Male Hemolymph Research Articles

An inhibitory mechanism over oviposition in the tobacco cutworm, Spodoptera litura by juvenile hormone analogue pyriproxyfen

The effects of a juvenile hormone analogue, pyriproxyfen, on the reproduction of tobacco cutworm, Spodoptera litura, were examined. Topical application of 0.3 ng to day-0 female pupae reduced the number of eggs oviposited. Ovarian development and egg maturation in pyriproxyfen-treated moths were normal in most females. The reduction in eggs oviposited was due to the lack of an oviposition stimulating factor which was present in female haemolymph but not in male haemolymph. A lower activity of the factor was present in virgin females and pyriproxyfen-treated females. Precipitation with ammonium sulphate, heat treatment, dialysis and protease treatment showed that the factor is a protein and its molecular weight is more than 14 kDa.

Regulation of vitellogenin synthesis by juvenile hormone in the corn earworm,Helicoverpa zea

Summary In the corn earworm Helicoverpa zea yolk deposition in oocytes begins 8–10 h after adult eclosion and is controlled by juvenile hormone (JH). Hemolymph from females at the time of yolk formation, analyzed by SDS-PAGE, contains polypeptides apparently identical to polypeptides from eggs; and these have been tentatively designated as apovitello-genins (apoVgs). ApoVgs were not found in male hemolymph. The hormonal control of production of the apoVg with an apparent molecular weight of 165 kDa (apoVg-I) was studied by SDS-PAGE. Decapitation of females immediately after eclosion did not prevent the appearance of apoVg-I whereas the same operation done 5–7 h prior to eclosion abolished its production. Treatment of the latter group of females with methoprene, a JH analogue, restored production of apoVg-I whereas injection of 20-hydroxyecdysone (20-HE) did not. When pharate adults were kept at 10°C from 10–12 h before the time of their anticipated eclosion, eclosion was delayed for 4–7 d. In such females...

Binding of lindane to locust haemolymph proteins

1. Within the scope of effects of sublethal doses of lindane on reproductive physiology of the migratory locust, binding of the insecticide to hemolymph proteins was studied after incubation of locust haemolymph with [ 14C]-lindane. 2. In male haemolymph, radiolabelled lindane is principally associated with the major plasma lipoprotein, high density lipophorin (HDLp), whereas in female haemolymph radiolabel is additionally recovered in vitellogenin. 3. Elevation of the haemolymph titer of vitellogenin by ovariectomy as well as by additional treatment of locusts with the juvenile hormone analogue methoprene (ZR-515) results in increased recovery of lindane in vitellogenin, whereas recovery in HDLp is consecutively decreased.

Development of male‐incubated ovaries in the gypsy moth, Lymantria dispar

AbstractOvaries from Lymantria dispar females were transplanted into an environment lacking vitellogenin, the male milieu, in order to determine how the presence of vitellogenin in the hemolymph affects the process of protein uptake by gypsy moth oocytes. When undeveloped ovaries from newly ecdysed last instar females were transplanted into males of the same stage, follicles detached from the germarium and increased in size, but the growth of oocytes proceeded more slowly than those from female controls. Although chorion fromation was delayed in male‐grown ovaries, scanning electron microscopy of chorionated eggs recovered from adult males showed that a chorion with normal surface architecture was formed by the adult stage. SDS‐PAGE analysis of the male‐grown ovaries and hemolymph from males receiving ovaries showed that vitellogenin production was not stimulated by the organ transplant and only male hemolymph proteins were internalized by the male‐incubated ovaries. Thus, in the absence of vitellogenin, endocytosis of male hemolymph proteins occurred, but the rate of oocyte growth was slowed.

Changes in Total Hemolymph and Ovarian Proteins During Oogenesis in Argas (Argas) hermanni (Acari: Argasidae)

Changes in the total protein concentrations in the hemolymph and ovaries of fed, mated female Argas (Argas) hermanni Audouin during oogenesis and in the hemolymph of males were studied. There was a decline in the total protein level in hemolymph of mated ticks for 2 d after feeding. This decrease was followed by an increase, reaching a maximum on day 6 in males and days 5 and 6 in females. Protein levels in the hemolymph of female ticks were higher than that of the male during the period of vitellogenesis and onset of oviposition (3–6 d after feeding). At the end of the oviposition period (day 20), the protein level in the hemolymph decreased, approaching that of the male. These data suggest an increase in the demand and use of proteins in the hemolymph of females by the developing oocytes. The increase of the hemolymph proteins in the females coincided with an increase of the ovarian protein level, ovarian weight, and the number of mature oocytes inside.

Ovarian development and vitellogenesis in the sawfly,Athalia rosae ruficornisJakovlev (Hymenoptera, Tenthredinidae)

Summary Ovarian development in Athalia rosae ruficornis Jakovlev (Hymenoptera: Tenthredinidae) is described. Number of nurse cells per egg chamber is most often around 60 (close to 63 according to the 2n–1 rule), but in many cases it deviates from this number significantly. Two major yolk proteins [vitellins: large (apparent molecular weight 160–170 kD) and small (48–50 kD] were identified by SDS-PAGE. Western blotting and immunochemical detection using polyclonal antibodies prepared against each of the vitellins revealed that adult female but not male (both haploid and diploid) hemolymph contains vitellogenins corresponding to these vitellins. Vitellogenins become detectable in the hemolymph of late pupae, and vitellins one day later in the oocytes of adults. Transplantation of immature ovaries into the adult male abdomen caused not only significant accumulation of vitellins in the oocyte but also appearance of small amounts of hemolymph vitellogenins in host males. Injection of homogenate of immature ovaries also caused appearance of small amounts of hemolymph vitellogenins in host males.

Electrophoretic studies on male haemolymph in two groups of Heteroptera: Chemical characterization of plasma proteins

Abstract SDS-PAGE on male haemolymph plasma from the three bugs, Lethocerus indicus, Ranatra filiformis and Antilochus coqueberti representing the two groups of Heteroptera, was carried out in order to determine the chemical nature of the prosthetic groups attached to the haemolymph proteins by using several chemical tests. These chemical characterizations indicate that the haemolymph contains a complex of glycoproteins, lipoproteins and ribonucleoproteins of varying electrophoretic patterns in the two groups. Most of the plasma proteins show glycosylation, and some of them further show attachment with the lipid and RNA molecules.

Storage proteins of the fall webworm, Hyphantria cunea drury

AbstractTwo storage proteins, storage protein‐1 (SP1) and storage protein‐2 (SP2), were found in hemolymph and fat body during the development of Hyphantria cunea, the fall webworm. Both storage proteins show similiar quantitative changes during development in males and females; however, SP1 is more abundant. The hemolymph of last instar larvae contains high concentrations of the storage proteins. However, following pupation, the storage proteins accumulate in fat bodies. SP1 peaks in the hemolymph of males and females late in last instar larvae (8‐day‐old 7th instar larvae).SP1 has a native molecular weight of 460,000 and consists of six identical subunits (Mr = 76,700), while SP2 has a molecular weight of 450,000 and is composed of two different subunits (Mr = 74,100 and 72,400). Both SP1 and SP2 are hexamers and are phosphorylated glycolipoproteins. The pl values of SP1 and SP2 were determined to be 5.70 and 5.50, respectively.Antibodies raised against SP1 react positively with vitellogenin and ovary extract, as well as with proteins in the hemolymph from last instar larvae and proteins in pupal fat bodies. Storage protein synthesis starts in fat bodies of a 4‐day‐old 7th instar larvae and in female peaks at 6–8 days of the 7th instar.

Stimulated synthesis of the female‐specific storage protein in male larvae of the silkworm Bombyx mori treated with juvenile hormone analog

AbstractApplication of methoprene to fourth (penultimate) instar larvae of the silkworm Bombyx mori induced the appearance of the feeding dauer larvae at the fifth (last) instar and prevented pupal metamorphosis. Methoprene also increased the protein concentrations of hemolymph last instar larvae by preventing sequestration of storage proteins by the fat body. Usually, the female‐specific storage protein 1 (SP1)* disappears from the male hemolymph at the time of the last larval instar. However, exposure of male larvae to methoprene at the penultimate instar enhanced the accumulation of SP1 in the hemolymph. The SP1 accumulated in males did not differ in molecular weight and immunoreactivity from the SP1 produced in female larvae. Both sexes of fourth instar larvae allatectomized on day 1 instantly accumulated SP1 in the hemolymph, and methoprene application after allatectomy suppressed the hemolymph accumulation of the SP1. In contrast, if allatectomy was carried out at a later stage of the fourth larval instar, SP1 concentration in hemolymph of fifth instar larvae did not increase, suggesting the different juvenile hormone action for regulation of SP1 synthesis in the penultimate instar larvae of silkworms.

Changes in the titer of the female‐predominant storage protein (81K) during larval and pupal development of the waxmoth, galleria mellonella

AbstractThe levels of an 81K storage protein in the waxmoth, Galleria mellonella, were monitored during the course of development using rocket immunoelectrophoresis. During the fifth and sixth larval stadia, 81K protein levels increased during feeding and growth but sharply declined at each larval molt. During the fifth and sixth stadia hemolymph levels of the 81K protein increased to about 1 and 2.5 mg/ml, respectively, with no discernible differences between levels in males and females. Neither the fat body nor the remainder of the carcass contained the 81K protein, indicating that the accumulation of this protein during the intermolt period was exclusively in the hemolymph and redistribution of the 81K protein into other tissues does not occur at the final two larval molts. During the seventh (final) larval stadium the absolute quantities of the 81K protein increased from 23 μg per insect to over 1,600 μg in females and to 300 μg in males. The hemolymph concentration of the 81K protein reached 28 mg/ml in females and 6 mg/ml in males with only low levels found in the remaining tissues. Shortly after pupal apolysis, marked by eyespot retraction, the fat body in both sexes rapidly and quantitatively sequestered the 81K protein from the hemolymph. The 81K protein in the hemolymph of both males and females rapidly dropped to nearly zero concentration by pupation. The 81K storage protein remained localized in the fat body cells after uptake occurred, even though the fat body cells disaggregate and reaggregate during metamorphosis. During pharate adult development the 81K storage protein disappeared from the fat body without entering the hemolymph. At adult eclosion 81K was virtually absent from the tissues of both males and females.

Electrophoretic patterns of male haemolymph plasma proteins in two groups of indian bug

Abstract SDS-PAGE on male haemolymph plasma proteins from the three bugs, Lethocerus indicus, Ranatra filiformis and Antilochus coqueberti of the Heteroptera was carried out in order to explore the differences in their protein patterns. All three predatory bugs show apparent differences in their haemolymph protein patterns which seem to be species-specific. These differences largely depend upon ecological niches. There were 19 protein bands in R. filiformis, whereas L. indicus showed the presence of 18 and A. coqueberti 15. Comparison of the electropherograms of haemolymph shows that only a few of these bands are present in all three insects.

Identification and analysis of the major yolk polypeptide from the Caribbean fruit fly, Anastrepha suspensa (loew)

AbstractA single major yolk polypeptide (YP) having a molecular mass of approximately 48,000 daltons (Da), was identified in the ovaries and oviposited eggs of the Caribbean fruit fly, Anastrepha suspensa. The polypeptide was partially purified from oviposited eggs using gel permeation and ion‐exchange chromatography. Analysis of YP synthesis in vivo and in tissues cultured in vitro indicated that the ovary was the major site of synthesis with very low levels of YP derived from the adult fat body. Using a monospecific polyclonal antiserum to 48 kDa YP in an immunoblot assay, low levels of vitellogenin were found in female hemolymph; slightly lower levels of an immunoreactive 48‐kDa polypeptide were detectable in male hemolymph. Although YP synthesis was detectable within 12 h after eclosion, the major increase in YP accumulation occurred at 3–4 days posteclosion coincident with the initiation of observable yolk deposition. The physical characteristics of YP from A. suspensa were similar to YPs from other dipterans in terms of molecular mass and antigenicity, yet the tissue‐ and sex‐specific regulation of the YP differed from other dipterans as well as most other insects.

Vitellogenesis in the insect Dacus oleae. Isolation and characterization of yolk protein mRNA

The two major yolk polypeptides (YP1 and YP2) of Dacus oleae have been isolated from egg extracts. Their molecular weights (as determined by SDS-PAGE) are 47,000 and 49,000, respectively, and they are synthesized in vivo by ovaries and fat bodies of female flies; none of the two polypeptides were detectable in male haemolymph. The mRNAs for the YP1 and YP2 polypeptides were isolated from female flies. Purification was achieved, using three successive cycles of sucrose gradient centrifugation under denaturing conditions and hybridization to yolk protein clones immobilized on DBM paper. The purity of the yolk protein mRNA was judged by translation in a cell-free protein-synthesizing system derived from rabbit reticulocytes. Purified yolk protein mRNA migrated, following denaturing agarose gel electrophoresis, as a single band, slightly faster than the 16S rRNA. Yolk protein complementary DNA (cDNA) was transcribed from purified yolk protein mRNA. The resultant cDNA was an almost full length copy as defined by denaturing gel electrophoresis following hybridization-arrested translation. Hybridization analysis of the yolk protein gene transcripts using the synthesized yolk protein cDNA as a probe, showed that these are present only in the ovaries and fat bodies of female flies but not in the fat bodies of male flies.

Identification and characterization of a novel postlarval hemolymph protein from Manduca sexta

AbstractThe identification, purification and characterization of a new postlarval specific hemolymph protein from Manduca sexta is described. Incorporation of [35S]methionine into Manduca sexta hemolymph proteins in vivo was investigated as a function of development. A major protein band of Mr ≈ 50,000 was highly labeled during the prepupal and adult stage but not in feeding larvae. This postlarval protein (PLP) was isolated from adult male hemolymph and its chemical and immunological properties determined. PLP is a basic protein (pI ∼8.6). Electrophoresis under denaturing conditions reveals a subunit Mr ≈ 50,000 while the native protein has an apparent Mr ∼ 85,000 by gel permeation chromatography. Anti‐PLP serum recognized PLP but not other hemolymph proteins on immunoblots. In vitro translation of fat body mRNA followed by immunoprecipitation revealed that fat body is the site of PLP synthesis. Quantitation of PLP levels in hemolymph throughout development was performed and suggests PLP may play a role in adult development of M. sexta.

Vitellogenin mRNA from Dacus oleae: Characterization, cDNA cloning, and dependence on sex and developmental stage

AbstractPoly(A)+ RNA was isolated from 5‐day‐old females of the olive fruitfly Dacus oleae. In vitro translation of mRNA, followed by immunoprecipitation with anti‐vitellin antiserum, demonstrated the synthesis of two vitellogenin polypeptides of 54,000 and 56,000 daltons. In sucrose gradient centrifugation, the vitellogenin mRNA sediments in the 14.4S region that corresponds to an RNA size of approximately 1,600 nucleotides. Translation of poly(A)+ RNA isolated from male flies revealed the presence of small quantities of vitellogenin mRNA, consistent with the finding of vitellogenin in male hemolymph of Dacus. Using the female poly(A)+ RNA population as template, we constructed a cDNA expression library in a lambda gt11 vector, which was screened with anti‐vitellin antiserum. Four recombinant clones were isolated containing inserts of 550–800 bp*, which were identified as vitellogenin cDNA clones by hybrid‐selected translation. All four cDNA clones showed crossreaction with vitellogenin genes of Drosophila melanogaster. The Dacus clones were used as probes to study vitellogenin expression during postembryonic development of Dacus. In females, vitellogenin mRNA appeared at the time of eclosion with increasing accumulation up to the 5th day of adult life; then it reached a plateau. On day 10, the mRNA titer had already started to decrease and on day 25, no vitellogenin mRNA was detectable. In males, vitellogenin mRNA was found up to the 8th day after eclosion, in very low concentrations. No vitellogenin mRNA was present during larval and pupal stages.

Effects of precocene II on female‐specific hemolymph polypeptides in Oncopeltus fasciatus

AbstractUsing polyacrylamide gel electrophoresis (PAGE) under denaturing conditions, two major polypeptides of 200,000 and 170,000 daltons were detected in the hemolymph of mature female Oncopeltus fasciatus, but they were not found in the hemolymph of males or newly emerged females. Those polypeptides constituted the two major bands of early vitellogenic oocytes; however, they were absent from the yolk of mature eggs. The slower‐migrating band (200,000 daltons) appears to correspond to a vitellogenic protein already identified in O. fasciatus, whose synthesis has been suggested to be independent of juvenile hormone (JH). Treatment of newly emerged adult females with the corpus allatum cytotoxin precocene II prevented the appearance of the female‐specific bands and induced an important accumulation of other proteins in the hemolymph. Yolk deposition was also inhibited in those animals. Topical application of JH to precocene‐treated females restored the appearance of the 200,000 and 170,000 dalton polypeptides in the hemolymph. These results suggest that JH is required for the synthesis of female‐specific polypeptides in O. fasciatus.

Sexual and developmental differences in the protein pattern of haemolymph and body tissues of Streptocephalus dichotomus Baird (Crustacea: Anostraca)

The protein pattern of haemolymph and body tissues of the freshwater fairy shrimp Streptocephalus dichotomus has been investigated in both sexes, using polyacrylamide disc gel electrophoresis. The electropherograms of four developmental stages show variations in number and intensities of protein fractions. In Stage III, two female-specific proteins of glycolipoprotein nature appear. This stage corresponds to maturity: females begin to possess mature oocytes in the ovary. These two vitellogenic proteins are well represented in the female haemolymph, ovary and freshly laid eggs, but are absent in the male haemolymph. A heterosynthetic mode of yolk formation is thus evident in this anostracan. The two sex-limited proteins are only faintly represented in shelled eggs, suggesting an early utilization of these compounds in embryogenesis.

Etude de la synthèse de la vitellogénine chez les mâles intersexués d' Armadillidium vulgare latreille (crustacé isopode oniscoïde): Comparaison avec les mâles et les femelles intactes ou ovariectomisées

In some natural populations of Armadillidium vulgare, intersex animals are genetic males which are feminized by maternally transmitted symbiotic bacteria. In these intersex males (iM) the fat body synthesizes vitellogenin, although their gonads are testes with hypertrophied—but nonfunctional—androgenic glands. Vitellogenin is present in the hemolymph of males changed experimentally into iM 90 days after inoculataion of the feminizing bacteria. During the molting cycle, vitellogenin synthesis in iM varies as in ovariectomized females or in vitellogenic females, with a peak at the stage D 1″. In A. vulgare, vitellogenin synthesis is a neutral character since it can be observed in a genetic male or in an ovareectomized female; however, it is inhibited by the androgenic hormone. In intersex males, vitellogenin synthesis is the result of their refractoriness to androgenic hormone.

Studies on electrophoretic protein patterns of fat body, haemolymph and ovary in an indian fresh water-bug, Laccotrephes rubra Linn. (Nepidae: Heteroptera)

—Polyacrylamide disc gel electrophoresis (PAGE) was performed on soluble proteins from the fat body, haemolymph (male and female), and ovary of the adult fresh water-bug, Laccotrephes rubra, in order to determine their protein patterns. The electrophoresis resulted in the separation of 16 and 17 protein components in male and female haemolymph respectively. In female fat body extracts 15 protein bands could be detected; whereas ovary extracts resolved into 11 protein bands only. Comparison of the electropherograms of protein pattern of fat body (FBPP), haemolymph (HPP) and ovary (OPP) revealed that the extra band present in the female haemolymph is common to both female fat body and ovary, which is suggestive of it being the female specific protein, i.e. vitellogenin. A careful examination of the various electropherograms further revealed that the majority of the protein bands of haemolymph and ovary are common to its corresponding fat body protein bands. This might suggest that the ovary derives most of its protein components from the surrounding haemolymph, which have presumably been synthesized and secreted by the fat body. The histochemical analyses demonstrated that the female-specific protein band is a glycolipoprotein; and most of the haemolymph protein zones stain as glycoprotein, lipoprotein or ribonucleoprotein. ‘Stains-all’ technique however, revealed the presence of four possible RNA bands in fat body extracts.

Sex- and cell-specific regulation of yolk polypeptide genes introduced into Drosophila by P-element-mediated gene transfer.

To find whether cis-acting regulatory sequences necessary for sex- and cell-specific expression of two yolk polypeptide genes (Yps) reside near the structural genes themselves, we introduced a 5.0-kilobase genomic DNA segment containing a 3' truncated Yp1 and a complete Yp2 into five different autosomal locations by P-element-mediated gene transfer. Transcripts from the introduced Yp1 were not found in male flies but appeared on a normal developmental schedule in adult females, accumulating in their body walls and ovarian follicles but not in guts or malpighian tubules. Protein from the introduced Yp2 allele was present in female hemolymph and vitellogenic ovaries but was lacking from male hemolymph. We conclude that sequences necessary for the correct stage-, cell-, and sex-specific expression of the Yp1 and Yp2 genes are included in this genomic fragment. These results combined with published work suggest that two tissue-specific, cis-acting, bidirectional, positive regulatory elements placed on either side of a centrally located HindIII site govern expression of both Yp genes--one element specific for fat body and the other specific for ovarian follicle cells.