A large amount of heme is produced upon digestion of red cell hemoglobin in the midgut of mosquitoes. The interaction between heme and the peritrophic matrix (PM) was studied in Aedes aegypti. By light microscopy, the PM appeared as a light brownish layer between the intestinal epithelium and the alimentary bolus. This natural color can be attributed to the presence of heme bound to the matrix. In histochemical studies, a diffuse peroxidase activity of the heme molecules was clearly observed between the erythrocytes and the PM at 14 h after the blood meal. This activity tends to increase and concentrate in the PM reaching its maximum thickness at 24 h after feeding. Most of the heme of the PM was found associated to with enormous number of small electron-dense granules. The amount of heme bound to the PM increased in parallel with the progression of digestion, reaching a maximum at 48 h after feeding, when 18 nmol of heme were found in an individual matrix. The association of heme with PM from insects fed with plasma is saturable, suggesting the existence of specific binding sites for hemin in the PM. Taken all together, our data indicate that the PM performs a central role in heme detoxification in this insect.
The insect myokinin (leucokinin-like) neuropeptide family includes peptides that have different physiological effects such as the induction of hindgut myotropic activity and stimulation of urine production. The C-terminal pentamer of myokinins Phe-X-(Ser/Pro/Ala)-Trp-Gly-amide [X=Phe, His, Asn, Ser or Tyr], had been previously determined as the minimum fragment able to elicit a functional response. The receptor(s) for these insect neuropeptides has not yet been identified. In order to characterize the Malpighian tubule leucokinin-like peptide receptor(s) from the yellow fever mosquito (Aedes aegypti), a leucokinin photoaffinity analogue (LPA) of sequence dAla-dTyr-Bpa-dLys-Phe-Phe-Ser-Trp-Gly-amide was designed based on structure/activity relationships for leucokinins. LPA caused depolarization of the transepithelial voltage (TEV) in female Malpighian tubule, confirming the activity of the peptide. The effective concentration to give half the maximum depolarization (EC(50)) was 17 nM. The (125)I-LPA was then used to characterize leucokinin binding proteins in female Malpighian tubule membranes. It specifically labeled and saturated a protein(s) of about 54 kDa as shown by SDS-PAGE/autoradiography and by competition experiments with excess unlabeled leucokinin analogues. (125)I-LPA bound to the 54 kDa protein(s) with a K(d) value of 13+/-3 nM in agreement with the EC(50) for the TEV bioassay. Altogether these data suggest that the 54 kDa protein is an Aedes-leucokinin receptor. This is the first characterization of an insect leucokinin receptor and reveals that LPA is a powerful tool to label insect myokinin receptors.
Previous studies reviewed here have indicated that juvenile hormone (JH) specifically binds to a 29 kDa protein in epidermal nuclei from Manduca sexta larvae. Also, a 29 kDa nuclear protein that showed the same developmental pattern bound specifically to a larval cuticle gene LCP14. These results indicate that the 29 kDa nuclear protein is likely a JH receptor. Two retinoids (SRI 5942-64 and Ro 13-6298) were found to be weak JH mimics with ED50s 60–100 times higher than that of JH III in the black Manduca larval bioassay. A genomic clone (Manduca “RAR”) then was isolated using the human retinoic acid receptor (hRAR) cDNA and the homologous region was sequenced. Thirteen out of 14 amino acids constituting the C-terminal half of the second zinc finger were identical in Manduca “RAR” and hRAR. Manduca “RAR” selected two mRNAs (3.8 and 4.5 kb) that are expressed at the peaks of the ecdysteroid titer during both the larval and the pupal molts, but not during the intermolt periods. When pieces of integument from day two 4th instar larvae were cultured with 4 × 10−6 M 20-hydroxyecdysone (20HE), Manduca “RAR” mRNA increased 13–15-fold by 6 h, then decreased after 12 h in the continuous presence of 20HE. The presence of 3 × 10−6 M JH slowed the rate of induction by 20HE. Thus, the “RAR” gene product is likely not the 29 kDa JH receptor but rather a transcriptional regulatory factor whose presence during a molt is modulated by JH.
The present study confirms previous reports of the presence of (Na+ + K+)-ATPase and anion-stimulated ATPase activity in Malpighian tubules of Locusta. In addition, the presence of a K+-stimulated, ouabain-insensitive ATPase activity has been identified in microsomal fractions. Differential and sucrose density-gradient centrifugation of homogenates has been used to separate membrane fractions which are rich in mitochondria, apical membranes and basolateral membranes; as indicated by the presence of succinate dehydrogenase and the presence or absence of non-specific alkaline phosphatase activity, respectively. Relatively high specific (Na+ + K+)-ATPase activity was associated with the basolateral membrane-rich fractions with only low levels of this activity being associated with the apical membrane-rich preparation. K+-stimulated ATPase activity was also associated, predominantly, with the basolateral membrane-rich fractions. However, comparison of the distribution of this activity with that of the (Na+ + K+)-ATPase suggests that the two enzymes did not co-separate. The possibility that the K+-stimulated ATPase was not associated with the basolateral plasma membrane is discussed.Anion-stimulated ATPase activity was found in the apical and basolateral membrane-rich fractions and in the fraction containing mainly mitochondria. Nevertheless, the fact that this bicarbonate-stimulated activity did not co-separate with succinate dehydrogenase activity suggests that it was not exclusively mitochondrial in origin. These results are consistent with physiological studies indicating a basolateral (Na+ + K+)-ATPase but do not support the K+-stimulated ATPase as a candidate for the apical electrogenic pump. The possible role of the bicarbonate-stimulated ATPase activity in ion transport across both the basolateral and apical cell membranes is discussed.
The effect of some inhibitors and activators of mammalian DT-diaphorase on diaphorase-1 (DIA-1) and diaphorase-2′ (DIA-2′) purified from Drosophila virilis was studied. The inhibitors and activators changed the activity of these diaphorases in a different way, revealing a similarity between mammalian DT-diaphorase and D. virilis DIA-1 on the one hand and on the other between the D. virilis DIA-1 and the diaphorase purified from Bombyx mori eggs. These effects also confirm the independent genetic control of DIA-1 and DIA-2′ in D. virilis and make possible the differentiation of these diaphorase activities in crude enzyme extracts.
Two carboxylesterases (TE-I and TE-II) from the mid-gut of the termite Odentotermes horni. W., have been purified to apparent homogeneity by means of ammonium sulfate fractionation, gel-permeation on Sephadex G-75 and Ultragel AcA-34 and ion-exchange chromatography on DEAE-Sephacel. The homogeneity of the preparations was confirmed by polyacrylamide gel electrophoresis (PAGE), gel-electrofocussing and Ouchterlony double immunodiffusion. The apparent molecular weights determined by gel-permeation on Sephadex G-200 was 78,350 and by SDS-PAGE 78,500. In the presence of 2-mercaptoethanol, the proteins were split into two subunits of equal size with subunit molecular weight of about 40,000. The enzymes were found to have a Stokes radius of 3.35 nm. The amino acid analysis of the purified enzymes revealed the presence of a greater number of acidic and neutral amino acids than in other insect carboxylesterases. The isoelectric points of the enzymes, TE-I and TE-II, were 5.4 and 5.6, respectively. The two enzymes were inhibited by organophosphates. The substrate preference and inhibition patterns classify these enzymes as carboxylesterases (EC 3.1.1.1), but the physiological function is unknown. The apparent Km, Vmax, ki and I50 values are listed. The product inhibition studies with the enzymes revealed the linear competitive inhibition with acetate and linear non-competitive inhibition with 1-naphthol. In addition, optimum temperature and pH, thermal stability, effect of temperature and pH on Km and Vmax of the two enzymes were determined.
Ca2+-dependent proteinase activity was detected in the neural tissue of the honeybee Apis mellifera. DEAE-chromatography revealed two Ca2+-dependent proteinase activities, with elution positions corresponding to vertebrate calpain I and II. Both activities yielded apparent M(r) = 80,000, had a strict requirement for Ca2+ and a SH-reducing agent. PMSF and trypsin inhibitor did not affect the proteinase activities. Alkylating agents and mercaptide forming compounds as well as the calpain inhibitor calpastatin purified from bovine brain inhibited the activity. The natural inhibitor protein detected in the neural tissue of the honeybee showed properties equal to calpastatin from other sources. All observed properties are in accordance with those of the Ca2+-dependent proteolytic system calpain-calpastatin.
Cuticle proteins are thought to be important in defining the structural and functional differences occurring in insect cuticle. In order to explain and better understand the structural similarities among the cuticle proteins of the cotton boll weevil, Anthonomus grandis Boheman, described in a previous study (Stiles and Leopold, 1990, Insect Biochem.20, 113–125) three series of monoclonal antibody producing hybridoma cell lines were produced. Larval, pupal or adult cuticle proteins were used as antigens. While some of the monoclonal antibodies were specific for one or two cuticle proteins from a single developmental stage, the majority showed multiple cuticle protein binding patterns on Western blots. To determine whether this cross-reaction was due to common oligosaccharide chains bound to the proteins, lectins were used to probe Western blots. Many of the cuticle proteins were found to be glycosylated. The majority of the Con A reactive carbohydrate could be removed from the protein by N-glycosidase F digestion (specific for N-asparagine linked carbohydrate). N-glycosidase F digestion did not reduce the multiple cross-reactions of the monoclonal antibodies, nor did periodate oxidation of the CP. The carbohydrate remaining after enzyme digestion is presumably O-linked to serine/threonine.
Follicle cells were isolated from their oocytes using 0.15% collagenase and low speed centrifugation. Incubation of the follicle cell pellet with [3H]2-deoxyecdysone yielded its 22-phosphate ester conjugate. Addition of ATP/Mg2+ or GTP/Mg2+ to follicle cell homogenate incubated with 2-deoxyecdysone increased the phosphotransferase activity by 4-fold for ATP and 2-fold for GTP. In the case of intact cells, only ATP was effective. The enzyme had a cytosolic subcellular localization and its Km for 2-deoxyecdysone was 3 μM. The phosphotransferase activity required the presence of both ATP and Mg2+, and had an apparent Km for ATP of 0.83 mM, with maximum activity being obtained in the presence of 10 mM Mg2+. The activity was strongly inhibited in the presence of Ca2+ with IC50 = 1 mM. The reaction rate was linear for 10 min and with increasing protein concentrations up to 1 mg/ml. Optimal pH was about 7.4 and the optimal temperature was 37°C. The phosphotransferase activity survived freezing at −20°C, but was totally abolished by heat at 60°C for 10 min. Investigation of the variation in activity of the phosphotransferase during ovarian development revealed a peak at the end of oogenesis in excellent agreement with the titre of ecdysteroid 22-phosphates found in the oocytes just before chorionation and egg-laying.
The transparent accessory reproductive gland of Rhodnius prolixus synthesizes and accumulates a variety of polypeptides. Ouchterlony immunodiffusion demonstrates that the hemolymph contains proteins which react with polyclonal antibodies against extracts of transparent accessory glands. Accessory glands and hemolymph contain a 170 kDa polypeptide with similar mobility on SDS-polyacrylamide gel electrophoresis. This polypeptide reacts with antibodies against extracts of accessory glands. Surgical removal of the accessory glands prevents the appearance of the 170 kDa polypeptide in the hemolymph. In vivo labeling of accessory gland proteins with a mixture of [14C]amino acids demonstrates that the newly synthesized TARG polypeptide appears in the hemolymph between days 2 and 3 after feeding. It is concluded that a specific polypeptide which is synthesized in the transparent accessory gland is exported to the hemolymph.
The synthesis and secretion of egg-specific protein (ESP) were investigated using the follicle cells isolated from the developing ovary of the silkworm, Bombyx mori. The follicle cells were isolated manually from a follicle into a cell layer by thoroughly extruding the oocyte contents through a small hole. Whole follicles and isolated follicle cells were incubated in vitro with [35S]methionine, and ESP and its precursors were immunochemically isolated using antiserum raised to ESP. The isolated follicle cells incorporated label into ESP but the incorporation rate was about one-fifth of that found in whole follicles. About 20% of the total radioactivity of ESPs were recovered from the incubation medium of the isolated follicle cells while only trace activity (<2%) was found in the incubation medium of whole follicles. These results clearly showed that follicle cells synthesize and release ESP to be taken up by the developing oocyte.
Phospholipase A2 from the venom of the European honeybee (Apis mellifera) consists of three isoforms with approximate molecular masses of 16, 18, and 20 kDa, respectively, as deduced from SDS-PAGE. These variants, termed PLA-16, PLA-18, and PLA-20, were isolated by lectin affinity chromatography and preparative polyacrylamide gel electrophoresis. The amino acid sequences of the N-terminal peptide portions of all three isoforms, as assessed by automated Edman degradation, were identical with that expected for honeybee phospholipase A2. Sequencing data suggest that, while PLA-18 and PLA-20 carry oligosaccharide residues at asparagine-13, PLA-16 has escaped glycosylation during biosynthesis. Release of the carbohydrate from PLA-18 and PLA-20 with peptide: N-glycosidase F abolished the molecular mass differences between the three isoforms of phospholipase. Differences in sensitivity to α-mannosidase and monosaccharide composition of PLA-18 and PLA-20 further indicate that their electrophoretic separation is based on structural features of the N-glycosidically linked oligosaccharide. Noticeably, PLA-20 contains N-acetylgalactosamine, a sugar not having yet been described as a constituent of insect glycoproteins.
The hemolymph juvenile hormone (JH) titer of third through fifth stadia Trichoplusia ni parasitized by the polyembryonic parasitoid, Copidosoma floridanum, was measured by radioimmunoassay and compared to the titers of unparasitized larvae. The JH titer of parasitized larvae fluctuated from 28 pg/μl to undetectable levels. Maximum levels of hormone were present at ecdysis to the fourth and fifth stadium, and at the prepupal stage. Qualitatively, similar fluctuations were observed in unparasitized larvae. However, the titers in unparasitized larvae were much lower than those of parasitized larvae in the third and early fourth stadia, and the titer fell to undetectable levels in the fifth stadium 24 h earlier (48 h) than in parasitized larvae (72 h). Preventing the JH titer from falling during the fourth and fifth stadia by topical application of (RS)-methoprene or JH II had a juvenilizing effect on parasitized T. ni, and inhibited C. floridanum embryo morphogenesis. The effect of exogenous methoprene and JH on C. floridanum development depended on timing of application and dosage. Application of 100 pmol per day of methoprene beginning at 2 h of the host fourth stadium, prior to the large drop in the endogenous JH titer, inhibited morphogenesis in the majority of C. floridanum embryos. Application of methoprene at later times of host development did not inhibit morphogenesis although other developmental alterations were observed. The potential significance of host JH and ecdysteroid titers on polyembryonic development are discussed.
Hemolymph proteins of the Eastern subterranean termite, Reticulitermes flavipes (Isoptera, Rhinotermitidae, Rhinotermitinae) were examined from sterile and reproductive castes using native and denaturing polyacrylamide gel electrophoresis (PAGE). A high-mass protein (ca. 700 kDa) exhibited specific, JH III-displaceable photoaffinity labeling with [3H]EFDA, a diazoacetate analog of JH III. This protein was present in each termite caste, and had the characteristics of a glycosylated lipoprotein, i.e. a lipophorin. The JH-binding subunit of this protein showed a molecular size of 230 kDa using SDS-PAGE. The differences in the hemolymph proteins present in the soldiers, workers, larvae, nymphs, and replacement reproductives of this rhinotermitid are discussed.
Inclusion of glucose or trehalose in the medium during the incubation of locust fat body in vitro leads to a reduction of the relative amount of active (AMP-independent) glycogen phosphorylase. The presence of adipokinetic hormone (AKH I) results in a rapid activation of phosphorylase, reaching a maximum within 5 min. This AKH effect is highly dependent on added Ca2+, and requires ⩾ 1 mM Ca2+ for maximal enzyme activation. Ca2+ alone has no effect on phosphorylase activity, but it does activate the enzyme when the ionophore A23187 is also included in the medium. In a cell-free system from locust fat body the activation of endogenous phosphorylase by phosphorylase kinase is stimulated by Ca2+. Activity of the latter enzyme can be increased further by high doses of calmodulin. Both in the presence and in the absence of external calmodulin, the calmodulin antagonist trifluoperazine has an inhibitory effect on phosphorylase kinase. Results are discussed in relation to the possible mechanisms underlying hormonal control of glycogenolysis.
Traditionally, endocrinologists have discovered hormones by identifying, thanks to a suitable bioassay, which effectors are involved in the control of a given biological function. By contrast, "Reverse Endocrinologists" first obtain the evidence for "hormones" by using available biochemical methods, and then try to determine what their function may be, usually by analogy with what has been formerly established in other animal groups by "Classical Endocrinologists".This problem is exemplified by ecdysteroids and vertebrate-type steroids, that are widespread among invertebrates. In many cases, it is not known whether these compounds are endogenously synthesized or if they originate from the food. Little is known concerning their possible hormonal functions, with the exception, of course, of ecdysteroids in Arthropods, where they were originally discovered.Up to now, searching for the possible hormonal function of molecules isolated with "heterologous" tools, i.e. performing "reverse endocrinology", has appeared a disappointing approach for the identification of physiological regulators.
This study was undertaken to determine the processing of vitellogenin (Vg) and the role of juvenile hormone (JH) in the regulation of vitellogenesis in the tick Ornithodoros parkeri. Ticks usually require a blood meal to induce vitellogenesis. However, we have shown that a pyrethroid, cypermethrin (CyM), can stimulate Vg synthesis in unfed Ornithodoros moubata females. Vg concentration and synthesis were analyzed by SDS-PAGE spotting-scanning and fluorography using [35S]-methionine. Although unfed females show high titers of Vg in the hemolymph, this is not due to new synthesis. Vg synthesis stimulated by engorgement increases beginning on day 2 after engorgement and reaches a maximum level on day 8. Vg is synthesized in the fat body, secreted into the hemolymph and then processed and incorporated into the ovaries as vitellin. JH I, II and III, methoprene (JHA), and CyM were topically applied to unfed females and Vg synthesis analyzed on day 5 by fluorography. JH and JHA did not stimulate Vg synthesis. CyM stimulated Vg synthesis but not ovarian development. These preliminary results indicate that JH does not function in the regulation of vitellogenin synthesis in this species.
Mechanical disruption of day 7 fifth instar Manduca sexta larval prothoracic glands prior to in vitro incubation with [3H]cholesterol resulted in a dramatic 10-fold increase in its conversion to [3H]7-dehydrocholesterol (20–50%), when compared to intact gland incubations (2–5%). Both procedures resulted in a 0.5–2% conversion to [3H]ecdysteroids. Endogenous cholesterol levels also decreased by this same 20–50% during the incubation, suggesting that the added tracer [3H]cholesterol was equilibrated with the total endogenous cholesterol pool. Glands from earlier fifth instar larvae were capable of similar conversion of [3H]cholesterol to [3H]7-dehydrocholesterol but without concomitant conversion to [3H]ecdysteroids, while in day 7 glands, conversion to [3H]ecdysteroids was temporally correlated with both the in vitro secretory activity of intact glands and the endogenous hemolymph ecdysteroid titer. These data suggest that the rate-limiting step in ecdysteroid biosynthesis occurs after the synthesis of 7-dehydrocholesterol. In addition to 7-dehydrocholesterol and ecdysteroids, four intermediate polarity metabolites were detected following the incubation of disrupted prothoracic glands. One, M1, appears to be an immediate precursor of ecdysone and 3-dehydroecdysone. Another, M3, while not a precursor of the ecdysteroids, may be a degradation product of a proposed epoxide intermediate of 7-dehydrocholesterol. A hypothetical scheme for the biosynthesis of ecdysteroids from cholesterol is presented.
Secretion from female reproductive accessory glands of the dipteran Ceratitis capitata was found to have antibacterial properties against E. coli. At least two basic polypeptides with mol. wt 15.5 and 4.7 kDa respectively, were identified as responsible for such activity. Furthermore, the 15.5 kDa protein is active against a number of Gram-positive and -negative bacterial strains. Lysozyme activity is also present in the secretion.
The following hydrocarbon classes have been identified in insect cuticular lipids: A, n-alkanes; B, olefins, comprising B1, alkenes, B2, alkadienes and B3, alkatrienes; and C, methylalkanes, comprising monomethyl-, dimethyl-, trimethyl- and tetramethylakanes. Biosynthetic pathways have been described for most of the main classes and hydrocarbon composition may be considered a product of an insect's genotype and thus available for taxonomic use. Olefins and methylalkanes usually occur as isomeric mixtures and hydrocarbon composition can be complex. This provides the chemotaxonomist with many potential characters. Preliminary investigations show that hydrocarbon composition not only separates species but also reflects links between species and between higher taxa. A hydrocarbon chemotaxonomy which also takes into account the elongation-decarboxylation pathways used by insects to synthesise their hydrocarbons could provide information on the evolution of hydrocarbon biosynthesis in the insects.
