles that connect to lateral oviducts, fused with a common oviduct. The size of the ovary in diapausing larvae for 9 months was determined. The length and width of the ovaries were the smallest in September larvae (0.343 ± 0.03 and 0.071 ± 0.01 mm, respectively). The ovaries were the largest during ovarian development in May (0.752 ± 0.08 mm long and 0.084 ± 0.01 wide). Additionally, ovarian size was significantly larger in adults than in pupae. The ovarian protein concentration of larvae in May was 0.59 ± 0.06 mg/ml and increased to 16.61 ± 7.5 and 37.42 ± 5.5 mg/ml in pupae (June) and adults (July), respectively. The results showed ovarian development in all life stages of this holometabolous insect, which has a longer life cycle than other lepidopterans.

Abbreviation: TEM: transmission electron microscope     

Toxicological impact of butralin,glyphosate-isopropylammonium and pendimethalin herbicides on physiological parameters of Biomphalaria alexandrina snails

ABSTRACT

Biomphalaria alexandrina snails have been used as bioindicators for freshwater qaulity and the effects of some herbicides such as butralin, glyphosate-isopropylammonium and pendimethalin). In the present study the effect of these three herbicides on snail biochemistry was examined. The results indicated that the herbicides increased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the haemolymph of B. alexandrina snails and significantly decreased total protein and albumin content. Light microscopical examinations of haemocytes monolayers of B. alexandrina snails showed three different cell types (small cells, granulocytes and hyalinocytes). All three herbicides caused abnormalities in cell shapes. Flow cytometric analysis of haemocytes from B. alexandrina demonstrated that circulating haemocyte populations could be divided into two main subtypes differing in their granularity (granulocytes or hyalinocytes) and size (large and small cells). In addition, the flow cytometric analysis showed that the total number of dead haemocytes in the haemolymph was significantly increased in treated groups compared to the control group. Phagocytosis in groups treated with the herbicides was highly significantly increased compared to the control indicating a very strong response of the treated snails. The results of the alkaline comet assay of DNA damage demonstrated that these herbicides have a genotoxic effect.     

Vitellogenin synthesis in blood-fed Aedes aegypti in the absence of the head,thorax and ovaries

A blood meal initiates oöcyte maturation in Aedes aegypti, and we have used rocket immunoelectrophoresis to investigate the function of midgut, ovaries, and head in the onset of vitellogenin synthesis. Non-blood-fed females and those fed blood (by enema) containing soybean trypsin inhibitor never contained vitellogenin. This demonstrates that the pressure of an undigested blood meal on stretch receptors of the midgut plays no role in the induction of vitellogenin synthesis, rather the stimulus is a digestion product of blood.When females were ovariectomized or decapitated and then fed blood, the haemolymph contained newly synthesized vitellogenin 24 h later. This was also demonstrated in isolated ovariectomized abdomens. Apparently, induction of vitellogenin synthesis does not require factors from either the head, thorax, or ovaries. When ovariectomy or decapitation was postponed after a blood meal, the level of vitellogenin in the haemolymph rose. Therefore, interaction of factors from the head and ovaries maintain the synthesis needed for oöcyte maturation.     

New structures of the O-specific polysaccharides of Proteus. 3. Polysaccharides containing non-carbohydrate organic acids

Four new Proteus O-specific polysaccharides were isolated by mild acid degradation from the lipopolysaccharides of P. penneri 28 (1), P. vulgaris O44 (2), P. mirabilis G1 (O3) (3), and P. myxofaciens (4), and their structures were elucidated using NMR spectroscopy and chemical methods. They were found to contain non-carbohydrate organic acids, including ether-linked lactic acid and amide-linked amino acids, and the following structures of the repeating units were established: 3)--L-QuipNAc-(13)--D-GlcpNAc-(16)--D-GlcpNAc-(1 (S)-Lac-(2–3) (1) 4)--D-GlcpA-(13)--D-GalpNAc-(14)--D-Glcp-(13)--D-Galp-(14)--D-GalpNAc-(1 L-Ala-(2–6) (2) 3)--D-GalpNAc-(16)--D-GalpNAc-(14)--D-GlcpA-(1 L-Lys-(2–6)--D-GalpA-(14) (3) 4)--D-GlcpA-(16)--D-GalpNAc-(16)--D-GlcpNAc-(13)--D-GlcpNAc-(1 (R)-aLys-(2–6) (4) where (S)-Lac and (R)-aLys stand for (S)-1-carboxyethyl (residue of lactic acid) and N-[(R)-1-carboxyethyl]-L-lysine (alaninolysine), respectively. The data obtained in this work and earlier serve as the chemical basis for classification of the bacteria Proteus.     

Effects of heavy metal stress on free amino acids in the haemolymph and proteins in haemolymph and total body tissue of Lymantria dispar larvae parasitized by Glyptapanteles liparidis

Heavy metal contamination of the forest pest insect Lymantria dispar (L.) (Lepidoptera; Lymantriidae), the gypsy moth, can alter its haemolymph composition, as has already been shown for carbohydrates and lipids in recent studies. L. dispar larvae are frequently parasitized by Glyptapanteles liparidis (Bouché) (Hymenoptera; Braconidae) larvae, which can—to some extent—regulate the population size of the pest insect. The parasitoids feed on the haemolymph of L. dispar larvae; hence, a different haemolymph composition of the host alters the trophic situation of the parasitoids. The aim of the present study was to investigate whether metal contamination also affects the concentrations of free amino acids in L. dispar haemolymph, and protein concentrations in their haemolymph and tissue. L. dispar larvae were parasitized on the first day of the second instar and then reared on diets contaminated with Cd, Pb, Cu or Zn at two concentrations each. Haemolymph and total body tissue of the larvae (fourth instar/third day) were analyzed. The concentrations of the free amino acids were elevated in five out of the eight contamination groups (Cd6, Pb4, Cu6, Cu10, Zn60), whereas haemolymph protein concentrations were significantly reduced in all contaminated individuals. The haemolymph protein concentration was 18 mg/ml in the control group and decreased to less than 10 mg/ml due to cadmium and zinc contamination at both concentrations and in the low copper contamination group. In contrast, total body proteins (136 g/mg dry weight in the control group) were elevated due to heavy metal stress. Analyses of haemolymph protein concentrations during the fourth instar demonstrated an increase of the proteins from day one to day four (followed by a decrease on the fifth day) in the control group and the cadmium contamination group. A steady increase of proteins from the first to the fifth day in the copper and zinc contaminated larvae indicated a retarded development in these groups. Thus, the present study along with other recent studies demonstrated, that heavy metal stress changes the concentrations of all main haemolymph compounds of L. dispar larvae.