Composition en glucides conjugués de l'hémolymphe de Blaberus craniifer: Variations au moment de l'ecdysis imaginale

Analysis of macromolecular carbohydrates performed by gas liquid chromatography on Gregarina blaberae haemolymph showed the presence of hexoses (galactose, mannose, glucose), methyl-pentose (fucose), and hexosamines (N-acetylgalactosamine, N-acetylglucosamine). No trace of pentose or sialic acid or uronic acid was found. Mannose was the main neutral sugar. A change in the molar ratio of mannose consisting of an enrichment of female haemolymph occurred during larval-adult ecdysis. There was a parallel increase in glycoprotein staining with periodate-fuchsin after cellulose acetate electrophoresis of female haemolymph.     

Haemagglutinin activity in Acrididae (grasshopper) haemolymph

The haemolymph of Acrididae causes haemagglutination of human and animal erythrocytes. Thirteen of seventeen species tested had detectable activity and gave agglutination titres in the range 2–64, Melanoplus bivittatus, and M. sanguinipes showed greatest activity. Haemagglutinin activity is continuously present in male and female insects from 4th instar and throughout adulthood. Females contain slightly more activity than do males. M. sanguinipes haemolymph agglutinates rabbit, calf, human (all ABO types) guinea pig, mouse, chicken, cat, pig and sheep erythrocytes. Rabbit red cells are agglutinated most strongly and sheep and chicken cells least. M. sanguinipes haemolymph also agglutinates the protozoan Nosema locustae, a natural grasshopper pathogen. Preabsorption of haemolymph with different erythrocyte types selectively removes haemagglutinin activity suggesting the presence of multiple or heteroagglutinins. M. sanguinipes haemagglutinin is inhibited by glycoproteins, simple carbohydrates and carbohydrate derivatives. The inhibitory pattern is complex and among the sugars tested only mannose and derivatives of mannose are exclusively non-inhibitory. Haemolymph haemagglutinin activity is destroyed by heat and EDTA. It is totally precipitated by dialysis against water and may be partially recovered in phosphate or Tris buffer. Activity is stable in frozen haemolymph.     

A study of uptake of radiolabeled host proteins and protein synthesis during development of eggs of the endoparasitoid,Microplitis croceipes (Cresson) (Braconidae)

The uptake of radiolabeled haemolymph and fat body proteins from fourth instar larvae of Heliothis zea (Boddie) by eggs of Microplitis croceipes (Cresson) was examined by SDS-polyacrylamide gel electrophoresis and by autoradiography. None of the ¹²⁵I-labeled haemolymph proteins was detected in eggs exposed to the proteins in vivo. Although several of the proteins were observed in eggs incubated with the labeled proteins in vitro, none of these proteins was degraded or resynthesized into new structural proteins during development of the embryo. Similarly, no significant uptake of labeled fat body proteins by the eggs could be detected in vitro. On the other hand, protein synthesis measured by incorporation of [³⁵S]methionine occurred throughout egg development. Proteins were synthesized at least 1 hr after the egg was deposited into the host. The protein patterns of eggs on one-dimensional SDS gels were complex and ranged in size from less than 18,500 to more than 330,000 mol. wt. The protein band patterns of the newly synthesized proteins showed some qualitative differences at 1–8, 16–32 and 40 hr after egg deposition. We conclude that eggs do not absorb or utilize the host apoproteins (or degradation products) but instead synthesize proteins de novo from free amino acids in the host haemolymph.     

Changes in haemolymph proteins of the fall armyworm, Spodoptera frugiperda (J. E. Smith), associated with parasitism by the braconid parasitoid Cotesia marginiventris (Cresson)

Changes in haemolymph proteins of the fall armyworm, Spodoptera frugiperda, associated with parasitism by the parasitoid Cotesia (= Apanteles) marginiventris were monitored by sodium dodecyl sulphate polyacrylamide gel electrophoresis. As early as hour 4 after parasitization treatment, several electrophoretically slow-migrating, high-molecular-weight proteins were detected in the host's haemolymph. These proteins were detected earlier in haemolymph from parasitized larvae than in haemolymph from control larvae, and their concentrations were higher in heavily parasitized host larvae (≥ 3 eggs/host) than in lightly parasitized larvae (1 egg/host). Additionally, unique proteins that migrated electrophoretically with bovine serum albumin appeared in the haemolymph of parasitized larvae at hour 8 after parasitization treatment and were evident in haemolymph collected through to hour 64.     

Haemagglutinin activity in the haemolymph of individual Acrididae (grasshopper) specimens

Twenty-four individual grasshopper specimens representing four Melanoplus spp. contained similar broad-spectrum haemolymphatic haemagglutinin. The agglutinin activity showed highest titre toward human ABO and rabbit cells among nine types of erythrocytes tested. Titre values differed between individual insects but agglutination specificity toward different erythrocytes was similar. Agglutination of type-O red cells by individual grasshopper haemolymph was inhibited by 34 of 41 tested carbohydrates, carbohydrate derivatives, alcohols and chelating agents. Individual insects showed similar patterns of haemagglutination inhibition. Non-inhibitory compounds were mannose and mannose derivatives (excepting N-acetylneuraminate), several glucose derivatives, amino sugars and ethanol. The observations indicated that haemolymph from an individual grasshopper contained complex heteroagglutinin activity similar to that found in haemolymph pooled from several insects. Determination of minimal effective inhibitor concentrations confirmed the presence of heteroagglutinin activity primarily directed toward galactose and glucose and related α-linked glycosidic derivatives.     

In vitro culture of hymenopterous larval endoparasitoids

The solitary endoparasitoids Cotesia (= Apanteles) marginiventris and Microplitis croceipes, which attack noctuid larvae, were successfully cultured from post-germband stage eggs to fully grown first-instar larvae in completely artificial (but undefined) media. However, growth rates were slower than in vivo, and while some larvae of each species attained apparent competence to moult (based upon characteristic size and form), none did so.Up to 75% of pregermband M. croceipes eggs developed and hatched when co-cultured with fat body from non-parasitized larvae of the habitual host, Heliothis zea. Heat-treated haemolymph plasma from a non-permissive host, Manduca sexta, also fostered development by ca. 40% of newly laid M. croceipes eggs, but plasma from H. zea larvae was not beneficial. Growth rates of C. marginiventris and M. croceipes larvae were not improved by haemolymph plasma from their habitual hosts (Spodoptera frugiperda and H. zea, respectively).Heat-treated foetal bovine serum was growth-promoting for post-germband eggs and larvae of both parasitoids. Treatment of foetal bovine serum by charcoal adsorption (to remove sterols and peptide hormones) diminished its value as a supplement. Two major proteins of the serum, foetuin and albumin, also promoted survival and growth for both parasitoid species. The potential relevance of these findings to in vitro rearing of endoparasitoids is discussed.     

Influence of mannose on the apoplasmic retrieval systems of source leaves

Experiments were conducted in which d-mannose was supplied to mature Beta vulgaris L. (sugar beet) leaves, via the transpiration stream, to perturb photosynthetic carbon allocation by sequestering cytosolic Pi. Biochemical and enzymic analyses conducted on this tissue indicated that mannose 6-P was present, that it was only slowly metabolized, and that after a 24-hour pretreatment sugar metabolism was slightly perturbed. However, sucrose retrieval by the mesophyll tissue was greatly impaired in 24-hour mannose-pretreated tissue, a response which was due in part to mannose acting as an osmoticum. Inhibition of glucose, fructose, and arginine uptake into mannose-treated sugar beet leaf discs indicated that mannose may elicit a general perturbation of all membrane transport processes. This conclusion was supported by our finding that sucrose efflux was increased from mannose-treated tissue. Analysis of adenine nucleotide levels showed that whereas these levels declined over the first 3 to 6 hours of the mannose treatment, by 24 hours they had recovered to near control values. Similar experiments conducted on Nicotiana rustica indicated that whereas mannose 6-P was present in mature leaves, it remained at a much lower level than that found in sugar beet. Sucrose uptake into N. rustica was insensitive to mannose pretreatment. However, glucosamine treatment, which is also thought to sequester cytosolic Pi, inhibited sucrose uptake in both N. rustica and B. vulgaris. Further, experiments conducted on N. tabacum L. var Xanthii showed that mannose caused an inhibition of sucrose uptake, indicating that a range of sensitivity to mannose exists between closely related species. These results are discussed in terms of possible mechanisms of inhibition.     

Physiological pattern and further characterization of the haemolymph violet carotenoprotein of the fly Rhynchosciara americana

• 1.1. An electrophoretic purification procedure for the haemolymph violet carotenoprotein of R. americana was described. The purified protein was used for obtaining a specific antiserum.
• 2.2. This carotenoprotein contains: (1) a high weight percentage of glutamic acid, threonine and proline and a low weight percentage of histidine; (2) mannose and/or glucose as suggested by the interaction with concanavalin A; (3) phosphoryl groups.
• 3.3. The concentration of the violet carotenoprotein in the haemolymph is approximately constant during all the life cycle of R. americana.
• 4.4. The haemolymph of four species of Rhynchosciara genus shows the presence of proteins immunologically related with the R. americana violet carotenoprotein.

     

The relationship between nutrition,vitellogenin, vitellin and ovarian development in the housefly, Musca domestica L.

The effect of adult nutrition on oögenesis during the first gonotropic cycle was studied in three strains of the housefly, Musca domestica. Two of the strains were anautogenous and the third was autogenous. In these strains, three subunits (51, 43 and 42 kdaltons) of vitellogenin and vitellin were electrophoretically identical using SDS-PAGE electrophoresis for haemolymph proteins of vitellogenic females and for egg extracts. Each developmental stage of the ovary in individual females flies of both autogenous and anautogenous strains fed on either sugar or protein clearly reflected the appearance of electrophoretic bands for vitellogenin and vitellin. Using immunological analysis, a very small amount of vitellogenin was detectable in the haemolymph of previtellogenic flies. The highest level of vitellogenin appeared in the haemolymph at the middle of vitellogenic phase and reached about 25% of the total haemolymph protein. There were differences in vitellogenin concentration in females with mature eggs between the two anautogenous strains: vitellogenin was not detectable in one strain, and the other showed 30% of the maximal level.     

Effects of the parasitization of a braconid, Apanteles, on the blood of its host, Pieris

Parasitization of a braconid wasp, Apanteles glomeratus, of larvae of a common cabbage butterfly, Pieris rapae crucivora, caused changes in differential haemocyte count (DHC), total haemocyte count (THC), and encapsulative capacity against dead eggs of Apanteles in the fourth and fifth instar host larvae.However, no correlation could be found between the number of Apanteles eggs deposited and THC of the middle fourth instar host larvae or between the number of parasitoid larvae and specific gravity of the haemolymph from the late fifth instar host larvae.From the changes in DHC and in THC of both non-parasitized and parasitized Pieris larvae, an increase in the number of plasmatocytes of non-parasitized Pieris larvae in the early fourth instar period was supposed to be due to transformation of prohaemocytes into plasmatocytes, and a low population of plasmatocytes of parasitized larvae in the comparable period was assumed to be due to a suppression of transformation of prohaemocytes by some factor released from the parasitoid eggs.Failure of the parasitized fourth instar Pieris larvae to encapsulate injected dead eggs of Apanteles indicated that the parasitoid embryos were, in some way, actively inhibiting the encapsulation reactions of the host.The increase in THC of the parasitized fifth instar larvae could not be ascribed to a decrease in the volume of host haemolymph. Rather it could be interpreted by a suppression of adhesive capacity of haemocytes in the host haemocoel to tissue surfaces.Reduced encapsulative capacity of the parasitized fifth instar larvae might be attributed either to a depression of the adhesive activity of plasmatocytes resulting from a depletion of energy source for haemocytes in the host haemolymph by parasitization, or from an active suppression of adhesiveness of the plasmatocytes by secretions from ‘giant cells’ (teratocytes) originated from the parasitoid.     

Temporal variation of vitellogenin synthesis in Ectatomma tuberculatum (Formicidae: Ectatomminae) workers

Workers of the ant species Ectatomma tuberculatum (Ectatomminae) have active ovaries and lay eggs that are eaten by the queen and larvae (trophic eggs). Vitellogenins are the main proteins found in the eggs of insects and are a source of nutrients. The aim of this study was to characterize the period of vitellogenin production in workers of E. tuberculatum. The vitellogenin was identified from queen and worker eggs by SDS-PAGE. Anti-vitellogenin antibodies were obtained and used to detect this protein in the fat body and haemolymph of workers at different ages. Vitellogenin from E. tuberculatum consists of two polypeptides of 31 and 156 kDa. In the eggs of queens, the 156 kDa polypeptide is cleaved into two subunits of 36 and 123 kDa. The analysis of the haemolymph of workers showed that the secretion of vitellogenin varies with age. The secretion is initiated around the fifth day after emergence, with peak production from days 20 to 60, and stops around day 100. The variation in production is related to the different activities performed by the workers within the colony, suggesting that vitellogenin may have an important role in maintaining age polyethism.     

Separation procedure and sugar composition of oligosaccharides in the surface glycoprotein of friend murine leukemia virus

The sugar composition of the surface glycoprotein from Friend murine leukemia virus was determined by gas-liquid chromatography of the alditol acetates and by the thiobarbituric acid method, respectively. N-Acetylglucosamine, mannose, galactose, sialic acid and fucose were found in a molar ratio around 15.2:11.6:7.4:3.3:1.0. Ten ogligosaccharide fractions were obtained from glycoprotein preparations by a suitable sequence of degradation (with pronase, endo-β-N-acetylglucosaminidase H, neuraminidase, and by hydrazinolysis) and separation procedures (concanavalin A-affinity chromatography and gel filtration). The qualitative sugar composition of these fractions was analyzed by in vivo labelling with D-[6-³H]glucosamine, D-[2-³H]mannose, D-[6-³H]galactose, or L-[6-³H]fucose, and their molecular weights were estimated from the gel elution volumina. Four fractions of N-glycosidically linked oligosaccharides of the oligomannosidic (‘high mannose’) type oligomannosidic₇-oligomannosidic₁₀, about seven to ten sugar residues), two of the mixed (M₁₁ and M₁₂), and four of the N-acethyllactosaminic (‘complex’) type (N-acetyllactosaminic₉, probably nine sugar residues; (N-acetyllactosaminic_a-N-acetyllactosaminic_c, size unknown) were thus identified.     

Region of Flo1 Proteins Responsible for Sugar Recognition

Yeast flocculation is a phenomenon which is believed to result from an interaction between a lectin-like protein and a mannose chain located on the yeast cell surface. The FLO1 gene, which encodes a cell wall protein, is considered to play an important role in yeast flocculation, which is inhibited by mannose but not by glucose (mannose-specific flocculation). A new homologue of FLO1, named Lg-FLO1, was isolated from a flocculent bottom-fermenting yeast strain in which flocculation is inhibited by both mannose and glucose (mannose/glucose-specific flocculation). In order to confirm that both FLO1 and Lg-FLO1 are involved in the yeast flocculation phenomenon, the FLO1 gene in the mannose-specific flocculation strain was replaced by the Lg-FLO1 gene. The transformant in which the Lg-FLO1 gene was incorporated showed the same flocculation phenotype as the mannose/glucose-specific flocculation strain, suggesting that the FLO1 and Lg-FLO1 genes encode mannose-specific and mannose/glucose-specific lectin-like proteins, respectively. Moreover, the sugar recognition sites for these sugars were identified by expressing chimeric FLO1 and Lg-FLO1 genes. It was found that the region from amino acid 196 to amino acid 240 of both gene products is important for flocculation phenotypes. Further mutational analysis of this region suggested that Thr-202 in the Lg-Flo1 protein and Trp-228 in the Flo1 protein are involved in sugar recognition.     

Côntrole neuroendocrine des protéines sanguines vitellogenes d'Anacridium aegyptium sain et parasite

The changing patterns of haemolymph proteins were followed in male and female adults of normal and parasitized Anacridium aegyptium during diapause (autumn, winter) or during activity (spring) of their endocrine system without or with electrostimulations of the pars intercerebralis (PI).The haemolymph protein concentration is high in winter and decreases in spring. It is comparatively depleted in locusts infected by the fly Metacemyia calloti. However, the depletion is significant only in ‘castrated’ females.Fifteen protein fractions were resolved by polyacrylamide disk gel electrophoresis in haemolymph of normal and infected locusts during diapause and activity. Some fractions decrease in quantity during activity in males, normal females, and parasitized females with complete ovarian development. One fraction disappears in females with mature eggs and seems correlated with formation of the eggshell. Eight others protein fractions exhibit electrophoretic mobility identical to the 7 protein fractions of homogenates of eggs. There is little doubt that these haemolymph protein fractions are involved in yolk synthesis and are thus ‘vitellogenic’. One of these ‘vitellogenic’ fractions (band 6) is larger in yolk than in blood.Five protein fractions were demonstrated by electrophoresis of homogenates of parasites. Their electrophoretic mobilities are similar to those of 5 of the 8 haemolymph ‘vitellogenic’ fractions of the host. There is little doubt that these 5 haemolymph protein fractions (one of them is the band 6) are involved in the nutritional requirements of the parasite.Electrostimulation of the PI, during diapause and activity, increase the haemolymph protein concentration and chiefly the protein concentration of the blood band 6. Thus, the median neurosecretory cells of the brain (M-NSC) regulate protein synthesis and chiefly the synthesis of ‘vitellogenic’ proteins.In parasitized females, the increase of the haemolymph protein concentration after electrostimulations of the PI is associated with an enhancement of ovarian development. The depletion of the haemolymph protein concentration in ‘castrated’ females is thus involved in the inability of the oöcytes to sequester available proteins from the haemolymph. The haemolymph protein deficiency may be attributed to (1) an impairment of protein synthesis, attendant upon the hypoactivity of the M-NSC, and (2) the nutritional requirements of the parasite.     

Development of the suboesophageal body during embryogenesis without diapause in Locusta migratoria (linnaeus) (Orthoptera: Acrididae)

The development and the ultrastructural changes of the suboesophageal body were studied during embryogenesis of Locusta migratoria (Orthoptera : Acrididae). The suboesophageal body develops from the mandibular coelomic cavities. It differentiates early, before the completion of germ band segmentation (stage IIIc), the other mesodermal cells remaining undifferentiated. The cells of the suboesophageal body rapidly develop a structure similar to that of nephrocytes. They consist of a peripheral transfer zone and a perinuclear zone, the site of synthesis and storage. Material absorbed by endocytosis is taken up by α-vacuoles, then stored in β-vacuoles. Golgi vesicles, tubules and vesicle complexes may be involved in the secretory activity of the cells. The activity of the suboesophageal body is maximal until stage VI, after katatrepsis, after which degeneration begins. Very few cells remain at eclosion and they are completely degenerated. The suboesophageal body may be involved in the regulation of embryonic haemolymph composition, and it develops according to its function. The suboesophageal body differentiates early and is thus functional when the haemolymph first forms in the subgerminative space. It degenerates after the differentiation of the pericardial cells and the fat body, which regulate haemolymph composition.     

Catabolism and disposition of exogenous 5-hydroxytryptamine in cockroach,Periplaneta americana,tissues

The concentrations of tryptophan, 5-hydroxytryptophan, 5-hydroxytryptamine, N-acetyl 5-hydroxytryptamine and N-acetyl dopamine were determined in the cerebral ganglia, haemolymph and Malpighian tubules of the cockroach Periplaneta americana, using high performance liquid chromatography with electrochemical detection. Injected 5-hydroxytryptamine was rapidly removed from the haemolymph with a concomitant elevation of circulating N-acetyl 5-hydroxytryptamine and little accumulation of 5-hydroxytryptamine in the cerebral ganglia. N-acetyl 5-hydroxytryptamine and N-acetyl dopamine were also rapidly removed from the haemolymph. Incubation of haemolymph from 5-hydroxytryptamine-injected insects and glucosidase or phosphatase, indicated that most of the injected 5-hydroxytryptamine had been converted to a sugar conjugate of N-acetyl 5-hydroxytryptamine. Whole haemolymph did not catabolize 5-hydroxytryptamine or N-acetyl 5-hydroxytryptamine whereas Malpighian tubules N-acetylated both 5-hydroxytryptamine and dopamine and metabolized N-acetyl 5-hydroxytryptamine. Injection of p-chlorophenylalanine (200 and 500 μg/g) had no effect on 5-hydroxytryptamine concentrations in the cockroach cerebral ganglia.     

Identification of the juvenile hormone from the cricket, Teleogryllus commodus, and juvenile hormone titre changes

In the cricket, Teleogryllus commodus, eggs, haemolymph of 7th and 8th (last)-larval instars, and haemolymph of adults of both sexes contain only juvenile hormone III. While in the male the hormone titre is independent of previous mating experience, juvenile hormone concentration in haemolymph taken from females 36–38 hr after mating (an event which is followed by oviposition) is at a level 5 times higher than that of virgin females. Based on data gleaned from several research groups the identification of juvenile hormone III as the exclusive juvenile hormone in the Order Orthopteroidea is discussed.     

Hormonal control of vitellogenin synthesis in Oncopeltus fasciatus

Previous work indicated the existence of two vitellogenins (A and B) in the haemolymph of Oncopeltus fasciatus, and that vitellogenin B was juvenile hormone (JH)-dependent whereas A was not (Kelly and Telfer, 1977). We have extended these results using several electrophoretic techniques in combination with limited proteolysis of key proteins to show that (1) vitellogenin B is present in eggs in a modified form while vitellogenin A cannot be detected in eggs. (2) Vitellogenin A may be a precursor of B since it has a molecular weight of 200,000D, approximately three times that of vitellogenin B (68,000D) and analysis by limited proteolysis shows that two proteins to be nearly identical. (3) Neither ovariectomy nor treatment with the anti-allatotropin, precocene II prohibits the appearance of vitellogenins A and B in the haemolymph. (4) Injection of ecdysone or 20-hydroxyecdysone into adult, male Oncopeltus fasciatus induces the appearance of both vitellogenin A and B in the haemolymph, suggesting the possible involvement of ecdysteroids in the control of vitellogenin synthesis in this species. (5) We have no evidence for JH control of the synthesis of vitellogenin, however, the ratio of vitellogenin A to B in the haemolymph is higher in the precocene-treated females.     

The fate of vicilins, 7S storage globulins, in larvae and adult Callosobruchus maculatus (Coleoptera: Chrysomelidae: Bruchinae)

The fate of vicilins ingested by Callosobruchus maculatus and the physiological importance of these proteins in larvae and adults were investigated. Vicilins were quantified by ELISA in the haemolymph and fat body during larval development (2nd to 4th instars), in pupae and adults, as well as in ovaries and eggs. Western blot analysis demonstrated that the majority of absorbed vicilins were degraded in the fat body. Tracing the fate of vicilins using FITC revealed that the FITC-vicilin complex was present inside cells of the fat body of the larvae and in the fat bodies of both male and female adult C. maculatus. Labelled vicilin was also detected in ovocytes and eggs. Based on the results presented here, we propose that following absorption, vicilins accumulate in the fat body, where they are partially degraded. These peptides are retained throughout the development of the insects and eventually are sequestered by the eggs. It is possible that accumulation in the eggs is a defensive strategy against pathogen attack as these peptides are known to have antimicrobial activity. Quantifications performed on internal organs from larvae of C. maculatus exposed to extremely dry seeds demonstrated that the vicilin concentration in the haemolymph and fat body was significantly higher when compared to larvae fed on control seeds. These results suggest that absorbed vicilins may also be involved in the survival of larvae in dry environments.     

Humoral inhibition of host-seeking in Aedes aegypti during oöcyte maturation

Female Aedes aegypti mosquitoes were given 1 μl blood enemas, and their subsequent host-seeking behaviour determined in an olfactometer. Those females failing to develop eggs consistently responded to a host stimulus whenever tested, but inseminated mosquitoes developing eggs were inhibited from host-seeking during the period of egg development. Gravid uninseminated mosquitoes were also inhibited, but not to the same degree as inseminated mosquitoes. Experiments involving surgical manipulations and haemolymph transfusions indicate that a haemolymph-borne substance, present during egg development, inhibits the response toward a host.