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Differential vitellin polypeptide processing in insect embryos
Abstract
This review focuses on the current status of knowledge regarding the process of vitellogenin (Vg) uptake and vitellin (Vt) storage in insect oocytes. We also consider the overall morphology of the yolk sac as an embryonic organ allowing for both temporal and spatial differentiation of yolk degradation to provide the primary food supply for the embryo. In this context we describe the evidence that demonstrates the occurrence of Vt polypeptide processing and how it may be affected by maternally derived proteases stored in the yolk granules and by the ubiquitin–proteasome system in the cytosol. The extent of Vt polypeptide processing induced by these proteases will be correlated with the structural modifications affecting yolk granules and vitellophages in developing insect embryos. To accomplish these goals the ultrastructural and cytochemical composition of yolk granules during vitellogenesis and embryogenesis will be reviewed. Last but not the least, our current understanding of the role played by acidification of yolk granules in the activation of maternally derived proteases will also be examined.
... Vg of most insect species is synthesized extraovarially in fat body cells in a tissue-, sex-, and stage-specific manner, released into the hemolymph and then incorporated into the developing oocytes via recepter-mediated endocytosis (see reviews: Byrne et al., 1989;Raikhel and Dhadialla, 1992). During this course, the Vg molecules are modified through cleavage, glycosylation, lipidation, and phosphorylation (see reviews: Raikhel and Dhadialla, 1992;Hagedorn et al., 1998;Giorgi et al., 1999;Tufail et al., 2005). After incorporation into eggs, the Vgs represent a major component of egg yolk proteins and serve as storage proteins to provide a source of amino acids for the developing embryo. ...
... One possible function of insect Vns oligomerized in eggs would be binding lipids, in which inactive ecdysteroids are enclosed. The ecdysteroids are then released as Vns and undergo proteolysis during embryogenesis (Hagedorn et al., 1998;Giorgi et al., 1999). It has been proposed that the DG residues Table 1 for references). ...
... Vg is synthesized along the ribosomes that are associated with the rough endoplasmic reticulum and subsequently transferred to the Golgi apparatus and eventually packaged into secretary granules emerging from the trans-Golgi network (Mazzimi et al., 1989;Snigirevskaya et al., 1997;Giorgi et al., 1989Giorgi et al., , 1998Giorgi et al., , 1999. During this process, the Vg is known to be modified post-translationally by glycosylation, phosphorylation, lipidation and proteolytic cleavage (Kunkel et al., 1980;Brennan and Mahowald, 1982;Osir et al., 1986b;Takahashi, 1987;Della-Cioppa and Engelmann, 1987;Don-Wheeler and Engelmann, 1997;Sappington and Raikhel, 1998;Giorgi et al., 1999;Tufail et al., 2005). ...
This chapter focuses on the current knowledge regarding the primary structure and mechanisms of vitellogenin (Vg)
biosynthesis in insects. So far, Vg sequences have been reported from 20 insect species; seven of them belong to
Hemimetabola and thirteen belong to Holometabola. Comparison of the coding sequences revealed that some features
like GLI/CG motif, cystein residues, and a DGXR motif upstream of the GLI/CG motif, were highly conserved near the
carboxy terminal of all insect Vgs. Moreover, a consensus (R/K)XX(R/K) or RXXR cleavage sequence motif existed at
the amino-terminal of all sequences outside the Apocrita except for Lymantria dispar where it existed on the C–terminal.
Furthermore, this chapter shows how Vg post-transcriptional processing exhibits a complicated pattern in insects
belonging to different orders. We also provide a brief account of hormonal regulation of Vgs and their uptake by the
oocyte. The Vg genes are large and specify a single transcript of 6-7 kb encoding a primary Vg precursor of ~200-kD,
which undergoes proteolytic processing to generate subunits ranging from ~50-180-kD. Recent molecular studies have,
however, shown that the proteolytic cleavage of Vg is more complicated in hemimetabolous insects (cleaved into several
subunits: small, medium and large) than in those of holometabolous insects, where Vg molecule is cleaved into two
subunits (one large and one small). Exceptions are Vgs of the Suborder Apocrita (higher Hymenoptera) where the Vg
primary precursor remains uncleaved. Nevertheless, the yolk proteins of higher Diptera (such as Drosophila melanogaster)
form a different family of proteins and are also not cleaved.
... One possible function of insect Vns oligomerized in eggs would be binding lipids, in which inactive ecdysteroids are enclosed. The ecdysteroids are then released as Vns and undergo proteolysis during embryogenesis [11][12]. It was proposed that the DG residues (of DGXR motif) together with the GL/ICG motif and cysteine residues might participate in forming the structure necessary for Vns to function properly during embryogenesis [33]. ...
... Biosynthesis of insect Vgs is accomplished along the ribosomes that are associated with the rough endoplasmic reticulum and subsequently transferred to the Golgi apparatus and eventually packaged into secretary granules emerging from the trans-Golgi network [12,[75][76][77][78]. During this process, the Vg is known to be modified post-translationally by proteolytic cleavage glycosylation, phosphorylation, sulphation and lipidation [see 2,12,[14][15][79][80][81][82][83][84]. ...
... Biosynthesis of insect Vgs is accomplished along the ribosomes that are associated with the rough endoplasmic reticulum and subsequently transferred to the Golgi apparatus and eventually packaged into secretary granules emerging from the trans-Golgi network [12,[75][76][77][78]. During this process, the Vg is known to be modified post-translationally by proteolytic cleavage glycosylation, phosphorylation, sulphation and lipidation [see 2,12,[14][15][79][80][81][82][83][84]. ...
The embryonic development of all oviparous animals occurs isolated from the maternal body. The egg should therefore contain all necessary supplies for the development of the embryo until eclosion. This supply is called yolk and is composed of nucleic acids, proteins, lipids and carbohydrates, stored in an organized manner inside the egg. Most of the yolk protein components are taken up during oocyte development via receptor-mediated endocytosis. Major proteins accumulated by insect oocytes are the two hemolymph lipoproteins, vitellogenin (Vg) and lipophorin (Lp). Once sequestered, the Vg is sent to yolk bodies for processing and stored as vitellin (Vn), the main nutritional reserves for the future embryo, whereas Lp (the high density Lp, HDLp) after unloading lipids (which are stored as lipid droplets, the energy source for the embryo) is stored as a very HDLp (VHDLp). This chapter reviews the current state of knowledge on the major yolk protein precuros (YPPs), the Vg and Lp, in insects and their role in oogenesis. Besides the biochemical and molecular aspects, the hormonal regulation and the uptake mechanism of these YPPs by the developing oocytes through receptor-mediated endocytosis will be addressed. Also, we point out some of the important areas for future research.
... reviews Raikhel and Dhadialla, 1992;Hagedorn et al., 1998;Giorgi et al., 1999;Sappington et al., 2002;. After incorporation into oocytes, the Vgs are stored in a crystalline form as Vns, a reserve food-source for the future embryo. ...
... One possible function of oligomerized insect Vns could be binding lipids, in which inactive ecdysteroids are enclosed. The ecdysteroids are then released as Vns undergo proteolysis during embryogenesis (Hagedorn et al., 1998;Giorgi et al., 1999). It was proposed previously that the DG residues (of DGXR motif) together with GL/ICG motif and cysteine residues at conserved positions might form a structure necessary for Vns to function properly during embryogenesis (Tufail et al., 2001). ...
... In addition to proteolytic cleavage, the Vg molecules are coand post-translationally modified to facilitate the transport of carbohydrates, lipids, sulphates, and other nutrients in the hemolymph to the ovaries (see reviews Raikhel and Dhadialla, 1992;Hagedorn et al., 1998;Giorgi et al., 1999;. These conjugates not only support the embryo but also are involved somehow in internalization of the carrier into the oocyte. ...
Vitellogenins (Vgs) are precursors of the major egg storage protein, vitellin (Vn), in many oviparous animals. Insects Vgs are large molecules (∼200-kD) synthesized in the fat body in a process that involves substantial structural modifications (e.g., glycosylation, lipidation, phosphorylation, and proteolytic cleavage, etc.) of the nascent protein prior to its secretion and transport to the ovaries. However, the extent to which Vgs are processed in the fat body varies greatly among different insect groups. We provide evidence by cloning and peptide mapping of four Vg molecules from two cockroach species (Periplaneta americana and Leucophaea maderae) that, in hemimetabolous insects, the pro-Vg is cleaved into several polypeptides (ranging from 50-to 180-kD), unlike the holometabolans where the Vg precursor is cleaved into two polypeptides (one large and one small). An exception is the Vg of Apocrita (higher Hymenoptera) where the Vg gene product remains uncleaved. The yolk proteins (YPs) of higher Diptera (such as Drosophila) form a different family of proteins and are also not cleaved. So far, Vgs have been sequenced from 25 insect species; 9 of them belong to Hemimetabola and 16 to Holometabola. Alignment of the coding sequences revealed that some features, like the GL/ICG motif, cysteine residues, and a DGXR motif upstream of the GLI/CG motif, were highly conserved near the carboxy terminal of all insect Vgs. Moreover, a consensus RXXR cleavage sequence motif exists at the N-terminus of all sequences outside the Apocrita except for Lymantria dispar where it exists at the C-terminus. Phylogenetic analysis using 31 Vg sequences from 25 insect species reflects, in general, the current phylogenies of insects, suggesting that Vgs are still phylogenetically bound, although a divergence exists among them.
... Additionally, at least in some models, follicle cells themselves synthesize yolk proteins (Bast and Telfer, 1976;Isaac and Bownes, 1982;Melo et al., 2000). Yolk granules are mobilized during embryogenesis by acid hydrolases that are stored by the oocyte during oogenesis and become active through acidification of these organelles during embryogenesis (Fagotto, 1995;Giorgi et al., 1999). At the final stages In this work we characterized the degenerative process of ovarian follicles of the bug Rhodnius prolixus challenged with the non-entomopathogenic fungus Aspergillus niger. ...
... In many insect models, yolk granules become acidified during normal embryogenesis (Giorgi et al., 1999;Motta et al., 2004) and atresia (Uchida et al., 2001), leading to yolk degradation (Fagotto, 1995;Uchida et al., 2001;Kotaki, 2003), whereas no reports of these phenomena are known during normal oogenesis. Considering the resorptive phenotype observed in Fig. 2B-D, the acidification status of yolk granules in atretic follicles was addressed. ...
... In order to address the mechanisms involved in yolk resorption, the presence of serine-and cysteine-protease activities in extracts of healthy vitellogenic and atretic follicles was tested, since these proteases have already been implicated in yolk processing during follicle atresia in arthropod and mammal models (Takahashi et al., 1993;Giorgi et al., 1999;Uchida et al., 2001;Sriraman and Richards, 2004). To address a possible interference of secreted proteases of fungal origin, atretic follicles induced by Zymosan A administration were also tested. ...
In this work we characterized the degenerative process of ovarian follicles of the bug Rhodnius prolixus challenged with the non-entomopathogenic fungus Aspergillus niger. An injection of A. niger conidia directly into the hemocoel of adult R. prolixus females at the onset of vitellogenesis caused no effect on host lifespan but elicited a net reduction in egg batch size. Direct inspection of ovaries from the mycosed insects revealed that fungal challenge led to atresia of the vitellogenic follicles. Light microscopy and DAPI staining showed follicle shrinkage, ooplasm alteration and disorganization of the monolayer of follicle cells in the atretic follicles. Transmission electron microscopy of thin sections of follicle epithelium also showed nuclei with condensed chromatin, electron dense mitochondria and large autophagic vacuoles. Occurrence of apoptosis of follicle cells in these follicles was visualized by TUNEL labeling. Resorption of the yolk involved an increase in protease activities (aspartyl and cysteinyl proteases) which were associated with precocious acidification of yolk granules and degradation of yolk protein content. The role of follicle atresia in nonspecific host-pathogen associations and the origin of protease activity that led to yolk resorption are discussed.
... They are then released into the haemolymph as oligomeric proteins with molecular weights ranging from 300 to 600 kDa (Tufail and Takeda, 2008;Wheeler et al., 1999). These protein aggregates are then transferred to oocytes via receptor-mediated endocytosis and stored in the form of crystals, at which time they are termed vitellins (Giorgi et al., 1999;Raikhel and Dhadialla, 1992). ...
... The difference in vitellin processing found between queen and worker eggs of E. tuberculatum may be due the absence of active proteases in worker's eggs or the absence of their activation inside the trophic eggs, which have no embryonic development (Giorgi et al., 1999;Khila and Abouheif, 2008). In the course of embryonic development, the vitellin peptides undergo specific cleavages inside the oocyte resulting in new smaller peptides. ...
... In the course of embryonic development, the vitellin peptides undergo specific cleavages inside the oocyte resulting in new smaller peptides. These cleavages occur due proteases associated with the yolk granules, that may be synthesized inside the oocyte or extraovarially, and activated during the embryonic development (Giorgi et al., 1999). ...
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.
... Among them, cathepsin B, cathepsin D and acid phosphatase have been frequently studied. In general, these enzymes are stored as an inactive form of a proenzyme in small yolk granules, but activated by the acidification of yolk granules, and then participate in digesting yolk proteins during embryonic development [33]. Analogous regulatory mechanisms in the degradation of yolk proteins are described in many species such as Spodoptera exigua [15], Dipetalogaster maxima [14], Blatella germanica [34], R. prolixus [16], Bombyx mori [8], Musca domestica [35] and Culex quinquefasciatus [9]. ...
... All three enzymes showed high activity in the eggs, but the activity patterns were not consistent with the expression profiles. The accumulation of three enzyme transcripts levels and high activity in the eggs may suggest their participation in the proteolytic process, where Vn is partially cleaved into small peptides and free amino acids [33]. ...
Background
Three main enzymes including cathepsin B, cathepsin D and acid phosphatase are involved in vitellin degradation, which is a major biochemical event of the embryonic development and can provide nutrients and metabolites for tick embryos. In the present study, the mRNA expression profiles and enzymatic activity of cathepsin B, cathepsin D and acid phosphatase were investigated during embryonic development in the tick Haemaphysalis longicornis.
Results
The results revealed that all three enzymes were expressed throughout embryonic development. Both cathepsin B and acid phosphatase transcripts were accumulated during the first four days. Cathepsin B reached its highest expression on day 5, whereas the peak expression of acid phosphatase and cathepsin D occurred on day 11. The highest activity of cathepsin B was observed on the first day of egg development, whereas cathepsin D reached its highest activity on day 13. Acid phosphatase activity increased gradually during the first five days and then remained stable until the end of egg development.
Conclusions
Three enzymes were expressed and activated in eggs, and also presented different dynamic changes with the development of embryos. The profiles of both mRNA expression and enzymatic activity of these enzymes indicate that they are controlled orderly and play multiple roles during embryonic development in ticks.
... Insect vitellogenins are proteins synthesized mainly in the female fat body and released into the haemolymph (Tufail and Takeda 2008, Azevedo et al. 2011), which are later transferred to oocytes via receptor-mediated endocytosis (Tufail and Takeda 2009) and stored as vitellins in yolk granules (Raikhel and Dhadialla 1992, Giorgi et al. 1999). ...
... The vitellins are the major egg yolk proteins in insects and are important for embryo nourishment (Raikhel and Dhadialla 1992, Giorgi et al. 1999, Tufail and Takeda 2008). ...
In the ant Ectatomma tuberculatum (Olivier, 1792), workers have active ovaries and lay trophic eggs that are eaten by the queen and larvae. Vitellogenins are the main proteins found in the eggs of insects and are the source of nutrients for the embryo in the fertilized eggs and for adults when in the trophic eggs. In social insects, vitellogenin titers vary between castes and affect reproductive social status, nursing, foraging, longevity, somatic maintenance, and immunity. In most insects, vitellogenin synthesis is mainly regulated by juvenile hormone. However, in non-reproductive worker ants, this relationship is poorly characterized. This study determined the effects of juvenile hormone on vitellogenin synthesis in non-reproductive E. tuberculatum workers. Juvenile hormone was topically applied onto workers, and the effect on vitellogenin synthesis in the fat body and vitellogenin titers in the haemolymph were analyzed by ELISA and qPCR. Juvenile hormone downregulated protein synthesis and reduced vitellogenin titers in the haemolymph, suggesting that in workers E. tuberculatum, juvenile hormone loses its gonadotrophic function.
... The Vgs have been studied extensively in various animals from vertebrates to invertebrates (Nardelli et al., 1987;Chen et al., 1997;Tufail and Takeda, 2008) and some highly conserved structures related with biological functions of Vgs Trewitt et al., 1992;Chen et al., 1997;Lee et al., 2000), such as polyserine tracts, DGXR motif, subtilisin-like endoproteases site (R/KXXR/K) and GL/ICG motif (Taborsky, 1991;Dhadialla et al., 1992;Mayadas and Wagner, 1992;Giorgi et al., 1999;Tufail et al., 2005) are found. The insect Vgs are synthesized mainly in fat body and have the tissue-, stage-and sex-specific characteristics Sappington et al., 2002). ...
... Vg is the precursor of vitellin and plays an important role in egg development. During this course, Vgs are processed by proteolytic cleavage, glycosylation, phosphorylation and sulphation at the post-transcriptional level Hagedorn et al., 1998;Giorgi et al., 1999). However, the process of Vg and the number of Vg gene vary in different insects (Tufail and Takeda, 2008). ...
In this study, the DNA sequence of vitellogenin from Antheraea pernyi (Ap-Vg) was identified and its functional domain (30-740 aa, Ap-Vg-1) was expressed in Escherichia coli BL21 (DE3) cells. The recombinant Ap-Vg-1 proteins were purified and used for antibody preparation. The results showed that the intact DNA sequence of Ap-Vg consisted of 8124 bp including 6 exons (2258, 205, 982, 879, 184 and 829 bp) and 5 introns (84, 78, 410, 1055 and 795 bp) and was highly homologous to the vitellogenin from Antheraea yamamai. SDS-PAGE and western blot analysis demonstrated that a 85 KD recombinant protein was successfully expressed in E. coli cells and its expression was not remarkably changed under induction by different IPTG concentration. The titre of antibodies raised against rabbits was about 1:7800 which was determined by ELISA.
... Como visto neste conjunto de dados, os estudos têm estabelecido quase conclusivamente que um sistema geral para degradação de vitelo durante o desenvolvimento embrionário, em uma ampla faixa de espécies de artrópodes, envolve a ativação de prócisteíno proteinases que se apresentam associadas a proteínas de reserva [38]. Menos conhecido é o mecanismo que leva à ativação destas enzimas. ...
... Além da ativação por acidificação, como citado anteriormente, existem outras hipóteses como o controle transcricional das enzimas responsáveis por esta hidrólise [44]; a proteólise limitada das pró-pro-teinases latentes, ou a remoção de inibidores específicos [31]. A fosforilação da vitelina parece ser um fator controlador de sua suscetibilidade à proteólise, sendo que em B. mori a fosforilação da Vt aumenta sua degradação por cisteíno proteinase [38]. ...
Background: Ticks are distributed worldwide, with impacts on human and animal health. The cattle tick Rhipicephalus (Boophilus) microplus is the main parasite that affects livestock in tropical and subtropical regions of the world, causing large economical losses. Tick control methods are based on the application of chemical acaricides, which has resulted in selection of resistant ticks and a potential risk of environmental pollution and food contamination. Vaccines have showed to be a feasible tick control method that offers a cost-effective, environmental friendly alternative to chemical control. However, more than ten years after the commercialization of the first vaccine against ticks, the identification of tick-protective antigens remains a limiting step in the development of an efficient formulation that would avoid the use of chemical acaricides. So, the study of parasite biology and understanding physiological mechanisms could be a good strategy to find new targets for an efficient vaccine. Review: It was reviewed the main insights about the reproductive process in ticks, emphasizing the hormonal control of vitellogenesis and enzymes involved in vitellin processing during embryogenesis. The processes of vitellogenesis and embryogenesis have been studied in various organisms, particularly in cockroaches, flies and ticks. Although the roles of 20-hydroxyecdysone (20E) and juvenile hormone have been well characterized for vitellogenesis in insects, we know much less about the hormonal control of vitellogenesis in ticks. Initially, it was hypothesized that juvenile hormone was involved in tick vitellogenin-synthesis. However, more critical studies uncovered no evidence for the occurrence of juvenile hormone or juvenile hormone-like molecules in several tick species. Current research shows that in ticks, it appears that ecdysteroids, and not juvenile hormone, regulate the expression of the vitellogenin gene and the synthesis and release of vitellogenin protein into the hemolymph. In general, the carbohydrate, lipid and amino acid composition of tick vitellogenin is similar to that of insect vitellogenin. Once in the hemolymph, oocytes uptake vitellogenin through receptor-mediated endocytosys. However, there are different strategies to control vitellogenin synthesis and uptake by ovary in ixodide ticks. In the oocytes, vitellogenin is partially processed in the endosomal compartment and then stored as vitellin, the main reserve of protein for embryo development, in specialized organelles, the yolk granules. Embryo development depends on the availability of yolk material stored into oocytes. So, the characterization of molecules involved in vitellogenesis and embryo development contribute to a better understanding of the tick parasite physiology. During embryogesesis, acidic enzymes are responsible for the availability of this material and embryo nutrition. The Vitellin-Degrading Cysteine Endopeptidase (VTDCE), Boophilus Yolk Pro-Cathepsin (BYC) and Tick Heme Binding Aspartic Proteinase (THAP) are enzymes involved in vitellin hydrolysis in R. microplus eggs. These enzymes are produced by gut and fat body and transported through the hemolymph to be internalized into the oocytes and then play their role in tick embryo nutrition. As VTDCE, BYC and THAP are involved in an important physiological process, their potential as targets in an anti-tick vaccine is an attractive research topic. With this objective, various enzymes have been tested in native or recombinant forms as candidate immunogens to a multiantigenic anti-tick vaccine. Conclusion: Significant advancements have been made in recent years on understanding the tick reproductive process, and some molecules that can be possible targets for development of new tick control strategies have been characterized.
... Como visto neste conjunto de dados, os estudos têm estabelecido quase conclusivamente que um sistema geral para degradação de vitelo durante o desenvolvimento embrionário, em uma ampla faixa de espécies de artrópodes, envolve a ativação de prócisteíno proteinases que se apresentam associadas a proteínas de reserva [38]. Menos conhecido é o mecanismo que leva à ativação destas enzimas. ...
... Além da ativação por acidificação, como citado anteriormente, existem outras hipóteses como o controle transcricional das enzimas responsáveis por esta hidrólise [44]; a proteólise limitada das pró-pro-teinases latentes, ou a remoção de inibidores específicos [31]. A fosforilação da vitelina parece ser um fator controlador de sua suscetibilidade à proteólise, sendo que em B. mori a fosforilação da Vt aumenta sua degradação por cisteíno proteinase [38]. ...
Background: Ticks are distributed worldwide, with impacts on human and animal health. The cattle tick Rhipicephalus (Boophilus) microplus is the main parasite that affects livestock in tropical and subtropical regions of the world, causing large economical losses. Tick control methods are based on the application of chemical acaricides, which has resulted in selection of resistant ticks and a potential risk of environmental pollution and food contamination. Vaccines have showed to be a feasible tick control method that offers a cost-effective, environmental friendly alternative to chemical control. However, more than ten years after the commercialization of the first vaccine against ticks, the identification of tick-protective antigens remains a limiting step in the development of an efficient formulation that would avoid the use of chemical acaricides. So, the study of parasite biology and understanding physiological mechanisms could be a good strategy to find new targets for an efficient vaccine. Review: It was reviewed the main insights about the reproductive process in ticks, emphasizing the hormonal control of vitellogenesis and enzymes involved in vitellin processing during embryogenesis. The processes of vitellogenesis and embryogenesis have been studied in various organisms, particularly in cockroaches, flies and ticks. Although the roles of 20-hydroxyecdysone (20E) and juvenile hormone have been well characterized for vitellogenesis in insects, we know much less about the hormonal control of vitellogenesis in ticks. Initially, it was hypothesized that juvenile hormone was involved in tick vitellogenin-synthesis. However, more critical studies uncovered no evidence for the occurrence of juvenile hormone or juvenile hormone-like molecules in several tick species. Current research shows that in ticks, it appears that ecdysteroids, and not juvenile hormone, regulate the expression of the vitellogenin gene and the synthesis and release of vitellogenin protein into the hemolymph. In general, the carbohydrate, lipid and amino acid composition of tick vitellogenin is similar to that of insect vitellogenin. Once in the hemolymph, oocytes uptake vitellogenin through receptor-mediated endocytosys. However, there are different strategies to control vitellogenin synthesis and uptake by ovary in ixodide ticks. In the oocytes, vitellogenin is partially processed in the endosomal compartment and then stored as vitellin, the main reserve of protein for embryo development, in specialized organelles, the yolk granules. Embryo development depends on the availability of yolk material stored into oocytes. So, the characterization of molecules involved in vitellogenesis and embryo development contribute to a better understanding of the tick parasite physiology. During embryogesesis, acidic enzymes are responsible for the availability of this material and embryo nutrition. The Vitellin-Degrading Cysteine Endopeptidase (VTDCE), Boophilus Yolk Pro-Cathepsin (BYC) and Tick Heme Binding Aspartic Proteinase (THAP) are enzymes involved in vitellin hydrolysis in R. microplus eggs. These enzymes are produced by gut and fat body and transported through the hemolymph to be internalized into the oocytes and then play their role in tick embryo nutrition. As VTDCE, BYC and THAP are involved in an important physiological process, their potential as targets in an anti-tick vaccine is an attractive research topic. With this objective, various enzymes have been tested in native or recombinant forms as candidate immunogens to a multiantigenic anti-tick vaccine. Conclusion: Significant advancements have been made in recent years on understanding the tick reproductive process, and some molecules that can be possible targets for development of new tick control strategies have been characterized.
... It is synthesized by the fat body and midgut and partially in the ovary. It is transported to the ovary by hemolymph, where it is processed into Vn by the oocytes, which are stored in the yolk granules that provide nutrients for the embryonic development of ticks (Coons et al. 1989;Giorgi et al. 1999;Rosell and Coons 1991). Cluster-339.20526 ...
The majority of ixodid ticks display host-specificity to varying extents. Feeding on different hosts affects their development and reproduction. Consequences can be analyzed at the level of the egg, as it is the initial stage of tick development. Tick egg proteins are abundant and diverse, providing nutrients for embryonic development. However, studies on tick egg profiles are scarce. In this study, we aimed to analyze whether feeding Haemaphysalis qinghaiensis ticks on the yaks (Bos grunniens) and domestic sheep (Ovis aries) has an impact on the variety and variability of the egg proteome. Detached engorged females were used to lay eggs, which were then collected, dewaxed, and subjected to protein extraction. The extracted egg proteins were enzymatically digested using Filter-Aided Sample Preparation (FASP), and the unique peptides were separated and detected by Liquid Chromatography-tandem Mass Spectrometry (LC–MS/MS). The MS data were searched against the previously constructed whole tick transcriptome library of H. qinghaiensis, and the UniProt database for the identification of tick-derived egg proteins. The analysis revealed 49 and 53 high-confidence proteins identified in eggs collected from B. grunniens (EggBg) and O. aries (EggOa), respectively. Of these, 46 high-confidence proteins were common to both egg types, while three were unique to EggBg and seven to EggOa. All the identified proteins mainly belonged to enzymes, enzyme inhibitors, transporters, and proteins with unknown functions. The differential abundance analysis showed that nine proteins were significantly more present in EggBg, while six were significantly more present in EggOa. Overall, enzymes were the most diverse group, while vitellogenin (Vg) was the most abundant. Blood meal uptake on different hosts has a certain effect on the egg proteome composition and the abundance of some proteins, but it may also lead to compensation of protein roles.
... 23 Actually, in the reproductive process of female insects, the production of Vg and its uptake by the oocyte are critical for successful reproduction. 24,25 This process is jointly regulated by multiple factors, including hormone pathways [mainly juvenile hormone (JH) and 20-hydroxyecdysone], nutrient signaling [amino acid/Target of Rapamycin (TOR)] pathways, growth pathways (insulin signaling pathway), energy pathways, transcription factors and others. 20,26,27 Reproduction in female insects, including S. furcifera, is a highly complex process that involves the coordinated action of multiple factors. ...
BACKGROUND
The white‐backed planthopper (WPH), Sogatella furcifera (Horváth), is a destructive rice pest with strong reproductive capacity. To gain insights into the roles of chitinases in the reproductive process of this insect species, this study represents the first‐ever endeavor to conduct an in‐depth exploration into the reproductive functions of four chitinase genes.
RESULTS
In this study, it was observed that four chitinase genes were expressed in female adults, with a relatively high expression level in the ovaries. SfCht2 and SfIDGF1 were highly expressed during later ovarian development. while SfENGase increased and then decreased with ovarian development. SfCht2, SfCht6‐2 and SfENGase were highly expressed in fat body on the first and second days after eclosion, whereas SfIDGF1 highest on day 7. Compared with control group, Silencing four chitinase genes inhibited ovarian development and significantly shortened the oviposition period of S. furcifera, reducing egg‐laying capacity but not affecting egg hatching. The detection demonstrated that the expression levels of SfVg, SfVgR and 70–90% juvenile hormone (JH) signaling pathway‐related reproductive genes was significantly down‐regulated. Moreover, SfCht6‐2 and SfENGase significantly affected the expression levels of Target of Rapamycin (TOR) signaling pathway genes. SfENGase had the ability to impact nutrient signaling pathways and fatty acid metabolism, repressing vitellogenin synthesis and ultimately influencing ovarian development of S. furcifera.
CONCLUSIONS
Overall, this study provides insight into the function of chitinases in insect fecundity and is of great significance for enriching the cognition of insect chitinase function. They will become the suitable target genes for controlling the most destructive rice planthoppers. © 2023 Society of Chemical Industry.
... A variety of proteolytic enzymes act precisely and orderly to ensure the embryo successfully develop (Giorgi et al. 1999). Cathepsin B, cathepsin D, and acid phosphatase were all involved in the process of tick embryo development. ...
The effects of temperature on the expression patterns and enzyme activity of cathepsin B (HlCatB), cathepsin D (HlCatD) and acid phosphatase (HlACP) during the embryo development of Haemaphysalis longicornis (bisexual population) were investigated in this study. Eggs were exposed to 20 °C (low temperature), 26 °C (normal temperature), and 30 °C (high temperature) immediately after laying, and collected on odd days of embryo development to measure HlCatB, HlCatD and HlACP gene expression using quantitative real-time PCR, as well as three enzyme activities using spectrophotometry. Then the associations between mRNA expression levels of three enzymes and their enzyme activities were assessed. Compared with normal temperature, the mRNA expression peaks of HlCatB were higher and appeared later at low and high temperatures and the activity of HlCatB increased on most days of embryonic development at high temperature. As for HlCatD, the expression peak appeared later at low temperature, but earlier at high temperature. The activity peaks of HlCatD were lower and appeared earlier at low and high temperatures. As for HlACP, the expression peak was higher and appeared later at low temperature, whereas it formed no prominent peak at high temperature. The activity peak of HlACP was higher at low temperature, but lower at high temperature. The linear regression analysis showed that activities of three enzymes were associated with their mRNA expression levels (P < 0.05). Three enzymes are involved in the embryo adaptation to temperature stress. Moreover, the mRNA expression level may be another factor affecting its enzyme activity.
... Similar to previous reports, SfVg had conserved domains of the LLTP superfamily, including the LPD_N, VWD, and DUF1943 domains [3,17,27]. The motifs GL/ICG and DGXR in the amino acid sequence of SfVg were also highly conserved domains in insect Vg proteins, which was regarded as a necessary condition for oligomerization [28,29]. The Vg protein can bind inactive ecdysone-containing lipids via oligomerization and release ecdysone during embryogenesis [30]. ...
The fall armyworm Spodoptera frugiperda is a highly polyphagous invasive pest. The strong reproductive capacity is an important factor in the rapid colonization and expansion of S. frugiperda. Vitellogenin (Vg) and vitellogenin receptor (VgR) play important roles in insect reproduction. As the precursor of vitellin (Vn), Vg provides essential nutrition for embryonic development, and VgR mediates the uptake of Vg by oocytes. In this context, we cloned and characterized these two genes of S. frugiperda (SfVg and SfVgR) and evaluated their expression profiles in different developmental stages and tissues. The RNA interference experiment was used to investigate their function in vitellogenesis. The ORF values of SfVg and SfVgR were 5250 and 5445 bp, encoding 1749 and 1815 amino acid residues, respectively. The qRT-PCR results revealed that both SfVg and SfVgR were highly expressed in female adults; SfVg was specifically expressed in the fat body, whereas SfVgR was highly expressed in the ovary. In addition, the depletion of either SfVg or SfVgR hindered oocyte maturation and ovarian development, leading to a significant decrease in fecundity. The present study reveals the importance of SfVg and SfVgR in the vitellogenesis of S. frugiperda, laying a theoretical foundation for the development of pollution-free pest control strategies with SfVg and SfVgR as new targets.
... We now know that A. mellifera produce only a single Vg molecule and that Vgs are distinct between species. They are absorbed into developing ova by a receptor-mediated processes specific enough even to exclude Vgs from closely related species (18,19). As such, it is highly unlikely that organisms as distantly related as an insect and its parasitic mite would have Vgs similar enough to utilize the same Vg receptor, making honey bees an implausible donor for mite egg yolk precursors (20,21). ...
The rapid reproductive capacity of Varroa destructor is among the most significant adaptations underpinning its success as a parasite. To exploit their honey bee host, the parasite must rapidly produce offspring that fully develop into adults and mate in an inflexible 9-day window. Inability to meet this deadline brings the fitness of the foundress mite to zero establishing heavy evolutionary pressure to accelerate reproduction & subsequent development of offspring. Our work fills in gaps in our understanding of a key pathway in this process. Varroa have a poorly-understood ability to pass heretofore unidentified host proteins through their body with minimal digestion/degradation. Via Native-PAGE, we were able to confirm that nine proteins shared with honey bee fat body tissue accumulate intact in the eggs of the foundress mite. As such, we hypothesized that the proteins were several egg yolk precursors synthesized and stored in the fat body. Using antibodies raised against honey bee vitellogenin (Vg) we positively identified this egg yolk precursor via SDS-PAGE and subsequent Western Blot. We then analyzed samples of honey bee fat body tissue, gravid Varroa, so-called phoretic Varroa, and Varroa eggs via HPLC MS/MS to identify the remaining host proteins and determine their relative abundance. We detected egg yolk precursors in the large lipid transfer protein superfamily, in addition to hexamerin storage proteins, and miscellaneous motor/transfer proteins integral to embryonic development (transferrin, myosin heavy chain, and heat shock protein 60). Varroa lack the capacity to produce some of these proteins and instead employ kleptoparasitism on a molecular level to provision their developing ova, a pathway not described in any other host/parasite relationship, hereafter referred to as kleptocytosis. These different families of proteins are normally produced by the female and conveyed to the vitellogenic egg cell through protein specific receptor-mediated pathways. Such pathways would exclude foreign proteins. We hypothesize that the need to rely on a receptor-mediated pathway is circumvented via the specialized nutritive reproductive tissue, the lyrate organ. Through microCT imaging we detail the connection between the developing ovum and this dual-lobed organ. Better understanding of this pathway presents a novel target for Varroa management as the treatment need only accomplish slowing acquisition or deposition of host proteins thereby disrupting the ability of the mite to meet the temporal demand of its host.
... The Vg gene encodes the precursor of vitellogenin, which is mainly synthesized in the adipocytes and then released into the hemolymph of female insects. After selective absorption by oocytes, Vg is modified in the egg and finally deposited as vitellin (Vt), which provides the nutrients needed for embryonic development [26][27][28][29][30]. In female BPH adults, it has been shown that elevated levels of Exu expression reduce ovary development, and that Exu and Mag have similar functions, whereas Vas gene expression increased during ovary development [31]. ...
Drought stress greatly impacts insect development and population growth. Some studies have demonstrated increased reproductive capacity in drought-stressed insects; however, physiological changes in the brown planthopper (BPH), Nilaparvata lugens (Stål), during periods of drought are unclear. In this study, BPH fed on drought- stressed rice had lower population numbers than BPH feeding on non-stressed rice. Water content, osmotic pressure of hemolymph and total amino acid content of BPH were significantly lower when BPH fed on drought-stressed rice compared to the non-stressed control; however, glucose content and glutathione S-transferase (GST) activity were significantly higher in BPH fed on drought-stressed rice. The expression of Vitellogenin and Exuperantia in BPH fed on drought-stressed rice was higher than that in BPH feeding on non-stressed control plants. The size of myofibrils and the abundance of mitochondria in BPH flight muscles were significantly lower in BPH fed on drought-stressed rice compared to non-stressed plants. These results indicate that water management impacts the physiology of BPH, which may be useful in understanding the relationship between drought stress and this damaging herbivore.
... Vitellogenin (Vg) is an indispensable reproduction-related protein that is traditionally identified as an appropriate parameter for appraising female fertility in insects [27]. In most insects, Vg is mainly synthesized by fat bodies or other tissues-for example, ovarian tissues [28]-secreted to the hemolymph, binds to the vitellogenin receptor, and finally enters the cell through endocytosis after reaching the oocyte's surface [29,30]. In our laboratory, a resistant strain (SE-Sel) of S. exigua was established through continuous selection with a sublethal dose of chlorantraniliprole, and a fitness cost accompanied with chlorantraniliprole resistance was found. ...
Spodopteraexigua, a multifeeding insect pest, has developed a high level of resistance to chlorantraniliprole, which is a benzoylurea insecticide that targets the ryanodine receptors (RyRs). Herein, the resistant strain (SE-Sel) and sensitive strain (SE-Sus) were obtained by bidirectional screening for six generations. The potential oviposited eggs and oviposition rate of the SE-Sel strain were dramatically lower than those of the SE-Sus strain; on the contrary, the weights of prepupae and preadult were significantly increased. As a post-mating response, the higher number of non-oviposited eggs in the SE-Sel strain was caused by a lower mating rate. In addition, the expression levels of vitellogenin (SeVg) and its receptor (SeVgR) in the SE-Sel strain were consistently lower than those in the SE-Sus strain. An RyRI4743M mutation, contributing to the resistance to chlorantraniliprole, was located in the S3 transmembrane segments and might have affected the release of calcium ions; it led to the upregulated expression of the neuropeptide SeNPF and its receptor SeNPFR, and the mating and oviposition rate were significantly recovered when the SeNPF was knocked down though RNA interference (RNAi) in the male adult of the SE-Sel strain. Moreover, the expression of the juvenile hormone-binding proteins SeJHBWDS3 and SeJHBAN in the male adult of the SE-Sel strain was significantly decreased, which proved the existence of a fitness cost from another angle. Therefore, these results indicate that the fitness cost accompanied by chlorantraniliprole resistance in S. exigua may be related to the decrease in mating desire due to SeNPF overexpression.
... 29 In most insects, Vg is mainly synthesized by the ovarian tissues, 30 or by the fat bodies, binds to the vitellogenin receptor (VgR) 31 and finally enters the cell through endocytosis. 32,33 In our laboratory, a resistant strain (Bis-SEL) of S. litura was established through continuous screening with increasing concentrations of bistrifluron. The resurgence associated with the insecticide resistance was found by Huang et al. 20 Identifying the resurgence associated with insecticide resistance can be useful for designing an integrated pest management (IPM) program that limits the spread of the resistant population. ...
BACKGROUND
Spodoptera litura is one of the major agricultural pests in China, and it has developed serious resistance to many traditional chemical insecticides. In the present study, the bistrifluron‐resistant (Bis‐SEL) strain accompanied by a higher oviposition, 113.8‐fold RR compared to the bistrifluron‐susceptible (Bis‐UNSEL) strain, was obtained by bidirectional screening. A comparison of their gonad coefficiency and genes related to oviposition or resistance was used to elucidate the resurgence mechanism.
RESULTS
The ovarian index, oviposition, and potential egg production in the Bis‐SEL strain of female adults were significantly higher than those in the Bis‐UNSEL strain, and the length of ovariole in the Bis‐SEL strain was also significantly elongated. The protein contents of vitellogenin (Vg) and vitellogenin receptor (VgR) in the Bis‐UNSEL strain were lower than those in the Bis‐SEL strain, consistent with their gene expressions levels, and there was a significantly positive linear correlation between Vg and VgR protein contents, further confirming that resistant strains have high reproductive fitness. Moreover, the chitin synthase A in the Bis‐SEL strain was clearly up‐regulated, and a mutation (H866Y) near the QRRRW in the catalytic domain caused a rise in the hydrogen bond between UDP‐GlcNAc and chitin synthase, and its chitin content was higher than that in the Bis‐UNSEL strain. Nevertheless, the sensitivity of the Bis‐SEL strain to bistrifluron was significantly recovered when it was knocked down though RNA interference.
CONCLUSION
The fitness advantages of bistrifluron resistance may be related to the up‐regulation and mution of chitin synthase A. © 2021 Society of Chemical Industry.
... Proteolytic enzymes involved in hydrolysis are temporally controlled to ensure the appropriate and orderly breakdown of yolk proteins in the embryos (Zhang et al., 2019). In general, these hydrolases are stored in small yolk granules in an inactive form and then activated to break down yolk proteins at specific stages during embryonic development (Giorgi et al., 1999). We speculate that KNRL plays an essential role in the embryo by coordinating the process of Vn breakdown and thus affecting the temporal expression of genes that encode proteolytic enzymes. ...
Vitellin (Vn) homeostasis is central to the fecundity of oviparous insects. Most studies have focused on the synthesis and transportation of Vn as a building block for developing eggs during vitellogenesis; however, less is known about how the utilization of this nutrient reserve affects embryonic development. Here, we show that the single ortholog of the knirps and knirps‐like nuclear receptors, KNRL, negatively regulates Vn breakdown by suppressing the expression of hydrolase genes in the brown planthopper, Nilaparvata lugens. KNRL was highly expressed in the ovary of adult females, and knockdown of KNRL by RNA interference resulted in the acceleration of Vn breakdown and the inhibition of embryonic development. Transcriptome sequencing analysis revealed that numerous hydrolase genes, including cathepsins and trypsins were up‐regulated after KNRL knockdown. At least eight of the nine significantly enriched Gene Ontology terms for the up‐regulated genes were in proteolysis‐related categories. The expression levels of five selected trypsin genes and the enzymatic activities of trypsin in the embryos were significantly increased after KNRL knockdown. Moreover, trypsin injection prolonged egg duration, delayed embryonic development, accelerated Vn breakdown and severely reduced egg hatchability, a pattern similar to that observed in KNRL‐silenced N. lugens. These observations suggest that KNRL controls Vn breakdown in embryos via the transcriptional inhibition of hydrolases. Generally, this study provides a foundation for understanding how embryo nutrient reserves are mobilized during embryogenesis and identifies several genes and pathways that may prove valuable targets for pest control.
... After that, a series of regulatory processes of vitellogenesis are carried out, which requires the coordination of various proteins, so as to achieve the rapid and efficient formation of eggs. Vn is hydrolyzed into raw materials such as amino acids needed by embryo development under the action of various hydrolytic enzymes in ovaries (Giorgi et al. 1999). In insect, many hydrolytic enzymes related to Vn degradation, such as aspartic protease, cysteine protease, serine protease, and carboxypeptidase, have been extensively investigated (Guo et al. 2019). ...
Haemaphysalis longicornis is an ixodid tick that can spread a wide variety of pathogens, affecting humans, livestock and wildlife health. The high reproductive capability of this species is initiated by the ingestion of a large amount of blood ingested by the engorged female tick. The degree of ovarian development is proportional to the number of eggs laid. Studying the regulatory mechanism of tick ovary development is relevant for the development of novel tick control methods. In this study, we used quantitative proteomics to study the dynamic changes in protein expression and protein phosphorylation during ovarian development of engorged female H. longicornis ticks. Synergistic action of many proteins (n = 3031) is required to achieve ovarian development and oocyte formation rapidly. Through bioinformatics analysis, changes in protein expressions and phosphorylation modifications in regulating the ovarian development of female ticks are described. Many proteins play an essential role during ovarian development. Also, protein phosphorylation appeared an important reproductive strategy to enable ticks to efficiently convert large amounts of blood in the ovaries into egg-producing components and ultimately produce many eggs.
... Vitellogenin (Vg), a yolk protein precursor, is mainly synthesized in the fat body and released into the hemolymph after being modified by glycosylation, phosphorylation, proteolytic cleavage, sulphated into glycolipoproteins and cleaved into large and small subunits (Tufail and Takeda, 2008). Vg is taken up into developing oocytes by vitellogenin receptor (VgR) mediated endocytosis (Giorgi et al., 1999). ...
The yolk protein precursor, vitellogenin (Vg), provides nutrition for embryonic development whereas the vitellogenin receptor (VgR) is responsible for the uptake of yolk protein by maturing oocytes. These two proteins are key reproduction-related proteins in insects. We cloned and characterized Vg and VgR genes in Sogatella furcifera, and investigated their function in oocyte maturation. Cloned SfVg and SfVgR have open reading frames of 6,114 and 5,796 bp, encoding 2,037 and 1,931 amino acid residues, respectively. Structural analysis indicates that SfVg has the three conserved LPD_N, DUF1943, and VWFD domains, SfVgR contains all conservative motifs of the LDLR superfamily. Both genes were highly expressed in adult females; SfVg was most highly expressed in the fat body whereas SfVgR was mainly expressed in the ovary. Knockdown of either gene reduced yolk protein deposition in oocytes and arrested oocyte maturation. However, silencing one of these two genes did not affect the transcript level of the other. These results demonstrate the role of SfVgR in transporting SfVg into oocytes. Both SfVg and SfVgR are essential for oocyte maturation in S. furcifera and both genes could potentially be targeted as means of controlling this pest.
... In hard ticks, Vg is a high molecular mass-precursor that is synthesized in the fat bodies and midgut [5][6][7][8][9][10]. Vg is released into the hemolymph and is taken up by oocytes via a receptor mediated endocytosis [11]. Vg accumulates inside oocytes yolk granules as vitellin, which is a critical nutrient for tick embryo development [12]. ...
Background
Rhipicephalus microplus is an efficient biological vector of Babesia bovis, a causative agent of bovine babesiosis. Babesia bovis is passed transovarially to the next generation of ticks, which then transmit the parasite to naïve animals. Due to the importance of the R. microplus ovary for tick reproduction and transmission of B. bovis, we investigated the hypothesis that silencing vitellogenin receptor gene expression in the ovary during tick feeding on B. bovis-infected cattle would affect parasite transmission to the next generation of ticks.
Results
Silencing expression of the vitellogenin receptor in the ovary by RNA interference, resulted in reduced tick fertility. We observed reduced egg production (i.e. reduced weight of eggs), a lower rate of embryonic development, and a reduction in hatching. Analysis of individual larvae by PCR confirmed that RNAi mediated downregulation of the R. microplus vitellogenin receptor and also interfered with transovarial transmission of B. bovis. None of the larvae (0/58) from the RmVgR dsRNA-injected group were PCR-positive, whereas 12% (7/58) and 17% (10/58) of larvae from the non-injected and buffer-injected control groups, respectively, were infected with B. bovis.
Conclusions
The combined effects of reduced fecundity and reduced infection in surviving larvae resulting from silencing indicate that vitellogenin receptor is essential for tick reproduction and may play a vital role in B. bovis transmission.
... Column was flushed at 100% B for 5 minutes and re-equilibrated in 100% A for 20 minutes. At each of the ten cycles, mobile phase during the salt pulse consisted of 10,20,30,40,50,60,70,85, or 100% C, or 90% C/10% B. Due to a slight increase in peptide hydrophobicity, for dimethyl labelling experiments, the cycle with 40% C was omitted, and an extra cycle was performed in the end with 90% C/10% B. ...
Ticks are arthropod ectoparasites of importance for public and veterinary health. The understanding
of tick oogenesis and embryogenesis could contribute to the development of novel control methods.
However, to date, studies on the temporal dynamics of proteins during ovary development were not
reported. In the present study we followed protein profile during ovary maturation. Proteomic analysis
of ovary extracts was performed by liquid chromatography-tandem mass spectrometry (LC-MS/
MS) using shotgun strategy, in addition to dimethyl labelling-based protein quantification. A total of
3,756 proteins were identified, which were functionally annotated into 30 categories. Circa 80% of the
annotated proteins belong to categories related to basal metabolism, such as protein synthesis and
modification machineries, nuclear regulation, cytoskeleton, proteasome machinery, transcriptional
machinery, energetic metabolism, extracellular matrix/cell adhesion, immunity, oxidation/
detoxification metabolism, signal transduction, and storage. The abundance of selected proteins
involved in yolk uptake and degradation, as well as vitellin accumulation during ovary maturation, was
assessed using dimethyl-labelling quantification. In conclusion, proteins identified in this study provide
a framework for future studies to elucidate tick development and validate candidate targets for novel
control methods.
... However, until recently it was taken for granted that several physiological processes well known in insects are the same in ticks. Vitellogenesis, and particularly studies with Vg receptors, fall under this classical dogma, since most of what is presently believed comes from studies with insects (Giorgi et al., 1999). Due to the importance of vitellin uptake in tick reproduction, in the present study we have determined and characterized the full-length open reading frame of Rhipicephalus microplus VgR (RmVgR) as well as the partial sequence of Rhipicephalus appendiculatus VgR (RaVgR). ...
The vitellogenin receptor (VgR), which belongs to the low-density lipoprotein receptors (LDLR) family, regulates the absorption of yolk protein accumulated in developing oocytes during oogenesis. In the present study, the full sequence of Rhipicephalus microplus VgR (RmVgR) and the partial sequence of Rhipicephalus appendiculatus VgR (RaVgR) ORF were determined and cloned. The RmVgR amino acid sequence contains the five highly conserved structural motifs characteristic of LDLR superfamily members, the same overall structure as observed in other species. Phylogenetic analysis separated VgRs in two major groups, corresponding to receptors from acarines and insects. Consistent with observations from other arthropods, RmVgR was specifically expressed in the ovarian tissue and its peak of expression occurs in females that are detaching from the host. Silencing with RmVgR dsRNA reduced VgR expression, which resulted in reduced fertility, evidenced by a decrease in the number of larvae. The present study confirms RmVgR is a specific receptor involved in yolk protein uptake and oocyte maturation in R. microplus, playing an important role in tick reproduction.
... In most insect species, Vgs are synthesized in female adults and secreted into the hemolymph, and ultimately captured and sequestered by the developing oocytes via receptor-mediated endocytosis Dhadialla 1992, Snigirevskaya and. During its synthesis in the fat body, Vg is hydrolyzed and cleaved by various proteolytic enzymes, modified by phosphorylation, lipidation, and glycosylation, sheared at the terminal amino acid, processed in the endoplasmic reticulum and Golgi body, and stored as a nutritional source for the developing embryo after incorporation into the oocytes (Hagedorn et al. 1998, Giorgi et al. 1999, Sappington et al. 2002, Tufail et al. 2005. ...
Vitellogenin proteins (Vgs) are precursors of the major egg yolk proteins in many oviparous vertebrates and invertebrates. Here we cloned the full-length cDNA of the Vg gene of Apolygus lucorum, an omnivorous mirid bug that feeds on various agricultural crops and small insects. The gene has 6,131 base pairs (bp) with an open reading frame (ORF) of 5,988 bp, encoding a protein with 1,996 amino acids, with molecular mass 218.3 kDa and isoelectric point 9.02. Phylogenetic analysis showed that Vg gene has a high similarity with that in Hemiptera insects and was most closely related to the rice leaf bug, Trigonotylus caelestialium. The expression of Vg gene was significantly higher in the fat body than in other tissues in females (ovary, hemolymph, gut, and malpighian tubule). Vg gene expression was extremely low in newly emerged adult females and increased with age. The effect of different food sources on the expression level of Vg in adult females was investigated after they were fed on green bean pods (Gb), H. armigera eggs (He), or a combination of green bean pods and H. armigera eggs (GbHe). The results showed that transcript level of Vg gene in females was significantly higher on the GbHe diet than on Gb or He. Our results demonstrate that A. lucorum can acquire nutrients from both plant and animal origin food sources, and the combined food sources significantly facilitated Vg synthesis.
... In insects, Vg as precursor of vitellin (Vn), is synthesized by FB as an extra-ovarian tissue in stage and time specific manners. In FB, Vg is cleaved proteolytically and post-translationally modified to facilitate the transport of lipids and other nutrients to the ovaries [8][9][10][11][12]. The primary Vg gene product of most insect species (200 kDa) is cleaved into large and small (about 50 kDa) subunits before secretion into hemolymph [4,[13][14][15]. ...
In silkworm, Bombyx mori, there are two types of fat body (FB) tissues, peripheral (PP) and perivisceral (PV)FB of which PPFB was commonly assumed to be the protein synthesis, and PVFB the storage site. The present study demonstrated that this hypothesis did not match experimental evidence for the lipid transfer protein vitellogenin (Vg). It showed that major biosynthesis of B. mori Vg takes place during the active feeding stage starting at day 3 V instar with PVFB as synthesis site. Maturation of Vg could also be observed in the increased appearance of its light and heavy chain at day 6 V instar. Vg was isolated from PVFB of day 3 of pupa. Vg subunits were purified and characterized as lipoglycophosphoproteins.
... These presumably would include Golgi, tubules, exocytotic vesicles, coated vesicles, early and late endosomes, and lysosomes, all of which are likely to have functional roles during such events as production of the embryonic cuticle (cf. Criel, 1991), formation of yolk platelets (Giorgi et al., 1999;Warner et al., 2002) and yolk degradation (Komazaki and Hiruma, 1999;Perona et al., 1988;Vallejo, 1985, 1989). Indeed, membrane-bound yolk platelets themselves should be considered, as they are acidified to varying degrees for both maintenance and degradation in other species (Abreu et al., 2004;Fagotto, 1995;Fausto et al., 2001) and contain very little P i in A. franciscana (Warner and Huang, 1979). ...
... Juvenile hormone (JH) and ecdysteroids are key hormones that are involved in the regulation of yolk protein synthesis and vitellogenesis in most insects (Hovemann et al., 1981;Giorgi et al., 1999). In contrast to the major regulatory role of JH on vitellogenesis for lepidopterans, ecdysteroids carry out the most important role in the regulation of dipteran reproduction by triggering the expression of ecdysoneresponsive genes, such as E74, E75, Broad-complex, ecdysone receptor (EcR), ultraspiracle (USP), and so on (Raikhel et al., 2002;Attardo et al., 2005), which are involved in the regulatory cascade of the vitellogenesis and oogenesis (Richard et al., 1998(Richard et al., , 2001. ...
... Vitellogenin is the precursor of major yolk proteins of oviparous insects. It is known to be synthesized mainly in the female fat body in sex and stage-specific manners, is secreted into the hemolymph, and then is sequestered by developing ovaries and oocytes (Raikhel and Dhadialla, 1992;Hagedorn et al., 1998;Giorgi et al., 1999). Unfortunately, with the exception of Athalia rosae (Hatakeyama et al., 1990), Nasonia vitripennis (Dong et al., 2008), and Apis mellifera (Wegener et al., 2009), information regarding the correlation between the secretion patterns of vitellogenin and ovarian development in hymenopteran insects is limited. ...
The Osmia cornifrons bee plays an important role in pollinating fruit trees, such as apple trees. To better understand diapause and oviposition in O. cornifrons, we investigated the correlation between ovarian development and the secretion level of OcVg protein in hemolymph. During ovarian development in wintering, the number of oocytes progressively increased compared with the length of the ovaries and the oocytes. After wintering, the oocyte and ovary sizes developed up to 6. days after emergence and declined after 6. days, but the number of oocytes decreased gradually. The secretion level of OcVg protein in the hemolymph revealed that during wintering, the secretion level increased from month 1 to month 2 and then stagnated after 2. months. After diapause, the secretion level increased gradually until day 6 of the newly emerged adult from the cocoon stage and thereafter gradually declined, remaining detectable until day 30 of the adult stage. The correction analysis between ovarian development and OcVg secretion level in the hemolymph showed that during wintering, the number of oocytes positively correlated with the OcVg secretion level. After diapause, the lengths of the ovary and first oocyte and the number of oocytes showed significant changes in the OcVg secretion level and strongly correlated with the OcVg secretion level, respectively. These results suggest that there is a significant interaction between ovarian development and the secretion level of OcVg protein and that the pattern of ovarian development and the secretion of OcVg protein are stage-specific in the O. cornifrons female. © 2015 Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society.
... These presumably would include Golgi, tubules, exocytotic vesicles, coated vesicles, early and late endosomes, and lysosomes, all of which are likely to have functional roles during such events as production of the embryonic cuticle (cf. Criel, 1991 ), formation of yolk platelets (Giorgi et al., 1999;Warner et al., 2002), and yolk degradation (Perona and Vallejo, 1985;Perona et al., 1988;Perona and Vallejo, 1989;Komazaki and Hiruma, 1999). Indeed, membrane bound yolk platelets themselves should be considered, as they are acidified to varying degrees for both maintenance and degradation in other species (Fagotto, 1995;Fausto et al., 2001;Abreu et al., 2004) and contain very little P i in A. franciscana (Warner and Huang, 1979). ...
... All insects provision their eggs with vitellin (Vn) as a major yolk protein, and the precursor of Vn is Vg. Vg molecules in the fat body are cleaved proteolytically and co-and post-translationally modified to facilitate the transport of nutrients to the ovaries (Giorgi et al., 1999;Hagedorn et al., 1998;Raikhel and Dhadialla, 1992;Tufail et al., 2005). After incorporation into oocytes, the Vg are stored in a crystalline form as Vn, a reserve food-source for the future embryo (Tufail and Takeda, 2008). ...
... In insects, the Vg molecules undergo proteolytic cleavage soon after synthesis in the fat body, and are co-and post-translationally modified. This posttranscriptional processing enables Vg molecules to carry with them the carbohydrates, lipids and other nutrients to the ovaries (see reviews in Raikhel & Dhadialla 1992;Hagedorn et al. 1998;Giorgi et al. 1999;Sappington et al. 2002;). Once Vg becomes part of the egg yolk, it is stored as vitellin (Vn), the main nutritional reserve for the developing embryo. ...
Vitellogenins (Vg) genes code for the major egg yolk protein precursor in insects and many other oviparous species. In insects, the Vg gene is expressed extra-ovarially in the fat body in sex-, tissue- and stage-specific manners. During the reproductive phase, the Vg mRNA is expressed in large quantities, which is then translated, secreted into hemolymph and ultimately taken up by the developing oocytes through receptor-mediated endocytosis. Once sequestered, the Vgs are stored as vitellin (Vn), the main nutritional reserve for the developing embryo. The regulation of Vg genes is directly under the control of hormones at the transcriptional level. Hormones involved in Vg gene transcription are juvenile hormone (JH), ecdysteroids and some neuropeptides. The overall understanding that has emerged is that the insects can be classified, based on the system of hormonal regulation of Vg gene transcription, into three groups: (i) insects (like most of hemipterans) that use only JH for Vg gene transcription; (ii) insects (like dipterans) that need both JH and ecdysteroids for Vg regulation; and (iii) insects like lepidopterans that require JH, ecdysteroids and additional hormones to regulate their reproductive biology. However, why insect species diverge in using different hormones to govern their reproductive physiology remains unclear. The present contribution focuses on the current status of knowledge regarding the regulation of Vg genes in insects. Besides a brief information on biochemical and molecular features, the role of Vg genes as a target of endocrine disruptors will be addressed. Also, the molecular mechanism of Vg gene regulation will be discussed.
... While the synthesis, transport, and processing of Vgs/YPs have been extensively studied (c.f. reviews by Engelmann 1979;Hagedorn and Kunkel 1979;Raikhel et al. 1992;Giorgi et al. 1999), for several insects the mechanisms specifically regulating the onset and continuation of endocytic activity by the oocyte remain in question. In several species of insects, ingestion of food (often a blood meal) begins a cascade of largely hormonal events culminating in production of Vgs, their release into the hemolymph, and receptor mediated endocytosis into yolk spheres in the terminal oocyte of each ovariole, while younger oocytes are inhibited from acquiring Vgs. ...
Abstract In Drosophila melanogaster M. (Diptera: Drosophilidae), a phospholipase-C to proteininase-C signal cascade leads to the endocytic uptake of yolk precursor molecules. The data suggest that D. melanogaster has a phospholipase-C/proteinkinase-C signaling pathway similar to that previously shown to be required for vitellogenesis in the milkweed bug, Oncopeltus fasciatus Dallas (Hemiptera: Lygaeidae). Calmodulin, derived from epithelial cells and transported to the oocytes via gap junctions, may trigger this pathway. To investigate this, a series of known antagonists to various elements of the pathway were used. W-7 (which prevents calmodulin binding to phospholipase-C), U-73122 (which prevents activation of phospholipase-C), verapamil (which blocks Ca(2+) release by IP3), HAG (which blocks diacylglycerol), and staurosporine (which inactivates proteinkinase-C) were each shown to inhibit endocytosis, thereby blocking formation of nascent yolk spheres.
... As oviparous animals, insect must allocate in their eggs sufficient nutrients to sustain embryogenesis, insect fat body synthesizes massive amounts of proteins that are posteriorly secreted to the hemolymph and delivered to the ovaries where they will be incorporated into the developing oocytes by receptor-mediated endocytosis (Raikhel and Dhadialla, 1992). Once inside the oocyte, yolk proteins accumulate in organelles called yolk granules (Snigirevskaya et al., 1997) wherein they are stored together with several hydrolases, such as acid phosphatase and proteases (Nussenzveig et al., 1992;Ribolla et al., 1993;Giorgi et al., 1999;Zhao et al., 2005;Oliveira et al., 2008). Subpopulations of yolk granules of various sizes, densities, and contents have been described in several oviparous models (Wallace, 1985;Fausto et al., 2001), and have been linked with the triggering of yolk degradation by hydrolases (Liu and Nordin, 1998;Cho et al., 1999;Fialho et al., 2002). ...
Despite being the main insect pest on soybean crops in the Americas, very few studies have approached the general biology of the lepidopteran Anticarsia gemmatalis and there is a paucity of studies with embryo formation and yolk mobilization in this species. In the present work, we identified an acid phosphatase activity in the eggs of A. gemmatalis (agAP) that we further characterized by means of biochemistry and cell biology experiments. By testing several candidate substrates, this enzyme proved chiefly active with phosphotyrosine; in vitro assays suggested a link between agAP activity and dephosphorylation of egg yolk phosphotyrosine. We also detected strong activity with endogenous and exogenous short chain polyphosphates (PolyP), which are polymers of phosphate residues involved in a number of physiological processes. Both agAP activity and PolyP were shown to initially concentrate in small vesicles clearly distinct from typically larger yolk granules, suggesting subcellular compartmentalization. As PolyP has been implicated in inhibition of yolk proteases, we performed in vitro enzymatic assays with a cysteine protease to test whether it would be inhibited by PolyP. This cysteine protease is prominent in Anticarsia egg homogenates. Accordingly, short chain PolyP was a potent inhibitor of cysteine protease. We thereby suggest that PolyP hydrolysis by agAP is a triggering mechanism of yolk mobilization in A. gemmatalis.
... Eggs of arthropods require large amounts of yolk proteins for development, most of which are synthesized outside the oocyte, mainly in the fat body, but also by ovarian follicular cells and midgut (Giorgi et al., 1999). A number of haemolymph proteins are endocytosed by oocyte and stored within the yolk granules during vitellogenesis. ...
An aspartic endopeptidase named THAP, from the eggs of the tick Riphicephalus (Boophilus) microplus, has been suggested to be involved in vitellin-degradation. Here we characterized this enzyme further, showing that THAP mRNA is present in the fat body, midgut and ovary of ticks, in two developmental stages (partially and fully engorged females). However, higher transcription levels were found in fully engorged vitellogenic females. The THAP protein was detected in the haemolymph, midgut and fat body and, in higher quantity, in the ovary of fully engorged females, and it was present throughout embryo development. The protein is synthesized as a higher molecular mass form and after the onset of embryogenesis THAP is converted into an active form by autocatalysis. We also produced a recombinant protein (rTHAP) in E. coli that was active in the fluorogenic peptide substrate and able to hydrolyze vitellin from 7-day-old eggs in a reaction that is heme-sensitive and inhibited by pepstatin A. However, rTHAP does not hydrolyze vitellin from 1 and 12-day-old eggs. As a result, we suggest a model for THAP synthesis, transport, storage and activation and for the role it plays in embryonic development by participating in vitellin processing.
... In contrast, effects of heavy metals on insect vitellogenesis, which involves the synthesis of yolk protein precursor, vitellogenin (Vg) in the fat body and the sequestration of Vg by ovaries as vitellin (Vt) (Raikhel & Dhadialla, 1992;Hagedorn et al., 1998;Giorgi et al., 1999), have received less attention except for reports on Oncopeltus fasciatus (Dallas) (Heteroptera: Lygaeidae) (Cervera et al., 2005(Cervera et al., , 2006. As mentioned above, metal transfer and its effects within producers and primary consumers (such as herbivorous or saprophagous species) have been considered in previous studies, whereas little attention has been paid to metal transfer over three trophic levels (producers, primary consumers and secondary consumers) and impacts of metals on secondary consumers, namely, parasites/parasitoids and predators (Bischof, 1995a(Bischof, , b, 1996Ortel, 1995a;Kazimírová & Ortel, 2000;Xia et al., 2006). ...
Effects of copper (Cu) accumulation by the flesh fly Boettcherisca peregrina (R.-D.) (Diptera: Sarcophagidae) on the ectoparasitic wasp Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae) were investigated experimentally by exposing host larvae to contaminated diets with final Cu concentrations of 400 μg/g and 800 μg/g diet fresh weight (DFW), respectively. Results showed that Cu can be transferred along food chains to secondary consumers (parasitoids) in small amounts, resulting in negative effects on parasitoid growth and development (body weight and developmental duration) as well as fecundity (number of offspring per female). Copper exposure also inhibited vitellogenesis of parasitoids from Cu-contaminated host pupae. It is suggested that the decreased fecundity and inhibition of vitellogenesis of N. vitripennis resulted from poor host nutritional state rather than from direct effects of Cu stress.
... With onset of development, Vt is gradually degraded to provide the growing embryo with an adequate food supply (Yamashita and Indrasith 1988). In recent years, a large body of evidence has clearly demonstrated that Vt undergoes a specific, developmentally regulated proteolysis during embryogenesis (Zhu et al. 1986;Masetti and Giorgi 1989;Nordin et al. 1990; Giorgi et al. 1999) and that the proteolytic enzymes required for this event are maternally derived gene products, stored in the oocyte's yolk granules as inactive, latent proproteases (Fagotto 1990;Cho et al. 1991Cho et al. , 1999Takahashi et al. 1993;Liu et al. 1997). Proteolytic cleavage of Vt is thought to be developmentally regulated by differential acidification of the yolk granules through activation of specific ion proton channels (reviews : Fagotto 1995;Takahashi et al. 1996). ...
A cysteine proprotease has been identified in developing embryos of the cockroach Blattella germanica and found to be a maternally encoded gene product that is transferred endocytically to the oocyte. The present study aims at establishing how this maternally derived proprotease is synthesized, packaged, and secreted during vitellogenesis. To this end, proprotease was localized immunocytochemically in the fat body of postmating females and its localization compared with that of vitellogenin over the same developmental periods. Fat bodies in cockroaches are comprised of two different cell types: trophocytes and bacteriocytes. Data show that proprotease and vitellogenin come to colocalize in compound granules of the fat body trophocytes. While synthesis of vitellogenin can be traced back to granules resulting from the coalescence of Golgi-derived vesicles in the trophocyte cytoplasm, proprotease appears to be localized predominantly on the cytolysosomes of both trophocytes and bacteriocytes. When probed with an anti-proprotease antiserum, bacteria are also positively labeled, regardless of whether they are segregated inside the cytolysosomes or free in the bacteriocyte cytoplasm. Since vitellogenin and proprotease colocalize within the same cell organelle, it is assumed that Golgi-derived vesicles, which contain vitellogenin, may fuse with cytolysosomes bearing proprotease to yield compound secretory granules. To account for the present observations, the origin and role of proprotease are discussed in relation to the turnover of bacteria in the fat body and to the requirements of endosymbiosis.
... In most cases in insects, Vg is synthesized extraovarially in the fat body, secreted into the hemolymph and then sequestered into oocytes via receptor-mediated endocytosis (Raikhel and Dhadialla 1992; Sappington and Raikhel 1998; Snigirevskaya and Raikhel 2005). Vg molecules are cleaved proteolytically and modified in the fat body to facilitate the transport of carbohydrates, lipids and other nutrients to the oocyte (Raikhel and Dhadialla 1992; Hagedorn et al. 1998; Giorgi et al. 1999; Sappington et al. 2002). After incorporation into oocytes, Vgs are stored in a crystalline form as vitellin (Vn), a nutrient reserve for the future embryo. ...
The giant water bug, Lethocerus deyrollei has become an endangered species in Japan. For conservation of this insect, hormonal regulation and the potential involvement
of environmental pollutants as endocrine disrupting chemicals (EDCs) in reproduction was investigated using vitellogenin (Vg),
the major yolk protein precursor, gene as a biomarker. For this, the cDNA encoding Vg of L. deyrollei was cloned and the amino acid sequence was deduced. The Vg polypeptide sequence was deduced from the 5,865bp cDNA, containing
several well-conserved features of insect Vgs. SDS–PAGE showed two Vg subunit polypeptides of about 193 and 56kDa in L. deyrollei. Northern blot analysis revealed that the Vg gene was transcribed specifically in the female fat body and Vg expression was induced when females in reproductive diapause were injected with juvenile hormone (JH) III and inhibited when
they were injected with 20-hydroxyecdysone (20E), suggesting that the Vg gene is regulated by the JH in L. deyrollei. Dramatic induction or inhibition of Vg gene transcript was detected when diapausing females were injected with EDCs. 17β-estradiol
and bisphenol A enhanced the expression but 4-t-octylphenol and 4-nonylphenol repressed it. These results indicate that EDCs
may disrupt the reproductive physiology of L. deyrollei in nature, providing a first indication that environmental pollutants could be the cause of the rapid disappearance of this
species from Japanese ecosystems.
KeywordsGiant water bug–
Lethocerus deyrollei
–Vitellogenin–cDNA cloning–Gene expression–Endocrine disrupting chemicals
... Insect eggs are cells rich in yolk, and in Lepidoptera they are composed of amino acids, lipids, carbohydrates, and abundant proteins (Raikhel 1984(Raikhel , 1992Cao et al. 1988; Barrett and Schmidt 1991;Yazlovetsky 1992;Ba and Phillips 1995;Giorgi et al. 1999). Accumulation of the aforementioned compounds occurs during oogenesis by means of various synthetic processes. ...
Embryonic development in Diatraea saccharalis F. (Lepidoptera: Crambidae) has been studied by means of whole mounts, histochemical techniques, and light microscopy. Three embryonic stages were identified up to larval eclosion. In the first stage, 0–10 h, intense multiplication of energids occurred, and significant changes in RNA levels were detected in distinct zones before the start of blastulation. During the second stage, 10–24 h, new protein and RNA sites were detected in the egg’s periphery. This was probably because of migration of energids and formation of the blastula. Gastrulation occurs during this stage and is characterized by intense synthetic and mitotic processes. The third stage, 25–168 h, showed ortochromatic and metachromatic regions at the posterior pole and in the yolk, egg periphery, and body of the embryo. Organogenesis closes approximately on the sixth day when the embryo starts feeding on the remaining yolk. Its development is complete by the end of the seventh day on which the larva ecloses.
... The cysteine endopeptidase activity detected in hemolymph increases by 90 times after inhibitor is removed. This agrees with the idea that VT hydrolysis and nutrient availability in arthropods is a carefully controlled process (see Giorgi et al., 1999 for a review). Since immunization of bovines with BYC inhibits embryo development, it is reasonable to suppose that vaccination with other internal organ enzymes like VTDCE can also interfere in tick embryonic and larval phases. ...
Rhipicephalus (Boophilus) microplus is one of the most widely distributed tick in the world. The control of the parasite is based mainly on the use of chemical acaricides, which are produced from a limited set of molecules. These drugs induce selection of acaricide-resistant ticks, and are an important source of environmental pollution. An approach based on anti-tick vaccines may circumvent these obstacles. Characterization of the physiological function of tick molecules may be useful to develop new vaccines. Previously, we reported the ability of some tick proteins as inducers of protective immune response. Vaccination studies using tick proteins like native (nBYC), recombinant (rBYC) egg-yolk aspartic endopeptidase and cysteine endopeptidase (VTDCE) from R. microplus and glutathione S-transferase (Hl-GST) from Haemaphysalis longicornis demonstrated the immunogenicity and antigenicity of these proteins in bovines. Eventually, immunization with these proteins triggered a partial immune response against R. microplus infestation in cattle, manifested mainly as a reduction in egg fertility (7.7% and 13.9% for nBYC, 5.9% for rBYC; 4.7% for VTDCE, 7.9% for Hl-GST), and in the number of fully engorged ticks (18.2% for rBYC, 14.6% for VTDCE, 53% for Hl-GST). The data so far obtained suggest that these proteins have potential to be used as antigens in an anti-tick vaccine. Other proteins involved in tick embryogenesis also have this potential, like THAP and BmCl1, which are enzymes with key roles in vitellin and hemoglobin hydrolysis. Moreover, the identification of analogous proteins present in other tick species may bring information about the way to develop a vaccine against multiple tick species which can help to solve the problem faced by numerous countries where animals are parasitized by more than one tick species. The aim of the present review is to comprehensibly summarize the data obtained in the last few years by our collaborative research, discussing the efforts we have made to find antigens efficient enough for a cattle tick-controlling vaccine. This review discusses tick physiology studies aimed at the selection of possible targets, characterization of the selected proteins with emphasis on their biochemical and immunological aspects and results of vaccine trials on bovines.
... In free living insects, embryo develops by consuming the maternally derived nutrients accumulated in vitellins [21], [22]. These reserves of nutrients, currently named yolk, are deposited in oocytes during the vitellogenesis. ...
Aphidius ervi (Hymenoptera: Braconidae) is an entomophagous parasitoid known to be an effective parasitoid of several aphid species of economic importance. A reduction of its production cost during mass rearing for inundative release is needed to improve its use in biological control of pests. In these contexts, a careful analysis of its entire development phases within its host is needed. This paper shows that this parasitoid has some characteristics in its embryological development rather complex and different from most other reported insects, which can be phylogenetically very close. First, its yolkless egg allows a high fecundity of the female but force them to hatch from the egg shell rapidly to the host hemocoel. An early cellularisation allowing a rapid differentiation of a serosa membrane seems to confirm this hypothesis. The serosa wraps the developing embryo until the first instar larva stage and invades the host tissues by microvilli projections and form a placenta like structure able to divert host resources and allowing nutrition and respiration of embryo. Such interspecific invasion, at the cellular level, recalls mammal's trophoblasts that anchors maternal uterine wall and underlines the high adaptation of A. ervi to develop in the host body.
Fipronil and amitraz are potentially toxic compounds used for controlling ticks infesting pet and livestock. The use of fipronil on large animals was limited because of its high costs while amitraz is still persisting in the market since its introduction over four decades ago. Though resistance in ticks against these pesticides has been reported worldwide since 2000, the toxicity of these chemicals at cellular level in ticks is still poorly understood. The present study aimed to examine the gross and cellular impact of fipronil and amitraz on the gut, ovaries and synganglion of engorged Rhipicephalus microplus females. Fipronil and amitraz treated tick groups showed formation of a large number of vacuoles of different size throughout the cytoplasm of generative cells whereas sessile, residual and detached digestive cells were very low in numbers. The treatment of ticks resulted in the formation of vacuolations at periphery of all oocytes. Ultra-thin sections of the synganglion revealed severe rupture of neural lamella and perineurium with apoptosis of neural cells after fipronil treatment whereas in the amitraz treated ticks, severe destruction of neuropile region and extensive vacuolation of type I and II cells of cortical region as compared to the unexposed ticks were noted.
Haemaphysalis flava is the vector of several pathogens and has medical and veterinary importance. Transcriptome information of the ovary of H. flava is unavailable and limits understanding of its molecular basis of reproduction. We studied the ovary transcriptome of partially engorged and fully engorged H. flava using high-throughput RNA sequencing technology. A total of 53,025,360 and 57,942,890 clean reads were obtained with 7.95 GB and 8.69 GB clean bases in partially engorged ticks (PETs) and fully engorged ticks (FETs), respectively. The clean reads were assembled into 138,711 unigenes. A total of 72,043 unigenes (51.93%) were annotated and 66,668 unigenes (48.07%) were unknown. A total of 38,487 differentially expressed genes (DEGs) were found between PET and FET with 19,031 upregulated genes and 19,456 downregulated genes. The RNA-seq results were validated by qRT-PCR, including six upregulated genes and three downregulated genes. Some unigenes coding for nutrient transporters, proteases, and protease inhibitors were found and analyzed. This study was the first time to perform the transcriptome sequences of the ovary of partially engorged and fully engorged H. flava. The results can benefit the understanding of the molecular basis of ovary maturation and oogenesis of the H. flava and boost the development of the strategies for control of H. flava.
The present study was carried out to assess the effects on the ovary of fed female Argas persicus following spraying of the ticks with spores of the fungus Beauveria bassiana suspended in triton X100 at a concentration of 10⁷ conidia/ml. Scanning and transmission electron microscopy observations provided evidence that B. bassiana invaded the ovary, causing extensive morphological damage and deterioration of the developing oocytes. Destruction of the shape and internal organelles of young and previtellogenic oocytes and complete inhibition of vitellogenesis was evident. This histopathological study is the first demonstration of ultrastructural damage in the ovaries of A. persicus after infection with B. bassiana. The data presented confirm that B. bassiana affects the ovary either directly by entering the oocytes and/or indirectly by producing toxins in the haemolymph that interfere with the development of oocytes, thus potentially contributing to the control of this tick in a way that is safe for its host and the environment.
Biochemical profile of silkworms can depict the profound body biomass conversion throughout the larval stages. In view of this, the determination of metabolite levels in the hemolymph of caterpillars ready to pupate may be interpreted in relation to the imminent silk filament synthesis. Due to different extents of silk production according to the genetic strain of silkworm, it was hypothesized that a potential effect of selection of desirable traits of economic importance could have conditioned levels of some biochemical parameters. To test this hypothesis, twenty silkworm strains from Iranian gene bank were used. Aspartate amino transferase (AST), Alanine amino transferase (ALT), Alkaline phosphatase (ALP), phosphorus (P) sodium (Na), potassium (K) and iron (Fe) were determined in the haemolymph of caterpillars ready to pupate (5th day of the 5th instar stage) of each genetic strain. Enzymes and electrolytes were selected in the light of acquaintances on the terminal larval metabolism and development, potentially involved in synthesizing and spinning fibroin and sericin filaments for cocoon creation. Biochemical profiles pointed to differences between strains that were used in the hierarchical clustering analysis. Results showed that the highest level of (AST) was found in strain 113-K (191.63 U/L) and the lowest in 31(94.81 U/L) reflecting the different activity of protein transamination, likely related to body protein conversion into fibroin and sericine. Significantly, Na⁺ (p = 0.032) and K⁺ (p = 0.022) displayed to vary in relation to cocoon shell weight categories among the genetic strains investigated. However, AST varied only closed to significance (p = 0.062). The canonical discriminant analysis (CANDISC) pointed to a good discrimination of high performance strains on the basis of the metabolic profile, from those with performance below the median value, but in a non significant way. Dendrograms of hierarchical clustering analyses pointed to 3 distinct groups with different apparent metabolic levels for silk synthesis.
Osmia cornifrons plays a major role in the pollination of orchards, but basic information on vitellogenin and oocyte development is limited. To better understand vitellogenin in hymenopteran insects, we cloned a cDNA encoding vitellogenin from the hornfaced bee O. cornifrons. Osmia cornifrons vitellogenin cDNA contains 5477 bp with an open reading frame of 1783 amino acid residues, and has a predicted molecular mass of approximately 200.21 kDa and a pI of 6.55. Osmia cornifrons vitellogenin possesses four consensus (RXXR/S) cleavage sites and has conserved DGXR and GL/ICG motifs in the C-terminus. The deduced amino acid sequence of the O. cornifrons vitellogenin cDNA showed a 66% identity with Megachile rotundata, 53% to Apis mellifera, 51% to Bombus ignitus and 42%–30% with other hymenopteran insect vitellogenins. Phylogenetic analysis showed that O. cornifrons vitellogenin clustered with vitellogenins from Megachilidae, Apidae, Vespidae and Formicidae species but not with those from Pteromalidae, Aphelinidae or Ichneumonidae species. The expression profile of O. cornifrons vitellogenin mRNA during development revealed that O. cornifrons vitellogenin was first detected in the pupal stage and was continuously detected during the adult stage. Interestingly, O. cornifrons vitellogenin mRNA expression was low in mid-diapause, then gradually increased beginning on day 3 of the newly emerged adult stage, and subsequently declined. These results suggest that the expression level of O. cornifrons vitellogenin mRNA is stage-specific.
Vitellogenins (Vgs) and mature yolk from some non-Dipteran insects can be recognized by Drosophila melanogaster oocyte Vg receptors and incorporated via receptor-mediated endocytosis into nascent yolk spheres (NYS). It had previously been assumed that only Vgs of Drosophila or other Dipterans could be so endocytosed. Drosophila ovarian follicles from 4-day old females were incubated in the presence of physiological salt solution (PSS) containing some fluorescent TexasRed-Dextran (Dex-red) or PSS-Dex-red in which either female hemolymph, or vitellin (mature yolk) from lysed oocytes was present from any of the following: (1) Drosophila (Diptera); (2) Oncopeltus (milkweed bug, Hemiptera); (3) Acteaus (luna moth, Saturniidae Lepidoptera); (4) Papilio (swallowtail butterfly, Papilionidae Lepidoptera); or (5) Xylocopa (carpenter bee, Hymenoptera). Under incubation conditions, any NYS would become fluorescent due to non-specific fluid-phase uptake. Ovarian follicles incubated in PSS-DexRed alone or in PSS with hemolymph from males did not carry out endocytosis detectable by this technique, but all other treatments listed above did.
A female-specific protein, vitellogenin (Vg), and its corresponding egg vitellin (Vt) are identified in the ectoparasitic wasp Nasonia vitripennis. Both native Vt and Vg have a molecular mass of about 350 kDa, which is composed of two subunits of approximately 190 kDa and 165 kDa under reducing and denaturing conditions (sodium dodecyl sulfate—polyacrylamide gel electrophoresis). An indirect sandwich enzyme-linked immunosorbent assay developed with both monoclonal and polyclonal antibodies against N. vitripennis Vt. Vg was first detected in the hemolymph on the 10th day after parasitism, and was first observed in oocytes on the 12th day. In adults deprived of food, the highest hemolymph Vg level occurred at the time of adult eclosion and the highest level of Vt in ovaries was found at 30 h after eclosion. In contrast, feeding adults with 20% sucrose resulted in the reduction of Vt uptake by ovaries and the extension of life span, but had little effect on Vg production. Deprived of hosts, starvation of female wasps had no significant effects on ovariole growth and oocyte maturation before the wasps died. However, starvation of female wasps supplied with hosts accelerated the wasps laying progeny into hosts, but resulted in a decrease of total progeny production by comparison with wasps fed with 20% sucrose.
A positive correlation between the activity level of cysteine proteinases in developing eggs of common silkworm moth (Bombyx mori L.), on the one hand, and a set of commercial characteristics, on the other, was found. This allows the determination of cysteine proteinase activities (pH optima of 3.0, 3.6, and 8.6) to be recommended as a biochemical test for an early prediction of potential productivity of silkworm breeds. A positive correlation between the activity level of acid cysteine proteinases in eggs of parental breeds and a set of commercial characteristics of their hybrids was detected, indicating the possibility of predicting the degree of heterosis.
The maturation of insect eggs requires the fat body production of large amounts of a protein, vitellogenin (Vg), that is secreted into the hemolymph, selectively accumulated by the oocytes and stored in yolk bodies as crystalline vitellin (Vn). The coordination of activity in fat body and oocytes during vitellogenesis is achieved through a complex regulatory mechanism. The mosquito, Aedes aegypti, has proven to be a valuable organism in the study of vitellogenesis.
Degradation of proteins that are retained in the quality control apparatus of the endoplasmic reticulum (ER) has been attributed
to a third proteolytic system, distinct from the lysosomal and the cytoplasmic ubiquitin-dependent proteosomal proteolytic
pathways. However, several recent studies have shown that ER degradation of a mutant membrane protein, CFTRΔF508, is at least
in part mediated from the cytoplasmic side by the 26 S proteasome. In this study, we examined the possibility that ER degradation
of mutant secretory protein α1-antitrypsin (α1-AT) Z, the mutant protein associated with infantile liver disease and adult-onset emphysema of α1-AT deficiency, is mediated by the proteasome. The results show that a specific proteasome inhibitor, lactacystin, inhibits
ER degradation of α1-ATZ in transfected human fibroblast cell lines and in a cell-free microsomal translocation system. Although
it is relatively easy to conceptualize how a transmembrane protein like CFTRΔF508 might be accessible on the cytoplasmic aspect
of the ER membrane for ubiquitination and degradation by the proteasome, it is more difficult to conceptualize how this might
occur for a luminal polypeptide. The results show that, once within the lumen of the ER, α1-ATZ interacts with the transmembrane molecular chaperone calnexin and specifically induces the polyubiquitination of calnexin.
The results, therefore, provide evidence that the proteasome, from its cytoplasmic localization, induces the degradation of
the luminal α1-ATZ molecule by first attacking the cytoplasmic tail of calnexin molecules that are associated with α1-ATZ.
Knowledge of the chemistry and biology of yolk proteins has expanded explosively in the past decade and promises to be equally as volatile in the present one. Some old issues have been partially resolved but new issues are arising with the application of new technology.
Yolk spheres in tick eggs contain a latent procathepsin L, which is activated in vivo, in parallel with yolk degradation, and in vitro by acid treatment (Fagotto, F., Arch. Insect Biochem. Physiol., 1990b in press). Mature cathepsin L hydrolyzes vitellin at acidic pH (Fagotto, F., Arch. Insect Biochem. Physiol., 1990a in press). Here, yolk spheres' pH has been estimated using acridine orange. In the early development, all yolk spheres are neutral, then an increasing number acidify, until hatching, where general acidification seems to occur. This fits well with vitellin utilized slowly during embryogenesis, more intensely at hatching (Chinzei, Y. and I. Yano, Experientia 41, 948, 1985), and can be related to sequential degradation of individual spheres observed during embryonic development, then extensive yolk liquefaction in the larva. Different yolk spheres populations have been separaded on Percoll density gradients. In freshly laid eggs, yolk spheres are dense, neutral, undegrated and contain exclusively the precursor of cathepsin L. In later stages, yolk spheres are progressively recovered in lower density fractions, displaying acidic interior and cytological signs of degradation. They cosediment with mature cathepsin L. It is concluded that acidification initiates yolk degradation through procathepsin L activation.
Multiple vitellogenins (VG) are found in species from all major families of cockroaches. Proof that observed multiple VGs are actually distinct proteins and not artifacts of differential processing requires careful examination. In Periplaneta americana two immunologically and electrophoretically distinct vitellins (VTs) of similar native size exist in the egg. VT1 is composed of four major peptides of molecular weights 170, 105, 92 and 78 K. VT2 is composed of three major peptides of molecular weights 105, 101 and 60 K. A peptide with molecular weight of about 105 K is found in both VG1 and VG2 and a similar sized peptide is also conserved in the VGs of all other Blatinae subfamily members examined despite the distant immunological relation of these proteins. The 170 K peptide of VT1 is likely to be a processing intermediate of the 92 K and the 78 K peptides. Each VT from a freshly ovulated egg is immunologically identical to and has essentially the same peptide substructure as its serum precursor. The cumulative of the constituent peptides of each VG is approx. 260 K, consistent with 17S native dimeric molecules of approx. 520 K . A specific and limited cleavage of VT major peptides occurs during early embryogenesis (at 30°C, 9 days after ovulation) resulting in a partial loss of immunological determinants. During subsequent yolk utilization the VTs undergo gradual degradation.
This volume was first published in 1980. Coated vesicles are distinct organelles whose existence has been recognized for many years. Since the original observations of the invaginations of erythroblast and oocyte membranes, coated vesicles have been found associated with a wide variety of membranes and they have been seen to be of importance in the uptake and transport of peptides, proteins and lipoproteins as well as in some cases as mediators of immune function. This book is a comprehensive treatment of coated vesicles and their involvement in the process of endocytosis (the engulfment of material in membranous enclosures).
Covalent modification of cellular proteins with ubiquitin represents a versatile intracellular signaling pathway that is linked to such diverse phenomena as DNA repair, organelle biogenesis, and protein turnover. Best understood among these is the role of the ubiquitin system in targeting cytoplasmic proteins for proteolysis by the 26 S proteasome complex (reviewed in Chapter 6). Although most of the substrates for the ubiquitin conjugation system studied to date have been cytoplasmic or nuclear proteins, this chapter reviews recent data that suggest a role for the ubiquitin system in degradation and trafficking of proteins within the central vacuolar system. By central vacuolar system, I refer collectively to the compartments of the secretory and endocytic/lysosomal pathways including the endoplasmic reticulum, Golgi apparatus, plasma membrane, lysosomes, and the various transitional compartments and transport vesicles.
At the first International CNRS Symposium on Biosynthesis, Metabolism and Mode of Action of Invertebrate Hormones in Lille in September 1975, we reported the presence of ecdysteroid-immunoreactive molecules in vitellogenic ovaries of adult females of Locusta migratoria [1]. These molecules were active in the Calliphora bioassay, but it was only 1 year later that we could assess by physicochemical methods their identify as ecdysteroids [2,3].
Malaria, dengue and other mosquito-borne infections are among the most devastating diseases (57, 144). The maintenance and dispersal of mosquito-borne disease depends upon the successful reproduction of the mosquito. The cornerstone of the reproductive cycle is vitellogenesis involving massive production of yolk protein precursors and their accumulation in developing oocytes. In anautogeneous mosquitoes, vitellogenesis is dependent on the availability of a blood meal and, as a consequence, is linked to transmission of pathogens. Pathogens acquired transovarially or from an infected host during an initial blood meal are transmitted during subsequent blood meals. Therefore, elucidation of vitellogenesis and other aspects of mosquito reproductive physiology is critical for the successful development of novel strategies in vector and disease management.
The precise function of the yolk platelets of sea urchin embryos during early development is unknown. We have shown previously that the chemical composition of the yolk platelets remains unchanged in terms of phospholipid, triglyceride, hexose, sialic acid, RNA, and total protein content after fertilization and early development. However, the platelet is not entirely static because the major 160-kD yolk glycoprotein YP-160 undergoes limited, step-wise proteolytic cleavage during early development. Based on previous studies by us and others, it has been postulated that yolk platelets become acidified during development, leading to the activation of a cathepsin B-like yolk proteinase that is believed to be responsible for the degradation of the major yolk glycoprotein. To investigate this possibility, we studied the effect of addition of chloroquine, which prevents acidification of lysosomes. Consistent with the postulated requirement for acidification, it was found that chloroquine blocked YP-160 breakdown but had no effect on embryonic development. To directly test the possibility that acidification of the yolk platelets over the course of development temporally correlated with YP-160 proteolysis, we added 3-(2,4-dinitroanilo)-3-amino-N-methyldipropylamine (DAMP) to eggs or embryos. This compound localizes to acidic organelles and can be detected in these organelles by EM. The results of these studies revealed that yolk platelets did, in fact, become transiently acidified during development. This acidification occurred at the same time as yolk protein proteolysis, i.e., at 6 h after fertilization (64-cell stage) in Strongylocentrotus purpuratus and at 48 h after fertilization (late gastrula) in L. pictus. Furthermore, the pH value at the point of maximal acidification of the yolk platelets in vivo was equal to the pH optimum of the enzyme measured in vitro, indicating that this acidification is sufficient to activate the enzyme. For both S. purpuratus and Lytechinus pictus, the observed decrease in the pH was approximately 0.8 U, from 7.0 to 6.2. The trypsin inhibitor benzamidine was found to inhibit the yolk proteinase in vivo. By virtue of the fact that this inhibitor was reversible we established that the activity of the yolk proteinase is developmentally regulated even though the enzyme is present throughout the course of development. These findings indicate that acidification of yolk platelets is a developmentally regulated process that is a prerequisite to initiation of the catabolism of the major yolk glycoprotein.
Soil development and plant commumtJes are described on three sand dunes estimated to be 130, 440 and 1,000 years old, respectively, at Tautuku Bay, Southeast Otago. A comparison of chemical and morphological properties of soils beneath a plant sequence from {itAmmophila armaria} through low forest of {itMetrosideros umbellata} and other broadleaved tree species to tall forest of {itWeinmannia racemosa} and {itM. umbellata} with occasional podocarps, shows an increase in organic matter from less than one to greater than 40%C, and a decline in pH from 8.0 to 4.3.
Three kinds of yolk proteins (vitellin, egg-specific protein and 30 k-proteins) are found in silkmoth eggs and have been well characterized. Essentially these proteins are considered to be amino acid reserves for developing embryos. Since at an early stage of egg development the cysteine proteinase accounts for the majority of the total proteinase activity, it may be involved in the degradation of yolk proteins. The enzyme is stored in the eggs as an inactive pro-form, indicating that the activation of the enzyme might be one of the key steps in yolk protein degradation. To investigate at the molecular level how yolk proteins degradation takes place, we have studied Bombyx acid cysteine proteinase (BCP) during an early period of embryonic development. We summarize how proteinases are regulated and are involved in the degradation of Bombyx yolk proteins during embryogenesis. These will be discussed mainly in light of recent results obtained from eggs of the silkmoth, Bombyx mori.
The accumulative pathway of yolk protein precursor, vitellogenin (VG), in the mosquito oocyte was studied by qualitative and quantitative VG immunolocalization, fixation with tannic acid, and lysosomal enzyme cytochemistry. VG binds to its receptors located on specific membrane microdomains at the base of and between the oocyte microvilli. The VG-receptor complexes then concentrate in coated pits and are internalized by coated vesicles. Dissociation of VG from its receptor occurs soon after transformation of coated vesicles into uncoated vesicular or tubular endosomes. The endosomes then coalesce into transitional yolk bodies (TYB), where VG accumulates and condenses. Narrow tubules without VG, connected to TYB, presumably represent a specific compartment for the recycling of VG receptors. TYB remain a “sink” for incoming VG until their transformation into mature yolk bodies (MYB), whereupon VG is transformed into a crystalline form, presumably vitellin. All compartments of the VG-accumulative pathway lack lysosomal enzymes and, therefore, the final VG destination, the MYB, is also endosomal and prelysosomal compartment, specialized for long-term storage of yolk protein.
The fat body produces vitellogenic proteins which are released to the haemolymph and incorporated into developing oocytes, where they are processed to vitellins. Endocrine and other physiological cues regulate vitellogenesis in a complex homeostatic pattern, in which the ovary may have a pivotal role. The present paper describes the effects of ovariectomy on vitellogenin production in the cockroach Blattella germanica. The results show that the absence of ovaries leads to a rising and saturable accumulation of vitellogenic proteins, both in the haemolymph and in the fat body, whereas in intact females the production of these proteins is cyclic. This suggests that the ovary is involved in terminating vitellogenesis in intact females. We also report that vitellogenin is processed in the haemolymph of ovariectomized females in a similar manner to that of vitellin in the ovary of intact specimens
A procedure has been developed in Drosophila by which haemolymph from a female sterile mutant can be used as a vitellogenic probe. This consists of labelling haemolymph proteins metabolically in flies homozygous for the female sterile mutation fs(3)H23. Developing ovarian follicles are then exposed in vivo to labelled mutant haemolymph by transfusing it into wild-type females. The criteria whereby mutant haemolymph can be used reliably instead of purified vitellogenin are as follows: yolk polypeptides should be the major protein fractions labelled after in vivo exposure to mutant haemolymph and, labelling in the recipient ovary should be confined to the yolk spheres. In this study we show that both of these criteria are fulfilled when mutant haemolymph is collected 48 h after [3H]leucine injection.
Oocytes and embryos of the cockroach Blattella germanica were examined by optical and electron microscopy to study yolk granule degradation during embryo development. During vitellogenesis, progressively larger yolk granules are formed in the ooplasm and by chorionogenesis, the mature granules are packed so tightly that their shape is highly distorted. Throughout ovarian development, endosymbiotic bacteria lie at the follicle cell/oocyte interface. Just prior to chorionogenesis the endosymbionts transit the oocyte plasma membrane and cluster at the periphery. Bacteria become more numerous over the ventral region of the egg by day 1 postovulation and begin to invade the interior of the yolk mass from the ventral periphery. At that time, lysis of the nearby yolk granules occurs while those in the central ooplasm remain intact and free of bacteria up to day 4. Vitellophages become evident by day 2 postovulation. These cells are also distributed over the egg's periphery but are most numerous in the ventral region. Vitellophages, in association with the endosymbionts, protrude toward the yolk granules and extend filo- and lamellipodia over the granule surface. Portions of the yolk granules are then engulfed and sequestered as large vacuoles in the vitellophage's cytoplasm. The vacuoles then become vesiculated. As embryo development proceeds, the vesiculated portions partition into smaller multivesicular bodies. This study describes the dynamics of yolk granule-vitellophage interaction in embryos of B. germanica and suggests that yolk utilization entails the cooperative efforts of both vitellophages and endosymbiont bacteria.
Various aspects of the processing of Blattella germanica vitellin (Vt) in the oocyte and egg have been investigated. Employing subunit specific antibodies, the precursor product relationships among the subunits of this Vt have been determined. After endocytosis of Vt by the oocyte, the Mr 160,000 subunit Vt is cleaved to products of Mr 95,000 and Mr 50,000. In association with an unprocessed Mr 102,000 peptide, these form the subunits of the Vt of freshly ovulated eggs. Between 4 and 5 days post ovulation (at 30°C), all three subunits of Vt are again processed proteolytically before use by the embryo. Although Vt's high mannose-type oligosaccharides are trimmed during embryogenesis, their modification occurs subsequent to the day 4–5 proteolysis, precluding the possibility that changes in oligosaccharide content or structure contribute to regulating this second proteolytic event. Although the predominant oligosaccharide of Vt is Man9GlCNAc2, the Mr 50,000 subunit of egg-borne Vt contains a much higher proportion of Man6GlCNAc2 than the other two subunits; therefore, this portion of the precursor vitellogenin must be more accessible to the processing mannosidases of the endoplasmic reticulum during its biosynthesis. A microtechnique for aspirating the yolk from individual eggs in an oothecapermits its isolation free of contamination by embryonic tissue. With this procedure, the specific activity profiles of exo-α-mannosidase, exp-β-N-acetylglucosaminidase, α-glucosidase and acid phosphatase were monitored during the first 6 days after ovulation, and some of their properties were also determined. Expression of the acid phosphatase and exo-β-N-acetyl-glucosaminidase activities coincide with the day 4–5 proteolysis, while α-mannosidase remains relatively constant throughout the first 6 days. Functions for these enzymes and the oligosaccharides of Vt during Vt storage and utilization are proposed.
In the eggs of the cockroach Blattella germanica, vitellin (Vt) utilization is initiated 4 days postovulation by the proteolytic processing of its three subunits. These reactions yield a specific set of peptides that are consumed by the developing embryo. A yolk proteinase activity, believed central to this processing event, has been investigated. First expressed at day 3 postovulation, just prior to Vt's processing, its specific activity with synthetic substrates increased four-fold to 18-fold through day 6. In addition, a mixing experiment showed that these proteinases(s) can also process Vt's large subunits in vitro. A relationship between Vt processing and proteinase specific activity was also noted with two B. germanica translocation heterozygotes, which displayed differences in the extent of Vt processing. One group of eggs (group A) failed to process any Vt subunit. A second group (B) processed the Mr 102,000 subunit but not the Mr 95,000. A third group (C) processed their Vt normally. Proteinase specific activities in the yolk of translocant's eggs at day 6 mirrored the extent of processing, being highest in group C eggs and effectively absent from the yolk of group A eggs. Eggs defective in Vt processing also contained arrested embryos. It is concluded that the yolk proteinase activity described here participates in Vt processing at day 4 postovulation. Microscopic examination of yolk obtained from eggs of wild type females showed that, as processing began in vivo (day 4), the yolk granules also underwent an abrupt decrease in size from diameters of 15–30 μm to 3–10 μm. Yolk granules of those translocant's eggs that were defective in Vt processing did not undergo this size decrease, suggesting that granule reorganization and Vt proteolysis may be linked functionally.
In eggs of the cockroach Blattella germanica, vitellin (Vt) utilization by the embryo is initiated at day 4 postovulation by the proteolytic processing of its three subunits to a specific set of peptides. A report from our laboratory (Nordin et al.: Archives of Insect Biochemistry and Physiology 15:119, 1990) described a yolk proteinase, activated at days 3–4, which processes the Vt. Further investigation of this event has focused on the yolk granules. Granules from eggs 4–6 days postovulation contained a significant subpopulation which accumulated high concentrations of the dye acridine orange (AO), a fluorescent probe of vesicle acidification, while those from eggs 0–3 days postovulation did not. AO accumulation was caused by proton translocation and was not due to dye binding or a Donnan equilibrium. The temporal correlation of granule acidification with Vt processing suggests a role for this event in yolk proteinase activation in B. germanica. This hypothesis was supported by the finding that incubation of yolk from freshly ovulated eggs in vitro at pH of 5 and below resulted in Vt processsing. Yolk granules of the blowfly Phormia regina also became acidified but this occurred in the oocyte prior to egg deposition.
Proteolytic processing of the vitellin in Blattella germanica eggs occurs 4 days postovulation and is correlated with both the onset of its utilization and the major portion of the embryo's growth. Yolk phosphatase is also expressed coincident with this event, and some aspects of its activation have been investigated. The enzyme is absent from the ooplasm (yolk) during the first 2 days following ovulation but increases approximately 20-fold in specific activity between days 3 and 4, when assayed at pH 3.9 or 4.8 and 9-fold at pH 6.5. No activation is observed for yolk-bound α-mannosidase, its specific activity remains elevated through the first 6 days following ovulation. This suggests that expression of the phosphatase is regulated independently of that of α-mannosidase in the yolk. Yolk with active phosphatase can dephosphorylate native vitellin, delipidated vitellin, and phosphocasein. Sucrose density gradient centrifugation of yolk obtained from eggs 4 days postovulation, revealed that phosphatase activity cosediments with material which reacts with antivitellin antibodies, while α-mannosidase and β-N-acetyl glucosaminidase are found near the top of the gradient. Oothecae derived from crossing certain translocational heterozygote males and wild-type females contain some eggs with severely depressed levels of yolk phosphatase in which embryos do not grow. Vitellin in these eggs fails to undergo proteolytic processing as late as day 5 postovulation and retains the subunit composition of freshly ovulated vitellin.
Two classes of vitellogenin binding sites with Kd-values of 7.3 nM and 290 nM were observed in follicle-membrane preparations of the cockroach Nauphoeta cinerea using a membrane-binding assay at pH 8. Separation of follicle cells and basal laminae from oocyte membranes prior to binding studies showed that the fraction consisting of follicle cells and basal laminae (FC/BL) contained high-affinity binding sites for vitellogenin (Kd=16.6 nM), whereas loweraffinity binding sites (Kd=200 nM) were found in the oocyte membrane fraction. The concentration of Ca2+ had a distinct effect on vitellogenin binding and uptake: maximal binding to the oocyte membrane fraction was observed at 0.3 mM Ca2+ and to the FC/BL fraction at 10 mM, whereas uptake of vitellogenin by oocytes in vitro was highest at 4 mM Ca2+. The calcium ionophore A23187 decreased vitellogenin uptake. This effect of A23187 could be counteracted by the calcium chelator Quin2. A hypothetical model for the uptake of vitellogenin into follicles of Nauphoeta cinerea is suggested.
Three major yolk proteins, vitellin, egg-specific protein and 30 kDa proteins, were purified from the same extracts of Bombyx mori eggs by high-performance liquid chromatography on a molecular sieving column. Each preparation was judged to be homogeneous by polyacrylamide gel electrophoresis. The subunit structure was estimated to be as follows: vitellin is a tetramer with a molecular mass of 420 kDa, consisting of two heavy subunits (178 kDa) and two light subunits (43 kDa); egg-specific protein is a trimer (225 kDa) of two heavy subunits (72 kDa) and one light subunit (64 kDa); 30 kDa proteins are a mixture of three monomers (1, 2 and 3) consisting of respective subunit molecular masses of 32.0, 31.0 and 29.5 kDa. The three yolk proteins contained the usual amino acids together with various lipids and carbohydrates. Antisera to each protein did not cross-react. The titration of vitellin, egg-specific protein and 30 kDa proteins on rocket immunoelectrophoresis showed a differential accumulation pattern during the course of oogenesis. In newly laid eggs, vitellin, egg-specific protein and 30 kDa proteins accounted for approx. 40%, 25% and 35%, respectively, in weight. The eggs developed in male hosts after implantation of ovary discs were deficient in vitellin but contained egg-specific protein and 30 kDa proteins at comparable levels to the normal female eggs. During embryogenesis, egg-specific protein was rapidly and completely utilized. Approx. 35% vitellin and 50% 30 kDa proteins remained unused and were carried over to the hatched larvae. Such accumulation and utilization of yolk proteins are correlated with the fates of the proteins during oogenesis and embryogenesis of B. mori.
1.1. Cathepsin-type activity of homogenates of Locusta migratoria migratorioides eggs is reported.2.2. Cleavage of haemoglobin was optimal at pH 3·5–4·1.3.3. Activity was related to age of eggs after oviposition and to embryonic stages.4.4. Occurrence and possible role of cathepsin-type enzymes in insects and in their eggs are discussed.
Phosphorylation of vitellogenin (yolk protein precursor) and vitellin (major yolk protein) polypeptides of Leucophaea maderae was studied by [32P]ortho phosphate labeling and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) autoradiography. The vitellogenin molecule was isolated from the hemolymph and fat body by antibody precipitation and high-performance liquid chromatography (HPLC), and shown to consist of at least five polypeptides (“subunits”) which had apparent molecular masses of 155, 112, 95, 92 and 54 kD. Labeling studies with 32P showed that the covalently attached phosphorus was distributed in an uneven fashion among the five polypeptides. The two heavily-phosphorylated polypeptides, 112 and 54 kD, corresponded to the large and small, mature vitellin subunits. Quantitative SDS-PAGE analysis of long-term 32P-labeled vitellin showed that these large and small “subunits” contained 55 and 30%, respectively, of the total radioactivity.
In vivo labelling of female Drosophila melanogaster with [32P]-inorganic phosphate, followed by immunoprecipitation of the vitellin and vitellogenin polypeptides, has shown that all three polypeptides contain covalently attached phosphate moieties. By transplanting ovaries into males, it was possible to demonstrate that the polypeptides produced in the ovary are phosphorylated in a manner that was not distinguishable from that found for haemolymph vitellogenin polypeptides. Two dimensional gel electrophoresis, however, of [32P]-labelled vitellin polypeptides suggests that phosphorylation alone probably does not account for the charge heterogeneity of the polypeptides.
1.1. Adult females of the stick insect Carausius morosus were injected with either [35S]methionine or [3H]leucine and eggs laid during the following 2 weeks collected for further analysis.2.2. After the injection, radioactivity started to appear in newly laid eggs with a time-lag of 9 days, reached a maximum incorporation on day 10 and then levelled off, due to dilution of the injected tracer.3.3. Newly laid eggs from injected females were analyzed by gel electrophoresis and the incorporated radioactivity revealed fluorographically.4.4. Data showed that vitellin polypeptides were the only egg constituent to become labelled under these conditions.5.5. In addition, light microscope autoradiography proved that the radioactivity incorporated in vivo into ovarian follicles and eggs was restricted to the forming yolk spheres or to the central ooplasm, respectively.6.6. Radioactive embryos were allowed to develop and the polypeptides appearing during development identified fluorographically on SDS gels.7.7. To establish whether these newly appearing polypeptides were due to de novo synthesis or to fragments of pre-existing vitellin polypeptides, embryos were also cultured in vitro in the presence of the above radioactive amino acids.8.8. These conditions allowed newly synthesized proteins to be identified in both native and SDS gels and to be distinguished from the vitellin polypeptides labelled in vivo.
Confocal scanning immunofluorescent microscopy and monoclonal antibodies were used to examine the route of uptake of vitellogenin (VG) by vitellogenic follicles and the ooplasmic localization of vitellin (VN) in the cricket, Acheta domesticus, and the stick insect, Carausius morosus. Uptake and cytoplasmic regionalization of a non-vitellogenic sulfated protein, sp 157/85, by C. morosus oocytes were also examined. By indirect immunofluorescence VG in both species and sp 157/85 were visualized in spaces between follicle cells and in peripheral yolk spheres. One cricket VG polypeptide had a regionalized distribution in the folliclular epithelium, and VN polypeptides in both species and sp 157/85 in C. morosus had regionalized distributions within the ooplasm. Localization of sp 157/85 to the anterior pole of the oocyte appeared to be stage-specific.
The combination of ion suppression, reverse phase high pressure liquid chromatography (IS-RPHPLC) and radioimmunoassay (RIA) employing two relatively non-specific, complimentary antisera was used to identify and quantify ecdysteroids of divergent polarities during embryogenesis of the tobacco hornworm, Manduca sexta. Newly layed eggs (0–1 hr) contained high levels (>30 μg/g) of a maternally derived, polar conjugate of 26-hydroxy-ecdysone (26E) but less than 0.6 and 0.2 μg/g of the polar conjugates of ecdysone (E) and 20-hydroxyecdysone (20E), respectively. The only free ecdysteroid detected was 26E (0.4 μg/g). Between 4 and 12 hr after oviposition, marked deconjugation activity occurred as titers of free 26E increased to 14 μg/g while levels of conjugated 26E fell to 19 μg/g. By this time there were only negligible levels of free E (less than 0.1 μg/g). After 12 hr, the deconjugation of the maternal polar 26E-26-phosphate conjugate slowed and levels of free 26E fell as it was metabolized. At 24 hr, low titers of free 20,26-dihydroxyecdysone (20,26E, 0.4 μg/g) and a β-D-glucose conjugate of 26E (0.8 μg/g) were detected, presumably formed by embryonic 20-monooxygenase and uridine diphospho-α-D-glucose (UDPG) glucosyltransferase activities, respectively. About the time of the deposition of the first larval cuticle (66–72 hr), these 26E metabolites reached high concentrations, (5 μg/g free 20,26E and 7 μg/g 26E-glucose conjugate) while free 26E fell to 2 μg/g. In newly hatched larvae, the 26E-glucose conjugate was the major detectable ecdysteroid. At no time during embryogenesis did the concentrations of free E and 20E exceed 0.5 μg/g and 0.3 μg/g, respectively.
A serine proteinase (molecular mass, about 25,000 Da) has been found in Drosophila mature oocytes. (1) The detected activity increases exponentially during embryogenesis. (2) The subcellular localization changes from the yolk granules, in oocytes, to the soluble fraction, in late embryos. (3) The proteinase cleaves by basic residues, but arginine is preferred (about 7-fold) over lysine.
Developing ovarian follicles of the silkmoth Hyalophora cecropia accumulate large amounts of ecdysteroids during oogenesis. As measured by an ecdysteroid radioimmunoassay (RIA), this accumulation begins near the end of vitellogenesis, just prior to nurse cell collapse, and continues through the beginning of chorion formation. Analysis of ovarian ecdysteroids by a combination of high-performance liquid chromatography and RIA demonstrates that the major proportion of these are present in a highly polar form, most likely as conjugates; ecdysone and 20-hydroxyecdysone were present as well, in much lower proportions. Light microscopic autoradiographs of photoactivated follicles after in vivo incubation with [3H]ecdysone indicate that within the oocyte ecdysteroids are associated with the yolk sphere membranes.
The occurence of the two major yolk proteins in vitellogenic follicles at various stages of development in the house cricket, Acheta domesticus, was studied electrophoretically. Follicles were staged into size classes ranging from 0.75 mm (stage I) to ≥2.0 mm (stage IV) for unfertilized eggs (chorionated follicles) and the molar ratio of the two vitellins within follicles of each stage determined. The ratio of vitellin I to vitellin II during the early stages of vitellogenesis was found to be greater than that during later stages, suggesting the possibility of changing selectivity of vitellogenin uptake by follicles as they progress through the vitellogenic phases of oogensis. The relative distribution of vitellins I and II within the cytoplasm of unfertilized, chorionated follicles was studied by sectioning stage IV follicles into thirds and determining the molar ration of vitellin I to II in each third. It was found that a differential distribution of each vitellin is established during oogenesis such that vitellin I is present in greater quantities in the posterior region and vitellin II in greater quantities in the anterior region of the follicle.
The molecular weights of the two vitellins were estimated using native polyacrylamide gels of varying percents acrylamide. The molecular weights of vitellins I and II were estimated at 352,000 and 327,000 daltons, respectively.
Formation of nuclear envelopes during the last cleavage mitosis and the formation of the cell membranes during the cellularization of the blastoderm have been studied ultrastructurally in the blowfly egg. Dense bodies arising from yolk granules by budding could contain membrane material destined to be incorporated into the new membranes of the blastoderm. The presence of transitional structures indicates that these bodies can be converted into dark multivesicular bodies. Large amounts of endoplasmic reticulum are found around the mitotic nuclei. Clusters or branched chains of vesicles associated with this are interpreted as evidence for the formation of endoplasmic reticulum by the breakdown of dark multivesicular bodies. Nuclear envelopes of mitotic daughter nuclei probably originate from endoplasmic reticulum. The egg contains both intranuclear and extranuclear annulate lamellae.
The main events of cytokinesis are furrow initiation and cell membrane growth during the slow first phase, but probably only cytokinetic movement during the rapid second phase. On the assumption that cell membrane growth occurs by incorporation of complete membrane pieces, the addition of coated vesicles and/or light multivesicular bodies is definitely most probable. Some intermediate profiles indicate that light and dark multivesicular bodies are related. The membrane needed for second phase cytokinesis could well be provided by the unfolding of surface microvilli and protuberances of the furrow canal.
Developing embryos of the stick insect Carausius morosus were examined ultrastructurally with a view to studying vitellophage invasion of the yolk mass during and after germ band formation. Newly laid eggs in C.morosus have a unique yolk fluid compartment surrounded by a narrow fringe of cytoplasm comprising several small yolk granules. Vitellophages originate mainly from a thin layer of stem cells, the so-called yolk cell membrane, interposed between the germ band and the yolk mass. Throughout development, a thin basal lamina separates the yolk cell membrane from the overlying embryo.
Vitellophages extend from the yolk cell membrane with long cytoplasmic processes or filopodia to invade the central yolk mass. Along their route of entrance, filopodia engulf portions of the yolk mass and sequester it into membrane-bounded granules. As this process continues, the yolk mass is gradually partitioned into a number of yolk granules inside the vitellophages.
Later in development, the yolk cell membrane is gradually replaced by the endodermal cells that emerge from the anterior and posterior embryonic rudiments. From this stage of development onwards, vitellophages remain attached to the basal lamina through long filopodia extending between the endodermal cells. Yolk confined in different vitellophagic cells appears heterogeneous both in density and texture, suggesting that yolk degradation may be spatially differentiated.
The sea urchin embryo is a closed metabolic system in which embryogenesis is accompanied by significant protein degradation. We report results which are consistent with a function for the ubiquitinmediated proteolytic pathway in selective protein degradation during embryogenesis in this system. Quantitative solid- and solution-phase immunochemical assays, employing anti-ubiquitin antibodies, showed that unfertilized eggs of Strongylocentrotus purpuratus have a high content of unconjugated ubiquitin (ca. 8 × 108 molecules), and also contain abundant conjugates involving ubiquitin and maternal proteins. The absolute content of ubiquitin in the conjugated form increases about 13-fold between fertilization and the pluteus larva stage; 90% or more of embryonic ubiquitin molecules are conjugated to embryonic proteins in hatched blastulae and later-stage embryos. Qualitatively similar results were obtained with embryos of Lytechinus variegatus. The results of pulse-labeling and immunoprecipitation experiments indicate that synthesis of ubiquitin in S. purpuratus is developmentally regulated, with an overall increase in synthetic rate of 12-fold between fertilization and hatching. Regulation is likely to occur at the level of translation, since others have shown that levels of ubiquitin-encoding mRNA remain virtually constant in echinoid embryos during this developmental interval. The sea urchin embryo should be a useful system for characterizing the role of ubiquitination in embryogenesis.
A perchloric acid extract of eggs of Hemileuca oliviae inhibits bovine trypsin, kallikrein and papain, as well as the native proteolytic activity (pH 7.0) of the developing embryo. Specificity is indicated by the lack of inhibition of other proteases. The amount of inhibitory activity changes during embryological development, reaching a maximum around 23 days, when the larva is fully developed. The inhibitory activity was lost by dialysis and was destroyed by ashing (450°C, 18 h) but was unaffected by exposure to 97°C for 3 min. The presence of two protease inhibitors was detected in the perchloric acid extract. The principal component has a molecular weight of approx. 9000 and its heat sensitivity is affected by pH. At the present time the role of these inhibitors in the developing embryo is unknown. Some trypsin-like native protease activity occurs in the egg during embryogenesis and may thus be the target enzyme in vivo.
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.
Two high mol. wt proteins have been identified and purified from newly laid eggs of Carausius morosus. Due to their apparent role in yolk formation, both proteins are taken to be vitellins, hereafter termed vitellin A and B.Upon dissociation by sodium dodecyl sulphate, vitellin A is shown to release two polypeptide subunits in equimolar ratio with a mol. wt of 180,000 and 70,000. When subjected to the same treatment, vitellin B releases three polypeptide subunits which range in mol. wt from 120,000 to 60,000.Antisera prepared against vitellins A and B show no sign of cross reaction with each other's antigen, whereas both react with haemolymph from adult females. Partial proteolysis of sodium dodecyl sulphate treated polypeptides reveals a number of electrophoretically similar low mol. wt fragments between the two. A model for the two egg proteins is proposed on the basis of the evidence available.
The action of homogenates of eggs of Locusta migratoria migratorioides (R. and F.) in various stages of development on casein and on leucylglycylglycine at various pH values was investigated. The pH values of the homogenates of eggs in different stages were found to vary from 6·0 to 6·6. In no case was hydrolysis of casein or leucylglycylglycine obtained with freshly laid eggs. At pH 5·6 a distinct cleavage of casein was obtained with batches of eggs varying from stage VIII to stage XII (i.e. during the polypod stage and the beginning of the oligopod stage). This action increased towards the end of development. At pH 7·8 a distinct action on casein appeared occasionally with batches of eggs varying from stage X to stage XX (i.e. from the advanced polypod stage to the stage when the head of the embryo reaches the anterior end of the egg after blastokinesis). This action was always found in the batches with eggs from stages XVIII to XXIII (i.e. immediately after the end of blastokinesis to shortly before hatching) and markedly increased towards hatching. At pH 6·5 a slight action on casein appeared with the batches containing eggs of stages VIII–XII. The cleavage became distinct with stages X–XX, and increased further towards the end of development. A distinct action on leucylglycylglycine, measured mainly at pH 7·8, occurred with stages III–XI and a greater one with eggs of all higher stages.It is concluded from the results that the homogenates of the eggs contain at least two kinds of endopeptidases.
Egg-specific protein was identified and purified from the eggs of the silkworm, Bombyx mori by DEAE-cellulose column and Sephacryl S-200 column chromatography. The final preparation was highly homogeneous as judged by a polyacrylamide gel electrophoresis and immunodiffusion test. Egg-specific protein was defined as glycolipoprotein with a mol. wt of 125,000. The molecule was composed of one subunit with mol. wt of 55,000, and contained about 2% carbohydrate and about 4% lipid. The rabbit anti-egg-specific protein serum cross-reacted with the extracts of ovaries and eggs but not with other tissues. The antiserum also reacted with the extracts of ovaries developed in male hosts. Immunohistochemical study revealed that this protein is localized in the follicle cells and yolk spheres of the developing ovaries and in the yolk cells of the early embryonic eggs. The egg-specific protein increased during oogenesis and was utilized more rapidly during embryogenesis.