Female Fat Body Research Articles

Characterization of a serine carboxypeptidase in the salivary glands and fat body of the orange wheat blossom midge, Sitodiplosis mosellana (Diptera: Cecidomyiidae)

A full-length cDNA encoding a serine carboxypeptidase (designated SmSCP-1) was recovered from an ongoing salivary gland EST project of the wheat midge. The deduced 461-amino acid sequence had a putative signal sequence at the amino terminus, indicating it was a secreted protein. The protein shared homology with serine carboxypeptidases from other insects, mammals, plants, and yeasts. SmSCP-1 mRNA was expressed in all stages of development and detected in salivary gland and fat body tissues but not in midgut tissue. Expression analysis and quantitative real-time PCR assays in male and female wheat midges and the fat body tissue of adult midges revealed that SmSCP-1 was up-regulated nearly four-fold in the female midges compared to males and nearly two-fold in female fat body compared to male fat body. The wheat midge serine carboxypeptidase (SmSCP-1) most likely has a dual function. As a secreted digestive enzyme, it could play a role in mobilizing host-plant seed reserves for feeding larvae and as expressed in fat body could function as an exopeptidase in degradation of vitellogenin and/or in post-translational processing of other enzymes.

Ornithine decarboxylase activity during the development of Anastrepha fraterculus (Diptera, Tephritidae)

Ornithine decarboxylase (ODC) (EC 4.1.1.17) is very important for polyamine biosynthesis, which is required for main biological events. In the present study, ODC activity was measured in samples of Anastrepha fraterculus's egg, larva, pupa body and abdomen, adult body, ovaries, and fat body of young females, and in ovaries of mature flies. The kinetic parameters (Km app and Vmax) for ODC activity were determined for pupa, larva, and young ovary. ODC activity showed fluctuations during A. fraterculus's life development. In its earlier stages, prior to emergence, the egg has high ODC-specific activity probably due to embryogenesis, which is characterized by a high rate of cell division. This enzyme activity is also significantly high in the ovary and fat body of young females possibly related to the increased oogenesis and vitellogenesis. The kinetic parameters (Km app and Vmax) had great variation. Our results using GTP showed that the great variation in kinetic parameters can be accounted for by post-translational modifications.

Nuclear Magnetic Resonance Microscopy of The Internal Structure of the Carrion Rolling Scarab Canthon cyanellus cyanellus (Scarabaeidae: Scarabaeinae)

Abstract Nuclear magnetic resonance microscopy was used to observe the internal structure of male and female carrion rolling scarab Canthon cyanellus cyanellus adults. Images were produced with a NMR microscope built at the University of Orsay, France. The NMR microscope operated at 360 MHz and produced images of live insects with an in-plan resolution of 30 μm × 30 μm from a slice 125 μm thick. NMR images of horizontal slices through head, thorax, and abdomen of males and females of different ages and physiological stages were obtained. Internal organs such as reproductive organs, gut, fat body, and pygidial glands were clearly observed in both sexes; ova at different stages and aedeagus were identified in females and males, respectively. Pygidial glands in both sexes and ova and fat body in females seem to be good indicators of the reproductive condition of the beetles. These results show the potential of NMR microscopy as a non-invasive method to study the relationships between the internal morphology,...

Fat body expressed yolk protein genes in Hyphantria cunea are related to the YP4 follicular epithelium yolk protein subunit gene of pyralid moths.

cDNA clones for two of the yolk proteins, YP1 and YP2, produced by the fat body of the moth, Hyphantria cunea, were sequenced and found to be homologous to the follicular epithelium yolk proteins of pyralid moths. Both cDNA clones coded for polypeptides of 290 residues and the deduced amino acid sequence identity between YP1 and YP2 was very high (79.0%). Analysis of the secondary structure of the predicted polypeptides suggests that YP1 and YP2 do not form heteromeric proteins because of differences in secondary structure due to the lack of alpha helices in YP1. Northern blot analysis showed that the transcripts for YP1 (1.2 kb) and YP2 (1.1 kb) were present primarily in the female fat body with only trace levels detectable in the ovary of the adult female. In a developmental study, the YP1 and YP2 transcripts were first detectable in 10-day-old pupae and increased into the adult stage. These results suggest that the YP1 and YP2 genes in H. cunea have been recruited to replace the vitellogenin gene as the primary source of yolk proteins. During this process they have acquired a modified pattern of expression that is different from homologous genes reported in pyralid moths. The assessment of the evolution of proteinaceous yolk in these moths should serve as an excellent model for the evolution of gene recruitment.

Cyclicity of mosquito vitellogenic ecdysteroid-mediated signaling is modulated by alternative dimerization of the RXR homologue Ultraspiracle.

In anautogenous mosquitoes, egg maturation requires a blood meal. As a consequence, mosquitoes are vectors of numerous devastating human diseases. Blood feeding triggers a 20-hydroxyecdysone (20E) hormonal cascade, which activates yolk protein precursor (YPP) genes in the female fat body, an insect metabolic tissue. An important adaptation for anautogeny is the previtellogenic arrest preventing activation of YPP genes. Equally essential is termination of their expression, so that another arrest is achieved after a batch of eggs is laid. Here, we report that mosquito Seven-up (AaSvp), a chicken ovalbumin upstream promoter-transcription factor homologue, is involved in regulating the cyclicity of vitellogenic ecdysteroid-mediated signaling through heterodimerization with a retinoid X receptor homologue Ultraspiracle (USP), the obligatory functional ecdysteroid receptor (EcR) partner. AaSvp inhibits 20E-dependent activation of the vitellogenin (Vg) gene in transfection assays. Two-hybrid and GST pull-down analyses demonstrate that in vitro AaSvp interacts with both AaUSP and AaEcR. However, the coimmunoprecipitation using fat body nuclear extracts reveals that at 33-36 h postblood meal, when the 20E titer sharply declines and YPP gene expression ceases, AaSvp replaces AaEcR in USP heterodimers. The chromatin immunoprecipitation assay indicates that protein-protein interaction rather than binding competition for the Vg ecdysteroid response element accounts for the inhibition of Vg expression by AaSvp.

Molecular cloning and characterization of the cathepsin B‐like proteinase from the cotton boll worm, Helicoverpa armigera

An enzyme purified from the ovaries of Helicoverpa armigera, as an active form with molecular mass of 30 kDa on SDS-PAGE, was identified as a cysteine proteinase because it could be inhibited by E-64, a specific inhibitor of cysteine proteinase, and required reducing conditions for activity. This enzyme was further identified as a cathepsin B-like cysteine proteinase by partial amino acid sequencing. A cDNA encoding this proteinase was cloned from H. armigera, using degenerate primers and RACE techniques. Results of Northern blots indicated that the mRNA encoding the proteinase was transcribed in the ovaries, the fat bodies of female and male adults, pupae and in the larvae. No mRNA was detected from the larval epidermis or from the midgut. Hence, transcription of the cathepsin B-like cysteine proteinase from H. armigera was tissue-specific, but not gender- or developmental stage-specific. However, proteolytic activities were only detected from ovaries, and adult female and male fat bodies. No activity was observed from pupal and larval fat bodies, from the larval epidermis or from the midgut. Only one form of mRNA of approximately 1100 bases was detected, and in situ hybridization showed that the transcripts were distributed in the adult female fat bodies, follicular cells and the oocytes. Since the proteinase expressed in ovaries was able to degrade vitellin in vitro, it may be involved in the degradation of vitellin during embryonic development.

A hexamerin protein, AgSP-1, is associated with diapause in the boll weevil

The objective of this research was to identify a reliable biochemical indicator for diapause (dormancy) in the boll weevil, Anthonomus grandis. Hemolymph polypeptides from reproductive and diapausing weevils were compared using denaturing sodium dodecyl sulpfate (SDS)-polyacrylamide gel electrophoresis (PAGE). A 77-kDa protein, which proved to be a hexamerin (AgSP-1), strongly correlated with morphological diapause characters in both male and female adult weevils. N-terminal sequence analysis identified the first 25 amino acids of the mature protein and was used to develop an antibody to AgSP-1. Anti-AgSP-1 reacted only with hemolymph from diapausing weevils of both sexes but not with hemolymph from reproductive weevils. Also, the yolk protein, vitellogenin (VG), inversely correlated with AgSP-1. When hemolymph VG was high, AgSP-1 was absent or barely perceptible. Juvenile hormone regulates VG synthesis in most insect species. Juvenile hormone is reported to stimulate reproductive maturation in the boll weevil (Physiological Entomology 22 (1997) 261) and to be absent during diapause (Physiological Entomology 22 (1997a) 269). Therefore, the juvenile hormone (JH) mimic, methoprene, was used to assess the role of JH activity in the boll weevil for terminating diapause, stimulating reproductive maturation and possibly influencing AgSP-1 titers. Application of methoprene was not effective in activating reproductive development. Hemolymph from methoprene-treated, females contained VG and AgSP-1 titers that were similar to acetone-treated and untreated control weevils. Using a genomic DNA library and 3′ RACE, two clones were isolated that yielded the complete sequence of AgSP-1 as well as a portion of the 5′ untranslated region. Northern blot analysis confirmed the presence of a 2.5 kB transcript for AgSP-1 in the fat body of diapausing weevils. AgSP-1 was also present in the fat body of reproductive weevils, but to a lesser extent. No sex-related differences in gene expression were observed; diapausing weevils of both sexes showed similar levels of AgSP-1 expression. An inverse correlation was observed between the VG transcript and AgSP-1 mRNA. VG was highly expressed in the fat body of reproductive females and only slightly expressed in tissue from diapausing females. Our data suggests that AgSP-1 is a diapause-specific protein in adult weevils and that JH, alone, is not effective in terminating diapause.

Vitellogenin of the cockroach, Leucophaea maderae: nucleotide sequence, structure and analysis of processing in the fat body and oocytes

A cDNA encoding vitellogenin (Vg) of the cockroach, Leucophaea maderae was cloned and sequenced . The deduced amino acid sequence consisting of 1913 residues (including 15 residues for a putative signal peptide) was obtained. Amino-terminal sequence analysis demonstrated that the pro-Vg was cleaved into four polypeptide ‘subunits’ following the three consensus RXXR cleavage site sequences, which were secreted as four Vg polypeptides (apparent molecular weights=112-, 100-, 92- and 55-kD), sequestered, and deposited in the egg as four respective vitellin (Vn) polypeptides. There was, however, an additional 90-kD Vn polypeptide existed in the egg. We show that this polypeptide is a processed product from 92-kD Vn polypeptide. Northern blot analysis of poly (A) + RNA reveals that mRNA coding for Vg is present only in the female fat body cells but neither in the ovary nor in the male fat body cells. The deduced amino acid sequence contained a serine-rich stretch at the C-terminal region. This stretch occurred also in Vgs of Periplaneta americana (Vg1 and Vg2) and Blattella germanica. The Vg of L. maderae had 26% and 31% homology with those of P. americana (Vg1 and Vg2) and B. germanica, respectively. Phylogenetic analysis (neighbour-joining) was made using four cockroach Vgs and the tree was compared with other molecular and conventional phylogenetic trees.

Effects of hypocholesterolaemic agents on the expression and activity of 3-hydroxy-3-methylglutaryl-CoA reductase in the fat body of the German cockroach.

In the fat body of adult Blattella germanica females, the expression of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) during the first reproductive cycle is parallel to that of vitellogenin, suggesting a functional link between the mevalonate pathway, and vitellogenesis and reproduction. We have studied the effects of compactin and fluvastatin, two inhibitors of HMG-CoA reductase, on the expression and activity of the enzyme in the fat body, and on the ootheca formation, ootheca viability, and number of larvae per viable ootheca. Short-term assays showed that both compounds reduce the protein levels and enzymatic activity of HMG-CoA reductase, and long-term experiments revealed that fluvastatin impairs embryo development.

ANALYSIS OF THE SOLUBLE PROTEINS IN THREE SPECIES OF PARASITOIDS AND MOLECULAR CHARACTERISTICS OF YOLK PROTEIN IN PTEROMALUS PUPARUM*

Abstract The results both from PAGE and capillary electrophoresis indicated that there was a female specific protein i.e. vitellogenin (Vg) or vitellin (Vt) in the female wasp of Pteromalus puparum (Hymenoptera: Pteromalidae). While there was no difference in the electrophoresis graph between soluble proteins of the female whole body and those of the male one both in two bracoids (Hymenoptera: Braconidae), i.e. Cotesia plutellae and Macrocentrus linears. According to the graph of the gradient SDS‐PAGE, it was clear that the Vg or Vt of P. puparum consisted of two subunits with approximate molecular weights, and their molecular weights were 74.4 and 52.8 KDa, respectively. Both immunological reactions between some main different tissues of the female wasps and the male whole body and the polyantibody against the Vt of this parasitoid, and the graph of the gradient SDS‐PAGE including soluble proteins sampled separately from hemolymph, fat body and ovary of the female and the whole body of the male demonstrated that Vg existed both in female fat body and hemolymph, and Vt deposited in the ovary, not in the male, as well as the Vg was synthesized in the female fat body.

Reproductive Correlates of Abdominal Fat Body Mass in Niveoscincus ocellatus, a Skink with an Asynchronous Reproductive Cycle

The relationship between the annual cycle of energy storage, in the form of abdominal fat bodies, and reproduction was studied in lowland and subalpine populations of the viviparous skink, Ni- veoscincus ocellatus. This species displays an unusual asynchronous reproductive pattern in which male and female preparations for reproduction are separated temporally, thereby providing an opportunity to clarify our understanding about the energy demands of squamate reproduction. Abdominal fat bodies in both sexes showed distinct annual cycles that were positively correlated with resource availability, peaking in summer. In males, fat body mass was correlated positively with testes development and negatively with plasma testosterone concentrations and mating behavior. This is in contrast to the patte obsernved in species with synchronous breeding cycles, where there is generally a negative relationship between testis size and lipid storage. We concluded that N. ocellatus is able to produce spermatozoa and develop abdominal fat reserves concurrently, and therefore testis size is unlikely to be constrained by energetic costs. In contrast, mating behavior appears to rely on stored energy, even though this results in reduced fat reserves for winter hi- bernation. Female fat bodies were smallest in midspring, coinciding with the end of vitellogenesis and early pregnancy. Vitellogenesis and early pregnancy appear to be energetically expensive and are reliant on stored energy in this species. Large fat bodies at the end of pregnancy in both populations are surprising given the reported high costs of reproduction. Two factors may contribute to this: resource availability may be greater in late summer, and the energetic costs associated with pregnancy appear to be highest in the early to midstages of gestation.

Cloning and characterization of a dopachrome conversion enzyme from the yellow fever mosquito, Aedesaegypti

In this study we describe the purification and molecular cloning of a dopachrome conversion enzyme (DCE) from the yellow fever mosquito, Aedes aegypti. DCE catalyzes the conversion of l-dopachrome to 5,6-dihydroxyindole in the melanization pathway. Melanin biosynthesis is involved with crucial protective phenomena in mosquitoes, including egg chorion and cuticular tanning, wound healing, and the melanotic encapsulation immune response. The enzyme was purified to homogeneity by various chromatographic techniques from A. aegypti larvae and has a relative molecular mass of 51 kDa as-revealed by SDS-PAGE analysis. Physiochemical analysis of DCE revealed a pH optimum of 7.5–8.0 and substrate activity for l-dopachrome and aminochromes generated from dopa methyl ester, α-methyl dopa and dopamine. Trypsin digestion of the isolated DCE and subsequent reverse-phase separation resulted in the isolation of several polypeptide fragments, from which two partial internal amino acid sequences were obtained by Edman degradation. PCR amplification, using a degenerate primer based on one internal amino acid sequence and an oligo-dT primer, produced a 650 bp DNA fragment. Subsequent screening of an A. aegypti pupal cDNA library resulted in the isolation of a 1.6 kb clone containing coding sequence for both internal DCE amino acid sequences, thereby confirming the identity of the isolated gene product (pAaDce1) as DCE. Northern analysis revealed the constitutive expression of DCE message in developmental stages and adults, with the majority of transcript localized in the fat body and ovaries of adult females. AaDce1 mRNA increased in abundance above constitutive levels in adult females when a melanotic encapsulation immune response was initiated by the intrathoracic inoculation of Dirofilaria immitis microfilariae.

Transcriptional regulation of the mosquito vitellogenin gene via a blood meal-triggered cascade

In anautogenous mosquitoes, a blood meal is required for activation of genes encoding yolk protein precursors (YPP). Vitellogenin (Vg), the major YPP gene, is transcribed at a very high level following blood meal activation. It is expressed exclusively in the female fat body, the tissue producing most of mosquito hemolymph and immune proteins. In this paper, we analyzed the upstream region of the Aedes aegypti Vg gene in order to identify regulatory elements responsible for its unique expression pattern. To achieve this goal, we analyzed the gene using transgenic Drosophila and Aedes as well as DNA-binding assays. These analyses revealed three regulatory regions in the 2.1 kb upstream portion of the Vg gene. The proximal region containing binding sites to EcR/USP, GATA, C/EBP and HNF3/fkh is required for the correct tissue- and stage-specific expression at a low level. The median region carrying sites for early ecdysone response factors E74 and E75 is responsible for hormonal enhancement of Vg expression. Finally, the distal GATA-rich region is necessary for extremely high expression levels characteristic of the Vg gene. The present work elucidates the molecular basis of blood meal-dependent expression of this mosquito gene, laying the foundation for mosquito-specific expression cassettes with predictable stage and tissue specificity.

Formation of lipid reserves in fat body and eggs of the yellow fever mosquito, Aedes aegypti

We examined the accumulation of lipids in adult females of the mosquito, Aedes aegypti. Females emerged with about 100 μg lipid in the fat body. With access to sugar water lipids increased over seven days to 300 μg. After a blood meal on day five, sugar-fed females accumulated 120–140 μg of lipids in their ovaries within 2 days. At the same time the lipid content of the fat body decreased by 100 μg, indicating transfer of lipids from fat body to oocytes. Experiments in which fat body lipids were prelabelled support this conclusion. Label was transferred to oocytes: in mature oocytes the specific radioactivity of lipids was 80% of the specific radioactivity of prelabeled fat body lipids. Components of blood meals are also used to synthesize oocyte lipids. Fat bodies of females starved for four days had only 27 μg of lipids left. When these females were given a blood meal, they matured oocytes, although the number of ooyctes was reduced and ovaries contained only half the amount of lipids found in ovaries of females which had first fed on sugar water. Fat body lipids of these females had only slightly increased to 36 μg. This demonstrates that female Ae. aegypti use sugar to synthesize lipids, but they can also use components of blood for this purpose.

A novel function of 20-hydroxyecdysone: translational repression of the lysosomal protease mRNA in the mosquito fat body

In the female fat body of the mosquito Aedes aegypti, lysosomes play important roles during the cessation of vitellogenesis by degrading the biosynthetic machinery and aiding the remodeling of the fat body cells. A detailed study of a mosquito lysosomal aspartic protease (AaLAP) has shown a unique expression pattern in the vitellogenic fat body: the level of AaLAP mRNA dramatically rises and peaks at 24 h post blood meal (PBM) correlating with the high titer of ecdysteroids; however, there is a 12 h lag before peak levels of AaLAP protein and its enzymatic activity has been observed. These observations suggest that the high titer of 20-hydroxyecdysone (20E) may hinder translation of the AaLAP mRNA. Here, we used an in vitro organ culture to study the effect of 20E on the protein synthesis of AaLAP in the fat body. The increase in the AaLAP protein level in the fat body, dissected at 24 h PBM and incubated for 6 or 12 h, was inhibited by the presence of 10 −5 M 20E in the medium. Incubation in the hormone-free medium did not effect accumulation of the AaLAP protein which proceeded at the levels comparable to the intact insect. Furthermore, the effect of 10 −5 M 20E on the AaLAP accumulation was reversible. These experiments support the hypothesis of the 20E-mediated repression of lysosomal protease mRNAs at the translational level in the regulation of vitellogenic and postvitellogenic events in the mosquito fat body. Analysis of the 5′ and 3′ -end untranslated regions (UTR) of AaLAP mRNA form secondary structures suggest that they may also contribute to mRNA stability and 20E-mediated translational inhibition.

Cells--analogues of the fat body in parasitic mites Myobia murismusculi (Schrank) (Trombidiformes: Myobiidae)

In body cavity of Myobia murismusculi females the cells with morphology similar to that in elements of fat body of other arthropods were found. They are characterized by large size, hypertrophied GER, and few secretory granules of two types. No such cells were revealed in males of the same mite species. Fat body in females at the nymphal stage is presented by poorly differentiated cells without secretory inclusions. A probable participation of the described tissue in vitellogenesis as well as the reason for its absence in other species of trombidiform mites are discussed.

A novel GATA factor transcriptionally represses yolk protein precursor genes in the mosquito Aedes aegypti via interaction with the CtBP corepressor.

In anautogenous mosquitoes, vitellogenesis, the key event in egg maturation, requires a blood meal. Consequently, mosquitoes are vectors of many devastating human diseases. An important adaptation for anautogenicity is the previtellogenic arrest (the state of arrest) preventing the activation of the yolk protein precursor (YPP) genes Vg and VCP prior to blood feeding. A novel GATA factor (AaGATAr) that recognizes GATA binding motifs (WGATAR) in the upstream region of the YPP genes serves as a transcriptional repressor at the state of arrest. Importantly, AaGATAr can override the 20-hydroxyecdysone transactivation of YPP genes, and its transcriptional repression involves the recruitment of CtBP, one of the universal corepressors. AaGATAr transcript is present only in the adult female fat body. Furthermore, in nuclear extracts of previtellogenic fat bodies with transcriptionally repressed YPP genes, there is a GATA binding protein forming a band with mobility similar to that of AaGATAr. The specific repression of YPP genes by AaGATAr in the fat body of the female mosquito during the state of arrest represents an important molecular adaptation for anautogenicity.

Molecular cloning of a female-specific cDNA with unique repeat sequences from the fat body of the adult locust, Locusta migratoria

A cDNA clone encoding a 25-kDa protein (25K) was isolated from a cDNA library made from RNA isolated from the adult fat body and ovaries of the locust, Locusta migratoria. The longest open reading frame of this cDNA clone encodes a 225-amino acid polypeptide, the N-terminal end of which was similar to the 21-kDa and 19-kDa juvenile hormone induced proteins identified in the locust hemolymph, but the C-terminal end was different. The C-terminal end of the 25K cDNA contained seven unique repeat elements of 10 amino acids each, most of which are polar residues. Expression of the 25K mRNA was tissue-, development- and sex-specific. A 1.2-kb mRNA was detected using the 25K cDNA as a probe only in the fat body of adult females. The mRNA started to appear at day 4 after the insect molted to the adult and rapidly increased by day 6. The mRNA was absent in the ovarian follicle cells and fat body of adult males. In vitro transcription and translation of the 25K cDNA produced a protein that migrated around 32 kDa on sodium dodecyl sulfate polyacrylamide gels. The 25K cDNA was expressed in a baculovirus expression system and the protein produced also migrated around 32 kDa.

A simple and rapid method for cloning insect vitellogenin cDNAs

We describe a simple and rapid method for cloning insect vitellogenin (Vg) cDNAs. The method relies on the facts that insect Vg amino acid sequences can be aligned confidently along their entire lengths and that a short, highly conserved GL/ICG motif and up to nine cysteine residues that follow at conserved locations are present near the C-termini. An adaptor-ligated double-strand cDNA library is constructed from poly(A) + RNA prepared from vitellogenic female fat body tissues using a commercial kit, and subjected to PCR with each of the degenerate nucleotide sequences for the GL/ICG motif and the adaptor sequence as primers. The PCR products (0.7–0.9 kb, representing the 3′ portion) are cloned, the nucleotide sequences are determined, and the deduced amino acid sequences are aligned with the known insect Vg sequences starting from the GL/ICG motif. Gene-specific primers corresponding to the sequences near the 5′-termini of the initial clones and the adaptor sequence are employed to obtain the remaining 5′ portion of the Vg cDNAs. The method was successfully applied to the bean bug Plautia stali (Heteroptera), revealing three Vg genes.

Cloning of vitellogenin cDNA of the American cockroach, Periplaneta americana (Dictyoptera), and its structural and expression analyses.

A cDNA expression library constructed from poly (A)(+) RNA prepared from vitellogenic female fat body cells of the American cockroach, Periplaneta americana (Dictyoptera) was screened using a polyclonal antiserum against the 100-kD polypeptide(s) from the egg extract. A partial Vg cDNA clone was obtained and sequenced. The 5' end portion of the cDNA was then obtained by the RACE method, cloned, and sequenced. The combined complete Vg cDNA was 5,854 bp long and contained a single ORF encoding 1,896 amino acids. The entire deduced amino acid sequence was aligned confidently with those of the known insect Vgs. A GL/ICG motif, a number of cysteines at conserved locations following this motif, and a DGXR motif upstream of the GL/ICG motif were present near the C-terminal. The chemically determined N-terminal amino acid sequence of the 170-kD polypeptide from the egg extract completely matched the deduced sequence starting from just after one of the consensus (RXXR) cleavage sites, indicating the occurrence of post-translational cleavage in the fat body cells. The Vg gene begins to be expressed in the 2-day-old adult female fat body cells but is never expressed in ovaries or in male fat body cells. Hemolymph Vg was first detected by immunoblotting in 4-day-old adult females, 2 days after the beginning of gene expression. Western blot analysis of major yolk polypeptides in nine cockroach species belonging to the two superfamilies, Blattoidea and Blaberoidea, using the antisera against P. americana major yolk polypeptides showed that the similarities in Vn antigenicity are basically limited to within a superfamily.