Subtle effect of Xenos vesparum (Xenidae, Strepsiptera) on the reproductive apparatus of its male host: Parasite or parasitoid?

Parasitic castration is an adaptive strategy where parasites usurp the hosts' reproductive physiology to complete their life cycle. The alterations in the host traits vary in their magnitude, from subtle changes in the host morpho-physiology and behaviour to the production of complex aberrant phenotypes, which often depend on the host gender. The strepsipteran macroparasite Xenos vesparum induces dramatic behavioural and physiological changes in its female host, the paper wasp Polistes dominula, while its effect on the male phenotype is largely unknown. In this study we investigated how a single X. vesparum parasite influences the functional morphology of P. dominula male reproductive apparatus. We performed morphometry and ultrastructure characterization of corpora allata, testes, seminal vesicles and accessory glands in parasitized and unparasitized males, and also in young and old males to control for the effect of age on the natural deterioration of these organs. Our results show that age significantly affects the development of male reproductive apparatus. A low parasite load - one parasite per host is the common prevalence in the field - has only a marginal impact on the reproductive morphology of P. dominula males, affecting quantitatively but not qualitatively the protein content of male accessory glands. Thus, in male P. dominula wasps, X. vesparum appears to behave as a true "parasite", in clear opposition to the role of "parasitoid" that it takes in female hosts where castration causes the reproductive death.

Fine structure of corpora allata of castes with different rates of juvenile hormone production in the termite Reticulitermes flavipes.

The aim of this work is to describe corpora allata (CA) of several castes of the termite Reticulitermes flavipes that have different rates of juvenile hormone (JH) synthesis, with respect to differences in fine structure, volume, and intensity of allatostatin immunoreactivity in their innervation. The castes chosen are workers and their potential derivatives, apterous secondary reproductives and pre-soldiers (the precursors of soldiers). These castes, at the stages chosen, produce JH at low, high and intermediate rates respectively. Hormone production is positively correlated with volume and negatively correlated with intensity of allatostatin immunoreactivity in axons within the glands. Characteristics of fine structure that correlate with increased activity are increase in abundance and width of mitochondria, decrease in ability to fix and visualize smooth endoplasmic reticulum. These features have previously been described for CA of cockroaches and other insects. Glycogen in the CA of all of the castes studied, especially the large amounts in highly active glands of physogastric apterous reproductive females, is the most striking difference between the CA cells of R. flavipes and previously described CA of cockroaches, in which glycogen is absent throughout the reproductive cycles. This suggests that glycogen is an important source of energy for hormone production by termite CA.

Phe-Gly-Leu-amide allatostatin in the termite Reticulitermes flavipes: content in brain and corpus allatum and effect on juvenile hormone synthesis.

In the subterranean termite Reticulitermes flavipes, allatostatins (ASTs) with the C-terminus Phe-Gly Leu-amide were localized by immunocytochemistry with antibody against a cockroach AST, Dippu AST-7. AST-immunoreactivity occurred in the corpus cardiacum and corpus allatum and in the lateral and medial neurosecretory cells of the brain that innervate these organs as well as in many other nerve cells of the brain. This was observed in workers, nymphs, soldiers and secondary reproductives. A radioimmunoassay, using anti-Dippu AST-11, demonstrated about 40 fmole equivalents of AST in brains of soldiers and secondary reproductives. The product of the corpora allata in this species was determined to be juvenile hormone III. Its synthesis by corpora allata of secondary reproductives, determined by in vitro radiochemical assay, was inhibited in a dose-dependent fashion by two cockroach allatostatins, Dippu AST-7 and Dippu AST-11. Thus, as in cockroaches and crickets, allatostatin-containing nerves innervate the corpora allata of this termite species and their production of juvenile hormone is inhibited by these neuropeptides.

Activity of the corpora allata of adult female Aedes aegypti: effects of mating and feeding.

The synthesis of juvenile hormone III (JH III) by the isolated corpora allata (CA) of Aedes aegypti adult female was studied using an in vitro radiochemical assay. We dissected the corpora allata-corpora cardiaca (CA-CC) complex attached to a piece of aorta. The complex was left connected to the intact head capsule to facilitate the visualization and transfer of the glands. A linear increase in the cumulative amount of biosynthesized JH III was found for at least the first 6 h of incubation; approximately 45% of the synthesized JH III was present in the medium. There was a dependence of JH III synthesis on exogenous methionine supply. Using reversed phase high performance liquid chromatography two major labeled products biosynthesized by the CA were separated. They co-migrated with JH III and methyl farnesoate (MF). The identity of the biosynthesized JH III was confirmed by gas chromatography-mass spectrometry. JH III synthesis was only 2.0 fmol/pair gland/h immediately after adult emergence, but increased to 32.6 fmol/ pair gland/h 18 h later in sugar-fed females. Two days after emergence, the CA biosynthetic activity slowly started to decrease, and reached values of around 5.3 fmol/pair gland/h by one week after emergence. Synthesis of JH was similar from either sugar-fed females mated or unmated. A blood meal resulted in a decrease of JH III synthesis in CA from mated females by 12 h after feeding and from virgin females by 24 h after feeding. JH III biosynthesis remained low for at least 96 h in mated females, but was back to higher levels 72 h after feeding in virgin females. Rates of JH III biosynthesis closely reflected the hemolymph levels of JH III both after emergence and after a blood meal described by Shapiro et al. (1986). The activity of the CA in Aedes aegypti females seems to be regulated by developmental changes and nutritional signals, and to be independent of mating stimulus.

Ecdysone agonist halofenozide affects corpora allata and reproductive physiology of the Formosan subterranean termite, Coptotermes formosanus.

Following a short swarming flight, winged adults of the Formosan subterranean termite, Coptotermes formosanus lose their wings and form tandem pairs. These dealates or primary reproductives then form incipient colonies. Topical application of 5 microg of the non-steroidal ecdysone agonist RH-0345 (halofenozide) in 0.1 microl DMSO to the primary reproductives during the 2000 season resulted in significant reduction in the number of eggs laid. There was however complete recovery of the treated females. Apparently the non-treated partner removed the treatment chemical while grooming indicating oral activity. In 2001 both topical application as well as feeding methods were tried. Significant effects were observed only in the topical treatment group, perhaps because of inconsistency in feeding. In this group, total progeny, the number of ovarioles in ovaries and the size of the female's corpora allata (CA) were all significantly reduced. Ultrastructure of the CA of treated females showed extensive vacuolation near the surface of the gland. The experiment was repeated in 2002 using both topical application and an improved oral feeding method. Whereas there was apparent recovery in the topically treated group after 90 days, the oral treatment was more persistent in its effect perhaps due to a higher amount of halofenozide consumed during feeding.

Ultrastructural changes in the corpus allatum after azadirachtin and 20-hydroxyecdysone treatment in adult females of Labidura riparia (Dermaptera).

In previous reports, we have shown that the injection of azadirachtin (AZA) as well as 20-hydroxyecdysone (20E) into vitellogenic females of Labidura riparia induces inhibition of vitellogenin synthesis and ovarian development. Juvenile hormone (JH) treatment rescues vitellogenin synthesis and ovarian growth (Sayah et al., 1995, 1996). In this work, we have studied ultrastructural changes of corpus allatum (CA) after injection of 200, 400, and 600 ng of 20E or 1, 3, and 5 microg of AZA. CA cells exhibit signs of inactivity in both AZA and females treated with 20E at doses of 3 microg and 400 ng, respectively. Conspicuous cytological effects consisting of multivesicular bodies with dense contents, abnormally large intercellular spaces comprising myelinic structures, and rare smooth endoplasmic reticula occurred in cytoplasm of CA glandular cells in both experimental females. However, the CA ultrastructure of females injected with 20E differs from CA cells of females injected with AZA in having a cytoplasm containing numerous electron-lucent intracellular areas and marked glycogen zones. They also differ in having abundant microtubules and well-developed junctional membranes. At a dose of 600 ng of 20E or 5 microg of AZA, the intensity of the cytotoxic effects is more apparent. CA cells display pycnotic nuclei, spherical mitochondria, large multivesicular bodies, and vacuolization of the cytoplasm. These results are discussed and compared with observations made on other insect species.

Cockroach allatostatin-immunoreactive neurons and effects of cockroach allatostatin in earwigs.

A monoclonal antibody to allatostatin I of the cockroach Diploptera punctata was used to demonstrate the presence of allatostatin-immunoreactive cells and fiber tracts in the neuroendocrine system of the earwig Euborellia annulipes. The corpora cardiaca cells were not immunoreactive, nor were the neurosecretory endings of fiber tracts from the brain to the corpora cardiaca. No immunoreactive material was detected in the corpus allatum, although the corpus allatum contained neurosecretory endings, and some cells of the brain, including medial and lateral protocerebral cells, showed immunoreactivity. In addition, the recurrent and esophageal nerves were allatostatin-positive. The last abdominal ganglion contained immunoreactive somata, and immunoreactive axons of the proctodeal nerve innervated the rectum, anterior intestine, and posterior midgut. We did not detect reactive endocrine cells in the midgut. Allatostatin I at concentrations of 10(-5) and 10(-7) M did not inhibit juvenile hormone biosynthesis by E. annulipes corpora allata in vitro. This was true for glands of low activity from 2-day females and brooding females, as well as for relatively high activity glands from 10-day females. In contrast, 10(-7) M allatostatin I significantly and reversibly decreased hindgut motility. Motility was decreased in hindguts of high endogenous motility from 2-day females and in those of relatively low activity from brooding females. These results support the notion that a primary function of allatostatin might be to reduce gut motility.

Endocrine and neuroendocrine effects of Azadirachtin in adult females of the earwig Labidura riparia.

In previous studies we have shown that injection of the insect growth regulator Azadirachtin (AZA) into young vitellogenic females induces inhibition of vitellogenesis in a dose-dependent manner. Juvenile hormone treatment rescues vitellogenin synthesis and ovarian growth. The cytopathological effects on ovaries and fat body are not linked to an inhibition of feeding. In this work we investigated the effects of AZA on the endocrine and neuroendocrine system. Enzyme immunoassay reveals that ovarian ecdysteroid levels are drastically reduced, in a dose-dependent fashion, by AZA. Ultrastructural study indicates that corpus allatum cells exhibit signs of inactivity and degenerative changes after AZA exposure. Using an antibody against allastostatin-3 of Blatella germanica (BLAST-3), we show the appearance of strong immunoreactivity of numerous cells and axons in the brain of AZA-injected females. We conclude that vitellogenesis inhibition by AZA consists of a direct cytotoxic effect as well as a generalized disruption of endocrine and neuroendocrine functions.

Fura-2 measurement of cytosolic free Ca2+ concentration in corpus allatum cells of larval Manduca sexta.

Cytosolic free Ca2+ has been implicated in the regulation of the larval corpus allatum (CA) of the tobacco hornworm Manduca sexta since agents presumed to cause changes in intracellular Ca2+ concentrations affect both basal and neuropeptide-modulated synthesis/release of the juvenile hormones. To determine whether differences in cytosolic free [Ca2+] in CA cells were associated with different levels of gland activity, methods were developed for dissociating CA cells with the retention of biosynthetic activity and for uptake by the dissociated cells of the fluorescent Ca2+ indicator Fura-2. Digitized fluorescence microscopy of the Fura-2-loaded cells enabled measurement of intracellular Ca2+ levels in individual cells. Intracellular free Ca2+ levels were measured in CA cells from selected days during the fifth larval stadium and were found to be highest when the CA were biosynthetically active. Treatment of CA cells from day 6 with the Ca2+ ionophore ionomycin elevated the intracellular Ca2+ level, corroborating the involvement of elevated intracellular [Ca2+] in ionophore effects on juvenile hormone (JH) and JH acid synthesis. The results of the study are considered in relation to our knowledge of the role of Ca2+ in the function of endocrine gland cells.

Metamorphosis of the corpus allatum and degeneration of the prothoracic glands during the larval-pupal-adult transformation of Drosophila melanogaster: a cytophysiological analysis of the ring gland.

The degeneration of the prothoracic glands of Drosophila melanogaster during pupal-adult metamorphosis was analyzed by light microscopy, scanning, and transmission electron microscopy. The ultrastructural observations were correlated with the ability of the ring gland to synthesize ecdysteroids in vitro. The ring gland is prominent during larval life and is identifiable until just before adult eclosion but undergoes dramatic changes in location, shape, size, ultrastructure, and function during pupal-adult development. Prothoracic gland degeneration is characterized by: a gradual decrease in its ability to synthesize ecdysteroids; a decreasing quantity of smooth endoplasmic reticulum (SER) and mitochondria; the absence of intercellular channels; cytoplasmic fragmentation; and the separation of the prothoracic gland from the corpus allatum and corpus cardiacum. An ultrastructural analysis of the corpus allatum during larval-pupal-adult metamorphosis and adult life was also correlated with function, i.e., juvenile hormone biosynthesis, using a radiochemical assay of ring glands and adult corpora allata in vitro. A relatively high concentration of SER, mitochondria, and mitochondrion-scalariform junction complexes are typical features of an active corpus allatum cell. The migration of the corpus allatum from the ring gland to its position as a separate gland in the adult fly was studied in detail. The capacity of the corpus allatum to synthesize juvenile hormone is at its peak in the ring gland of the early wandering third instar larva, whereas the corpus allatum of 2-day-old female adults displayed the greatest synthetic activity during adult life. The physiological significance of the alterations in gland activity is discussed.

Aspectos ultra-estruturais do corpus cardiacum, corpus allatum e aorta de Panstronggylus megistus (Hemiptera: reduviidae) / Ultrastructural aspects of corpus cardiacum, corpus allatum and aorta of Panstrongylus megistus (Hemiptera: Reduviidae)

Em P. megistus o corpus cardiacum e a aorta têm uma estrutura semelhante. Ambos apresentam axônios de neurônios secretores cerebrais. A presença de grânulos de neurossecreçäo e o revestimento por uma bainha conjuntiva reforçam a idéia da funçäo neurohemal da aorta. O corpus allatum mostra células de formas diferentes. Os núcleos säo volumosos e muitos deles têm nucléolos desenvolvidos. Numerosas mitocôndrias granulares e filamentosas säo vistas concentradas em determinadas regiöes das células. Os contornos das células e dos núcleos säo regulares, características de células do corpus allatum ativo

Ultrastructure of corpora allata of known activity during the vitellogenic cycle in the cockroach Diploptera punctata.

Ultrastructure was correlated with rates of juvenile hormone synthesis in corpora allata from females of the viviparous cockroach Diploptera punctata at seven daily intervals during the first vitellogenic cycle. Synthetic activity of the glands was determined by in vitro radiochemical assay before the glands were fixed for electron microscopic analysis. The cycle in rates of juvenile hormone synthesis progressed from about 20 pmol h-1 per gland pair (oocytes 0.60 mm long) to a maximum mean rate of 140 pmol h-1 per pair (oocytes 1.40-1.47 mm long) and declined to about 20 pmol h-1 per pair at ovulation (oocytes about 1.65 mm long). Conspicuous ultrastructural changes occurred with changing synthetic rates. In glands with increasing rates of synthesis, mitochondria showed less electron-dense matrix, greater diameter and more irregular shape. Smooth endoplasmic reticulum changed from easily seen to obscure tubules, networks, and vesicles. Rough endoplasmic reticulum appeared in longer, more curved segments. Newly formed autophagic vacuoles appeared in all glands of highest activity rates. In glands with decreasing rates of synthesis, the mitochondrial matrix became denser, width smaller, and shapes less irregular. Smooth endoplasmic reticulum again appeared tubular and distinct. Golgi complexes were more conspicuous. Rough endoplasmic reticulum in whorls and large numbers of autophagic vacuoles continued to be present.

Correlations between endocrine gland ultrastructure and hormone titers in the fifth larval instar of Manduca sexta.

Correlations were made between endocrine gland ultrastructure and circulating hormone titers of Manduca sexta to investigate the mechanisms of hormone biosynthesis and secretion. Both the prothoracic glands (PTG), which secrete ecdysone, and the corpora allata (CA), which secrete juvenile hormone (JH), were studied. In the prothoracic glands, the intracellular spaces increase in area and reach their maximum size following the major ecdysteroid peak in the fourth and fifth larval instars. Within the intercellular spaces are multivesicular sacs (MVS), structures which are clusters of vesicles bounded by another membrane. Since these sacs are largely depleted of their internal vesicles after the second cycle of tropic hormone stimulates the PTG to secrete ecdysone, the MVS probably release a gland cell product at this time. In the CA, concentric whorls of smooth endoplasmic reticulum are present in larval glands, when the JH titer is high, but are absent from pupal CA when the JH titer is low. The peak of JH at Days 6-8 of the fifth larval instar occurs after an increase is seen in the neurosecretory cell axon diameters suggesting that the CA are stimulated by a brain hormone to release JH. The number of Golgi complexes increases in pupal CA and dense bodies are present in pupal but not larval glands. These Golgi complexes may be involved in the manufacture of lysosomal enzymes which degrade JH within the gland itself.

Activity of corpora allata, endocrine balance and reproduction in female Labidura riparia (Dermaptera).

The reproductive activity of Labidura riparia females involves, after a 5-day maturation stage, a regular alternation of ovarian cycles and egg-care stages averaging 10 days each. Vitellogenesis is characterized by an increase in the size of the corpora allata (CA) where structured SER bodies appear, and by a rise of juvenile hormone (JH III) content in the hemolymph which is followed by an increase in the level of ecdysteroids. During the egg-care periods, the CA are inactive; structured bodies generate autophagic vacuoles, the titer of JHs and later that of ecdysteroids in the hemolymph decreases and remains stationary. Ovariectomy causes hypertrophy and hyperactivity of the CA for about two months. Subsequently, the titer of JH decreases and old females may display parental behaviour; the level of ecdysteroids falls and remains unchanged. After cauterization of the pars intercerebralis (PI) of the protocerebrum, the ovarian activity stops, the ovary shrinks, the JHs rapidly disappear but ecdysteroids remain at the same or even higher levels than those of normal females of the same age. On the basis of these data, we postulate the existence of a center located in the PI, inhibiting the production of ecdysteroids, and of a stimulating center located outside this area. The PI also exhibits an allatotropic function.

Correlative changes in the neurosecretory cells, corpus cardiacum and corpus allatum during egg maturation in the buffalo-fly, Lyperosia exigua (De Meijere) (Diptera: Muscidae).

The neurosecretory cells of pars intercerebralis, the corpus cardiacum and the corpus allatum are examined in histological sections of Lyperosia exigua females, fixed at daily interval during first cycle of egg maturation. The median neurosecretory A cells exhibit secretory activity by undergoing cyclical changes of synthesis and release with the advancing age and egg maturation. Similarly, a single corpus allatum also undergoes cyclical changes in volume and histological appearance in accordance with the yolk deposition in the developing oocyte.

Histological study on the retrocerebral-endocrine complex with special reference to neurohaemal involvement of aorta and pericardial cells in Coccinella septempunctata (L.) (Coccinellidae-Coleoptera).

About nine median neurosecretory cells (NSC) are situated in the antero-dorsal position on either side of the pars intercerebralis of the brain. No lateral NSC could be detected. The axonic bundles, after their decussation, emerge out of the brain in the form of a single nervi corporis cardiaci (NCC) on either side. These soon undergo a dichotomy--one set of nerve fibers enter the lobulated corpus cardiacum (CC) and the other set ramify over the surface of the aorta and sends out distinct branches to the individual large, oval-shaped, pericardial cells all along the length of the aorta. Presence of neurosecretory material (NSM) could be demonstrated both in aortal wall as well as along the nerve branches that innervate the pericardial cells. Traces of NSM could be detected within the cytoplasm of the pericardial cells. While the involvement of aorta and pericardial cells, as an additional neurohaemal organs was well documented for several species of Hemiptera, the present report for Coleoptera seems to be new and of considerable interest. Thus it would seem that the role of the aorta and pericardial cells as accessory neurohaemal organ is perhaps of wider occurrence than was hitherto believed. The irregularly lobulated CC contains copious amounts of thick clumps of NSM which, by and large, tends to show a peripheral distribution. Few intrinsic secretory cells are noticeable. The CC, at its postero-lateral border forms a cap- or hood-like projection that partially overlaps the corpus allatum (CA) below. At the mesial border, the CC sends out a lobe which seems to fuse with the aortal wall. The shape of CA shows individual variation. In some, it is oval while in others it is spherical, but always with a well defined outline. No allatal nerve is demonstrable. Instead, the CA is broadly connected with the hood region of the CC. AF-positive material is also detectable in the CA, which also tends to be peripherilly distributed.

Precocene-induced necrosis and haemocyte-mediated breakdown of corpora allata in nymps of the locust Locusta migratoria.

Light and electron microscopic studies, including the use of the vital dyes trypan blue and acridine orange, indicate that the topical application of precocene II rapidly triggers degenerative processes in the corpora allata (CA) leading within a few days to the virtual disappearance of the parenchymal cells of these glands. The following sequence of events was observed: 1) Within 2 h, many of the cells in fixed nymphal specimens showed increased electron density. During the next few hours, they decreased considerably in volume (shrinkage necrosis). Intercellular spaces increased simultaneously. Although a variable number of cells remained electron lucent, they showed nuclear and cytoplasmic degeneration later on (coagulative necrosis). 2) Haemocytes arrived at the CA sheath and invaded the CA in large numbers after 12 h. 3) CA cells became increasingly necrotic, and cell fragments were budded off and were phagocytosed most noticeably after 1 to 3 days. Thereafter no CA parenchyma remained. 4) Haemocytes dispersed.

Pathways and fine structure of neurons forming the nervi corporis allati II of the cockroach Periplaneta americana (L.).

By back-filling the nervus corporis allati II (NCA2) with Co2+ and precipitating the sulfide, two groups of somata (A and B) are revealed on the ipsilateral side of the subesophageal ganglion (SG). These occur anteroventrally, adjacent to the midsaggital plane. Group A consists of two cells; group B of five. Their processes form two discrete tracts issuing dorsoposteriorly into the neuropile between and slightly behind the circumesophageal connectives (CEC). After producing separate arborization fields in the dorsal neuropile, the tracts circumscribe the base of the ipsilateral CEC, unite, and their seven fibers enter NCA2 anteriorly. Prograde diffusion reveals 4--6 NCA2 axons penetrating the corpus allatum (CA) near a cap-like neurohemal organ. These axons form the transverse allatal tract (TAT), from whence they branch amongst the CA cells, and into the "cap", the postallatal nerves, and the opposite CA. Electron microscopy of transverse sections demonstrates nine neurosecretory axons entering the SG through NCA2. Proximal to the CA, NCA2 consists of a central bundle of neurosecretory axons and a peripheral zone confluent with the CA "cap". Depending upon the level of sectioning, there are 7--20 axons at the center, and seven pass into the TAT. The peripheral zone has the structure of a neurohemal organ.

Ultrastructure of the corpus cardiacum and corpus allatum of the house cricket Acheta domesticus.

The ultrastructure of the corpus cardiacum (CC) and corpus allatum (CA) of the house cricket, Acheta domesticus, is described. Axon profiles within the CC contain neurosecretory granules 160-350 nm in diameter which are indistinguishable from those found in type I neurosecretory cells of the pars intercerebralis and in the nervus corporis cardiaci I. The CC itself contains two cell types: intrinsic neurosecretory cells and glial cells. Intrinsic NSC cytoplasm contains Golgi bodies and electron dense neurosecretory granules 160-350 nm in diameter. Synaptoid configurations with 20-50 nm diameter electron lucent vesicles were observed within axon profiles of the CC. The structure of the CA is relatively uniform with one cell type predominating. Typical CA cells possess large nucleoli, active Golgi complexes, numerous mitochondria, and occassional microtubules. Groups of dark staining cells scattered throughout the CA of some animals were interpreted as evidence of cellular death.