Development of accessory reproductive glands and its control by the corpus allatum in adult male Melanoplus sanguinipes

Changes in the protein content of and the rate at which labelled protein appears in the accessory reproductive glands (ARG), fat body, and haemolymph were studied in normal and allatectomized (CA^?) males of the migratory grasshopper, Melanoplus sanguinipes. In addition, the effects of treatment of CA^? insects with synthetic juvenile hormone (SJH), copulation, and removal of the ARG were examined.In normal males the protein content of the ARG increases linearly during the first 14 days after emergence. Incorporation of label by the ARG is maximal at day 7 and then decreases until, at day 14, it is the same as at day 1. The protein content of the fat body and haemolymph increases up to day 10 then declines, whereas changes in the uptake of label by the fat body and haemolymph parallel those of the ARG.Removal of the corpora allata (CA) prevents the normal increase in protein content of the ARG, but the protein content of the fat body and haemolymph increases steadily throughout the 14 days. Incorporation of label into the ARG, fat body, and haemolymph remained low throughout the experiment. Treatment of CA^? insects with SJH, or copulation, stimulates the uptake of label by the ARG, fat body, and haemolymph and also results in an increase in their protein content.Removal of the ARG leads to an increase in the protein content of the fat body and haemolymph. Uptake of label by the fat body remains low after the operation. Although the rate at which labelled protein appears in the haemolymph is high initially, it declines steadily to day 14.We conclude that the CA regulate ARG development. It is suggested that the fat body, under CA control, synthesizes proteins which are incorporated into secretions of the ARG. Further, it is proposed that the primary effect of copulation is activation of the CA.     

Accumulation of secretory proteins in the accessory reproductive glands of the male migratory grasshopper, Melanoplus sanguinipes: A developmental study

Production of secretion in the accessory reproductive glands of male Melanoplus sanguinipes has been examined by electrophoresis and radiolabelling. The secretion of each group of tubules (long hyaline glands, white glands, short hyaline glands, and seminal vesicles) can be resolved into more than 20 protein bands and includes several glycoproteins and, in the long hyaline and white glands only, lipoproteins. Each group of tubules has a characteristic pattern of synthesis and accumulation of proteins; that is, specific proteins appear in the secretion at particular times during sexual maturation. In allatectomized insects, the long hyaline glands accumulate very little secretion; the white glands and short hyaline glands accumulate about one-third the normal amount; and accumulation in the seminal vesicles is not affected by the operation. Allatectomy exerts its effect by inhibiting the synthesis of particular proteins. The observations are discussed in terms of juvenile hormone-specific protein synthesis in the accessory reproductive glands.     

Male accessory gland substance of Melanoplus sanguinipes: An oviposition stimulant under the control of the corpus allatum

The accessory glands of male Melanoplus sanguinipes contain an oviposition stimulant. Injection of gland extracts from mature males induces oviposition in 75 per cent of capable virgin females within 24 hr. Injection of gland extracts from allatectomized males produces no stimulatory effect. Gland extracts from mature males contain two antigens which cannot be detected in gland extracts from allatectomized males. However, both antigens can be detected in gland extracts from allatectomized males 3 days after treatment with juvenile hormone.Anion-exchange chromatography of mature gland extracts yielded two fractions which, when injected into virgin females, induced oviposition in 71 per cent of capable insects within 24 hr. These two fractions are immunologically identical to the two antigens which are absent from gland extracts of allatectomized males. We suggest that synthesis of the oviposition stimulant in Melanoplus is controlled by the corpus allatum.Injection of brain extract also induces oviposition in 100 per cent of capable virgin females within 24 hr. A possible rôle for the brain in the oviposition process is discussed.     

Contribution of male-produced proteins to vitellogenesis in Melanoplus sanguinipes

The transfer during copulation of radioactively labelled male accessory reproductive gland (ARG) protein and its accumulation by the ovary of Melanoplus sanguinipes have been studied. Most of the transferred material leaves the spermatheca within 24 hr and enters the haemolymph from which it can be accumulated by the ovary. Injection of labelled male ARG protein into vitellogenic females demonstrates that during the first 24 hr after injection, accumulation by the ovary is rapid. Immunoprecipitation and immunoelectrophoretic studies indicate that some of the ARG protein is accumulated unchanged. It is proposed that when the male transfers several spermatophores during copulation, he may make a significant contribution of protein to the developing oöcytes.     

Infection of Locusta migratoria with entomopoxviruses from Arphia conspersa and Melanoplus sanguinipes grasshoppers

Entomopoxvirus (EPV) occlusion bodies isolated from Arphia conspersa and Melanoplus sanguinipes grasshoppers were fed to 3rd and 4th instar Locusta migratoria nymphs. Locus mortality induced by A. conspersa EPV was first detected 18 days after addition of virus to the diet, and reached a level of approximately 68% of the colony population by 60 days after virus inoculation. In a similar population of L. migratoria nymphs, mortality induced by M. sanguinipes virus reached 90% 60 days after virus inoculation. Entomopoxvirus was isolated from M. sanguinipes EPV infected locust nymphs and the viral DNA was cleaved with several restriction endonucleases. The DNA fragment patterns obtained after agarose gel electrophoresis were compared with the fragment patterns from the original sample of M. sanguinipes EPV DNA cleaved with the same restriction endonucleases. No differences in the cleavage patterns were detected between the two virus DNA samples. Virus structural proteins of M. sanguinipes EPV purified from infected locust nymphs were compared by polyacrylamide gel electrophoresis with virus proteins isolated from the original sample of M. sanguinipes EPV. A total of six different virus protein bands were detected between the two poxvirus preparations.     

Control of diapause induction by a change in photoperiod in Melanoplus sanguinipes

The effect of photoperiod on diapause induction, developmental time and body size was examined in Melanoplus sanguinipes, the lesser migratory grasshopper. Contrary to what is found in most insects, facultative diapause-egg production is found to be controlled by changing rather than constant photoperiods. In addition, developmental time is shown to be faster under short-day photoperiods than long-day photoperiods. And finally, body size is larger under long-day photoperiods and smaller under short-day photoperiods. Implications of these results for the regulation of the seasonal life cycle of M. sanguinipes in the field are discussed.     

Structural proteins of Amsacta moorei,Euxoa auxiliaris, and Melanoplus sanguinipes entomopoxviruses

The structural proteins of Amsacta moorei, Euxoa auxiliaris, and Melanoplus sanguinipes entomopoxviruses (EPVs) were separated by electrophoresis on sodium dodecyl sulfate (SDS)-polyacrylamide gels. More than 35 structural proteins were detected in each virus. Based on the distribution and the variation in the molecular weights of the virus structural proteins little homology was detected between the EPVs and vaccinia virus. The molecular weight of Amsacta EPV occlusion body matrix protein (110,000) was determined by SDS-acrylamide gel electrophoresis. The occlusion body matrix protein of Amsacta EPV occluded virus isolated from infected E. acrea larvae was rapidly degraded at pH 10.6 to peptides of approximately 94,000 and 60,000 daltons. After 2 hr incubation at alkaline pH, Amsacta EPV occlusion body protein was degraded to approximately 56,000 daltons. Proteolysis of occlusion body protein was inhibited by SDS. No proteolytic degradation was detected in occlusion body matrix protein isolated from Amsacta EPV infected BTI-EAA cells. Amino acid analysis indicates that entomopoxvirus occlusion body matrix protein consists of approximately 20% acidic amino acids and 9% of the sulfur-containing amino acids cysteine and methionine.     

Development of secretory ability in the spermatheca of the migratory grasshopper, Melanoplus sanguinipes

During the first week of adult life, the protein content of the spermatheca increases by about 45% but the carbohydrate content does not change significantly. Incorporation of radiolabelled leucine into protein is high in newly emerged females but by day 1 has declined to about two-thirds the initial level, where it then remains. With SDS-PAGE about 30 protein bands separate, including six glycoprotein fractions. All bands are present throughout sexual maturation and except for one (at 25 Kd), show little change in quantity during this period. Allatectomy or hormone replacement therapy has little effect on the parameters measured and it is concluded that the development of secretory activity in the spermatheca is not controlled by juvenile hormone.     

Molecular weight and base composition of DNA isolated from Melanoplus sanguinipes entomopoxvirus

The DNA genome of the orthopteran entomopoxvirus (EPV) isolated from Melanoplus sanguinipes was released from the virus by treatment with proteinase K and sodium dodecyl sulfate (SDS). The average length of the virus DNA molecule was determined by electron microscopy to be 62.8 μm, corresponding to a molecular weight of 124.3 × 10⁶ daltons (80 kb). The buoyant density of Melanoplus EPV DNA in cesium chloride was calculated to be 1.678 g/cm³, which corresponds to a base ratio of 18.6 mole% guanine + cytosine.     

The neuro-endocrine control of protein metabolism in the migratory grasshopper, Melanoplus sanguinipes

In normal females, distinct fluctuations in the protein content of the fat body and haemolymph are evident during each gonotrophic period. These fluctuations partly reflect changes in the protein requirements of the developing oocytes. Almost one half of the total protein deposited in the mature ovary is sequestered during the final stages of vitellogenesis when protein accumulated in the fat body and haemolymph is rapidly depleted. Although similar amounts of protein are deposited in the ovary during the first and subsequent gonotrophic periods, significantly less extraovarian protein is present throughout the latter periods.The accumulation of large amounts of protein in the fat body and haemolymph of ovariectomized females suggests that most yolk protein is of extraovarian origin. As the total protein content of these insects is comparable to that of vitellogenic females, ovariectomy apparently has no immediate effect on protein synthesis.Allatectomy or cautery of the median neurosecretory cells (mNSC) prevents vitellogenesis. Although protein gradually accumulates in the fat body and haemolymph of allatectomized females, the total protein content of these insects is significantly lower than that of controls. Treatment of allatectomized females with juvenile hormone analogue leads to a temporary but significant increase in the protein content of the fat body. However, the subsequent decline in fat body protein is paralleled by a pronounced increase in the protein content of the ovary. These findings suggest that the corpora allata (CA) stimulate both yolk protein synthesis in the fat body and its uptake into the ovary. The total protein content of mNSC-cauterized females is less than that of allatectomized females. This observation supports the proposal that the mNSC have not only an allatotropic effect but also a direct effect on protein synthesis.     

Incorporation of labelled dietary n-alkanes into cuticular lipids of the grasshopper Melanoplus sanguinipes

Dietary hydrocarbons are incorporated into cuticular lipids of the grasshopper Melanoplus sanguinipes. Dietary secondary alcohols and ketones, however, are not incorporated into the cuticular lipids. In typical experiments from 8 to 28 per cent of the fed labeled n-alkanes are recovered in the cuticular lipids. Most of the radioactivity recovered from feeding the C₂₃ n-alkane and a significant amount from the C₂₅ was found as a secondary alcohol in the form of a wax ester. The C₂₉ and C₃₁ n-alkanes were recovered primarily unchanged as the n-alkane. Eighty-five per cent of injected acetate incorporated into the hydrocarbon fraction is in the branched hydrocarbons. These results show that the insect synthesizes its branched hydrocarbons, whereas a large part of the normal hydrocarbons can be dietary.     

The hormonal control of accessory reproductive gland development in female Schistocerca gregaria

Allatectomy of adult female Schistocerca gregaria prevents the normal development of the accessory reproductive glands and no secretion is produced. Development of the glands can be restored by the administration of synthetic juvenile hormone and the response is dose-dependent. A continuous supply of hormone is required for maintenance of secretory activity. In the normal developmental sequence the total protein content of the glands remains constant until the time at which vitellogenesis occurs in the terminal oöcytes. As maturation proceeds there is a linear increase in protein content of the glands. The initial increase occurs as a result of cellular changes in the glands and is then followed by an increase due to an accumulation of secretion in the lumina.     

Effects of severance of stomatogastric nerves on egg-laying,feeding, and the neuroendocrine system in Melanoplus sanguinipes

The rate of egg-laying by female Melanoplus sanguinipes was reduced to about 60 per cent of that for normal mated females following severance of the recurrent nerve or the frontal ganglion connectives or removal of the frontal ganglion, but it was still somewhat higher than the rate for normal virgin females. Although the fat body was markedly reduced in size, the fact that eggs and some faeces were produced suggests that protein digestion and synthesis did not stop in our experimental insects. Feeding was not affected but food passage along the gut was severely restricted following these operations; however, the corpus allatum was not affected.     

Ultrastructure and nature of secretory proteins in the male accessory gland of Drosophila funebris

The ultrastructural differentiation of the secretory cells and the nature of secretory proteins in the male accessory gland of Drosophila funebris have been studied by electron-microscopic and immunological methods. (1) In the pupae at $\text{1}\text{1}\text{2}$ days before eclosion, secretory products can be detected in the lumen, even though most glandular cells are at the initial phase of differentiation. At the time of eclosion both main and secondary cells are fully differentiated, but the whole set of five immunologically active proteins are detectable only on the second to third day of adult life. (2) The secondary cells contain giant protein granules, the so-called filamentous bodies, which become partially fused and the filaments assume a twisted form. Randomly dispersed filaments and closely packed filament bundles are also visible in the gland lumen. Antigenic labelling of ultrathin sections and immunoreplica electrophoresis yielded no evidence for the microtubular nature of these filaments. The secretion stored in the lumen contains in addition a large quantity of flocculent proteins which have their origin in the main cells. (3) During the period of high secretory activity in the 7-day-old male flies no vacuolization and disintegration of either the main or secondary cells have been observed. We conclude that both types of cells have the merocrine secretory mechanism. (4) Ultrastructural alterations in the glandular cells confirmed our previous observation that copulation stimulates RNA and protein synthesis.     

An electrophoretic study of proteins of the ovary,fat body,and haemolymph in the migratory grasshopper, Melanoplus sanguinipes

Electrophoretic separation of saline extracts from the ovary revealed 14 proteins. Twelve proteins were detected in the fat body, of which seven had electrophoretic mobilities identical to those in the ovary. Similarly, eight of 16 proteins in the haemolymph of vitellogenic females ahad electrophoretically identical counterparts in the ovary. As these proteins accumulate in the haemolymph of ovariectomized females, the findings suggest that most yolk proteins are synthesized in the fat body. Although most female haemolymph proteins are present in males, two of the predominant yolk protiens are absent and represent female-specific proteins.Although certain proteins accumulate in the haemolymph of allatectomized females, the major ovarian proteins are absent or present in low concentrations. However, 48 hr after allatectomized females are treated with a juvenile hormone analogue, the haemolymph protein pattern resembles that of a normal female. This suggests that the corpora allata stimulate the synthesis of female-specific and other vitellogenic proteins. The median neurosecretory cells (mNSC) are also necessary for synthesis of female-specific proteins. Furthermore, proteins which are present in allatectomized females are absent in mNSC-cauterized insects suggesting that the mNSC stimulate general protein synthesis.     

The accessory gland and metathoracic scent gland function in Oncopeltus fasciatus

The paper provides further clues as to the physiological function and biological significance of the ‘accessory gland’ in the metathoracic scent apparatus of the lygaeid Oncopeltus fasciatus. From various lines of evidence (gas chromatographic, cytochemical) it is concluded as probable that the accessory gland secretes small quantities of a mucopolysaccharide secretory product together with water. The difficulty of reconciling these data with other data indicating that the accessory gland is involved in the biosynthesis of the scent aldehydes is discussed. It is suggested that the water secreted into the median scent reservoir by the accessory gland provides O. fasciatus with a means of volumetric compensation for a diminished output of scent repellent.     

A male accessory gland peptide that regulates reproductive behavior of female D. melanogaster

The adult male accessory glands of D. melanogaster synthesize and secrete a peptide that represses female sexual receptivity and stimulates oviposition. Normally, this peptide is transferred to females during copulation; however, the peptide shows the same biological activity after purification and subsequent injection into the abdominal cavity of female virgins. Amino acid sequencing of the purified peptide and oligonucleotide-directed cDNA cloning established that the peptide consists of 36 amino acids. It appears to be synthesized as a precursor with a hydrophobic signal sequence of 19 residues at its N-terminal end. The precursor peptide is encoded by a short mRNA that accumulates exclusively in the male accessory gland. The gene has been localized by in situ hybridization to polytene chromosomes at 70A.     

Ultrastructure of the male accessory ducts and prostate gland of Diclidophora merlangi (Monogenoidea)

The vas deferens, seminal vesicle, penis and common genital atrium of the monogenean, Diclidophora merlangi are lined by a very flat, lamellate epithelium. The structure is apparently syncytical, although nuclei or perikaryons have not been observed. The epithelium extends to just inside the gonopore where a septate desmosome marks the union with body tegument. There is minor regional variation in structure. The terminal portion of the seminal vesicle and the penis lumen are lined in part by the luminal cytoplasm of the prostate gland which surrounds this part of the reproductive tract. The prostate gland cells are synthetically active and produce a characteristic secretory body that is released either singly by exocytosis, involving membrane fusion, or in bulk via apocrine secretion. The secretion is acidophilic, PAS-positive and reactive for protein. The penis is sucker-like in structure and armed with a ring of 16 genital hooklets. Cilia have not been observed in any part of the male reproductive tract, and sense receptors are not apparent in the tegument surrounding the gonopore.     

Juvenile hormone regulation of male accessory gland activity in the red flour beetle, Tribolium castaneum

Male accessory gland proteins (Acps) act as key modulators of reproductive success in insects by influencing the female reproductive physiology and behavior. We used custom microarrays and identified 112 genes that were highly expressed in male accessory glands (MAG) in the red flour beetle, Tribolium castaneum. Out of these 112 identified genes, 59 of them contained sequences coding for signal peptide and cleavage site and the remaining 53 contained transmembrane domains. The expression of 14 of these genes in the MAG but not in other tissues of male or female was confirmed by quantitative real-time PCR. In virgin males, juvenile hormone (JH) levels increased from second day post adult emergence (PAE), remained high on third day PAE and declined on fourth day PAE. The ecdysteroid titers were high soon after adult emergence but declined to minimal levels from 1 to 5 days PAE. Feeding of juvenile hormone analog, hydroprene, but not the ecdysteroid analog, RH-2485, showed an increase in size of MAGs, as well as an increase in total RNA and protein content of MAG. Hydroprene treatment also increased the expression of Acp genes in the MAG. RNAi-mediated knock-down in the expression of JHAMT gene decreased the size of MAGs and expression of Acps. JH deficiency influenced male reproductive fitness as evidenced by a less vigor in mating behavior, poor sperm transfer, low egg and the progeny production by females mated with the JH deficient males. These data suggest a critical role for JH in the regulation of male reproduction especially through MAG secretions.     

Isolation and properties of Acheta accessory gland polysomes

Polysomes have been prepared from Acheta male accessory gland. The following factors are important in ensuring maximum yield: (1) homogenization at a large buffer to tissue excess (40 : 1), (2) use of 200 mM NH₄Cl as a nuclease inhibitor, (3) addition of either sodium deoxycholate or Triton X−100 to the buffer before homogenization and (4) use of the Dounce homogenizer. Inclusion of NH₄⁺ gives no deleterious effects with respect to dissociation or activity of the ribosomes.The occurrence of cold-induced ‘run-off’ of polysomes has been confirmed by direct measurement. Polysomes reassemble within 15 to 30 min after return of the animals to higher temperatures, with restoration of endogenous protein synthetic capacity to control levels; the ribosomes appear to retain an elevated capacity for poly(U)-directed protein synthesis.