Synthesis and storage of a neurohormone in insect brains in vitro

The build-up of neurosecretory material in the median neurosecretory cells and fibre tracts of cultured cockroach brains was demonstrated by staining and bioassay. Examination of the cultured brains by electron microscopy showed active production of neurosecretory granules after 3 days in vitro. The close correlation of the results obtained by these different methods of assay leaves little doubt that a neurohormone is being synthesized and stored.     

The neurosecretory cells of the brain and suboesophageal ganglion of Chironomus riparius

The neurosecretory cells of the supra- and suboesophageal ganglia of young, unmated, adult male midges, Chironomus riparius, have been examined by both light and electron microscopy. The 5 cell types recognized have been placed in three major categories on the basis of their ultrastructural characteristics:—α₁ cells, of which there are 8 in each medial neurosecretory cell (MNC) group and 3 in each group of ventral neurosecretory cells (VNC), contain electron-dense granules, 150 to 200 nm in diameter; α₂ cells containing irregular, electron-dense granules, 70 to 120 nm in diameter comprise the remaining 3 cells in each VNC group and the 2 or 3 cells in each outer neurosecretory cell (ONC) group; α₃ cells, of which there are 1 or 2 on each side of the midline in the ventral cortex of the sub-oesophageal ganglion (SNC₂), contain electron-lucent, spherical granules, 70 to 120 nm in diameter. The β cells contain spherical or ellipsoidal, electron-lucent granules, 80 to 100 nm in diameter, and make up the lateral neurosecretory cell (LNC) groups, each of three or four cells. The γ cells contain both spherical and flattened, electron-dense granules, 130 to 160 nm in diameter and 150 to 250 by 70 to 150 nm in size respectively, only 1 cell of this category being found in each half of the suboesophageal ganglion in the dorsal cortex (SNC₁). Axons from the MNC and VNC form the nervi corporis cardiaci I (NCCI) and those of the LNC and ONC, the nervi corporis cardiaci II (NCCII). Those of the SNC₁ appear to enter the wall of the stomodaeum but axons of the SNC₂ could not be traced.     

A comparative ultrastructural study of in vivo and in vitro derived adults of Microphallus similis

Davtes C. 1980. A comparative ultrastructural study of in vivo and in vitro derived adults of Microphallus similis. International Journal for Parasitology10: 217–266. The ultrastructure of in vitro cultured adults of Microphallus similis was examined by TEM and SEM and compared to that of metacercariae and in vivo grown adults. In cultured flukes the most conspicuous abnormalities were observed in secretory cells, especially those of the tegument, digestive caecum, forebody glands and vitellaria. In the tegumental cells and the forebody gland cells, the secretory granules appeared to lose some of their contents within the cell bodies suggesting that there may be some defect in the transport and/or packaging of secretory granules in vitro. In the vitellaria of cultured flukes some of the granules lost their characteristic appearance, becoming ragged in outline and very electrondense. The premature tanning of the vitelline secretions within the vitellaria is correlated with abnormal egg production in vitro. The caecal cells of cultured flukes differed from those of the metacercaria and the normal adult in several important respects which suggested that their function was probably impaired.     

Ultrastructure of the neurosecretory cells in the brain of diapausing pupae of the tobacco hornworm, Manduca sexta (l)

The ultrastructure of seven different types of neurosecretory cells (NSC) found in the medial and lateral areas of the brain of diapausing Manduca sexta is described. The five different types of NSC in the medial area have characteristic differences in their shape, size, neurosecretory granules (NSG), and the morphology of their organelles. The cell types of the medial area accumulated the NSG, but did not appear to be synthesizing and packaging new NSG, whereas the NSC in the lateral region were synthesizing and packaging NSG during diapause. The possible significance of the relationship between the lateral and medial cells is discussed.     

Moulting and ecdysone release in response to electrical stimulation of protocerebral neurosecretory cells in Locusta migratoria

Locusta migratoria larvae were submitted to electrical stimulation of the protocerebral neurosecretory cells (median neurosecretory cells of the pars intercerebralis and lateral neurosecretory cells), during the last larval instar. The effects of the treatment were observed both on the duration of the stage and on the variations in haemolymph ecdysone levels. In untreated larvae, there was an initial ecdysone peak at the beginning of day 5, which was followed by 4 larger peaks between days 6 and 8. Stimulation of the median neurosecretory cells at the beginning of the instar resulted in the formation of one very large hormonal peak at the end of day 3: a day and a half earlier than in the control groups. Moulting was likewise accelerated. Stimulation also increased the size of the peaks, as compared with the controls. Stimulation of the lateral neurosecretory cells had a weaker ecdysiotropic effect; neither the number nor the size of the peaks were changed, though, like ecdysis, they occurred earlier. Stimulation of the deutocerebrum had no effect on either ecdysone titres or moulting. Electrical stimulation of the median neurosecretory cells at the end of day 5, that is after the occurrence of the first ecdysone peak, shortened the larval stage while having no significant effect on ecdysone levels in the haemolymph. The neuroendocrine control of ecdysis in Locusta is discussed.     

Light and electron microscopic studies on the neurosecretory control of diapause incidence in Pieris rapae crucivora

The incidence of diapause was shown to be determined humorally during the larval-pupal ecdysis by means of brain extirpation experiments.On the basis of this observation, light and electron microscopic changes in the neurosecretory type II cells in the pars intercerebralis-corpus cardiacum system during pharate pupal and early pupal stages were examined in insects reared under long day-length (non-diapause individuals) and in insects reared under short day-length (diapause individuals). In the diapause individuals, neurosecretory granules in NS-II cells increased during the pupal instar and large aggregates of granules packed the cytoplasm. Thereafter, inclusion bodies showing cytoplasmic breakdown of the granules appeared.In the non-diapause individuals, on the contrary, electron micrographs suggesting the release of neurosecretory material from axon terminals were obtained just after the pupal ecdysis. There were very few granules, with many Golgi bodies and much rough ER 8 to 12 hr after the ecdysis.It is concluded that adult development is determined by the release of neurosecretory material from the axon terminals of NS-II cells at the larval-pupal ecdysis. If release does not occur, the pupae enter diapause. It is also thought that differences in day-length during the larval stages influence the activities of NS-II cells before pupation.     

Brain hormone carrier haemocytes in the moth Monema flavescens

The movement of neurosecretory substances released from the neurosecretory B cell in the pars intercerebralis to the haemolymph was examined with the progress of the termination of diapause in the slug moth pharate pupa, Monema flavescens.The injection of precipitates in the haemolymph of the pharate pupa just before the termination of diapause into diapausing pharate pupae reduced the numbers of days required for them to pupate. In the precipitates, seven types of haemocytes were present. The number of haemocytes, especially the granular cell, increased just before the termination of diapause. AF and CHP positive substances not detected in the haemocytes of diapausing pharate pupae appeared in the granular cells just before the termination of diapause. The period also coincided well with the releasing period of the neurosecretory B cell. Histological examination showed that granular haemocytes gathered around the pars intercerebralis at this period and exchange of neurosecretory substances occurred between granular haemocytes and neurosecretory B cells. Then granular haemocytes migrated to the region of the prothoracic gland. From digestion tests of the neurosecretory substances with rabbit serum and from the implantation tests of the neuroendocrine system, the substances detected in both the neurosecretory B cell and the granular haemocytes seemed to be the same. The dye injection caused a delay in larval-pupal ecdysis emergence. Droplets of black ink are incorporated into the granular haemocytes. This seems to be caused by blocking of the transport of neurosecretory substances released from cytoplasmic processes of the neurosecretory B cell.From these experiments, it is suggested that neurosecretory substances of the prothoracotropic hormone are transported to the prothoracic gland, along with granular haemocytes, after being released directly from the neurosecretory B cell to the haemolymph.     

Neuroendocrine regulation of corpus allatum activity in Manduca sexta: The endocrine basis for starvation-induced supernumerary larval moult

When newly-ecdysed 5th instar larvae of Manduca sexta were starved for 3 days and thereafter fed on standard diet the majority (90%) of the surviving larvae moulted into 6th instars. Allatectomy prior to starvation abolished the supernumerary moult, while denervation of the corpora allata (CA) had no effect.Cautery of medial neurosecretory cells, but not of the lateral cells, prevented supernumerary moulting and pupation ensued. Transplantation of brains from young 5th instar donors into larvae, whose medial neurosecretory cells were cauterized prior to starvation, restored the extra larval moult. Neither CA nor corpora cardiaca (CC) could be substituted for the medial neurosecretory cells.For induction of the supernumerary moult the medial neurosecretory cells are required only until day 1 after refeeding whereas the CA are required until day 3 after refeeding. Allatectomy on day 3 after refeeding resulted in the production of black 6th instar larvae.We conclude that starvation-induced supernumerary moulting is due to activation of the CA by allatotropin produced by medial neurosecretory cells in the brain. The anteromedial cells (group II) appear to be the source of allatotropin.     

The ultrastructure of the tegument and the digestive caeca of in vitro cultured metacercariae of Fasciola hepatica

Davies C. 1978. The ultrastructure of the tegument and digestive caeca of in vitro cultured metacercariae of Fasciola hepatica. International Journal for Parasitology8: 197–206. The ultrastructure of the tegument and digestive caeca of metacercariae of Fasciola hepatica grown in vitro in two different media is described and compared with the development of these two systems during maturation in vivo. Although the tegument of metacercariae grown in Medium RC showed no development, that of flukes cultured in Medium CS began to produce T-1 and T-2 granules typical of the liver phase of development in vivo. The gastrodermal cells showed some degree of conversion to an adult-like morphology in vitro with the production of typical secretory granules, a limited amount of orientation of the GER and the development of junctional complexes with adjacent parenchyma cells—this was particularly evident in flukes from Medium CS. The growth achieved in each of the culture media is correlated to the degree of development of the tegument and the digestive caeca.     

Neural control of the corpus allatum in the Colorado potato beetle, Leptinotarsa decemlineata: An electron microscope study utilizing the in vitro tannic acid Ringer incubation method

The release of material from neurosecretory synapses in the corpora allata of the Colorado potato beetle, Leptinotarsa decemlineata was visualised by an electron microscope procedure which involved tissue incubation in tannic acid. Using morphometry, the frequency of exocytosis phenomena was quantified in beetles kept under two different photoregimes. The number of exocytosis phenomena in the neurosecretory synapses in the corpora allata of beetles kept under short days was significantly higher than that of beetles reared under long-day conditions. In addition, the corpus allatum gland cells appeared to be more richly innervated by neurosecretory synapses under short-day than under long-day conditions. Previous studies using the in vitro radiochemical assay showed that the corpus allatum activity of short-day beetles is at least partly restrained by neurally mediated factors. The present morphological data strongly imply that this corpus allatum inhibitory substance is released from the neurosecretory synapses.     

An ultrastructural in vitro study on the regulation of neurosecretory activity in the freshwater snail Lymnaea stagnalis (L.) with particular reference to Caudo-dorsal cells

The neurosecretory Caudo-Dorsal Cells (CDC) in the cerebral ganglia of the freshwater pulmonate snail Lymnaea stagnalis produce an ovulation stimulating hormone. Previously it has been shown that neuronal and non-neuronal inputs are involved in the regulation of their activity. The degree of autonomy of these cells has been investigated by studying with morphometric methods the ultrastructure of CDC maintained in vitro. CDC of isolated cerebral ganglia which were cultured for 7 days show a considerable rate of synthesis, transport and release of neurohormone. Apparently these processes can proceed in the absence of neuronal and hormonal inputs from outside the cerebral ganglia. Completely isolated CDC, however, do not show neurosecretory activity in vitro; active Golgi zones, indicating the formation of neurosecretory elementary granules, are absent from such cells. Isolation does not seem to affect general cell functions such as protein synthesis and respiration. It is suggested that a neuronal input, originating within the cerebral ganglia, is necessary for the stimulation of CDC neurosecretory activity. Techniques are described for the isolation and culture of neurosecretory cells of L. stagnalis.     

Neurosecretory cells in the frontal ganglion of the tobacco hornworm, Manduca sexta

The frontal ganglion of the tobacco hornworm, Manduca sexta (L.), was found to contain two neurosecretory (NS) cells (max dia = 40–45 μm). The cytoplasmic inclusions of the NS cells were stained purple with paraldehyde fuchsin, and marked fluctuations in amounts of NS material in the perikarya were observed, depending upon the developmental status of the insect. The perikarya of NS cells in the frontal ganglia of starved larvae and diapause pupae contained large accumulations of NS material, whereas feeding larvae and developing pharate adults showed relatively low amounts of neurosecretion. Electron microscopy revealed large accumulations of NS granules (dia = 80–240 nm) in the frontal ganglia of diapause pupae, but only slight accumulations of granules were seen in the NS cells of developing larvae and pharate adults.It was concluded that axonal transport and release but not synthesis is shut down during starvation and diapause, leading to accumulation of NS material in the perikarya. It is also suggested that the failure of many investigators to differentiate NS cells in the frontal ganglion of various insects may have been due to the selection of very active stages when the amount of available NS material was too low to be visualized by conventional staining techniques.     

Autolysis in axon terminals of a new neurohaemal organ in the cockroach Periplaneta americana

An ultrastructural investigation showed that there was a neurohaemal organ in the wall of the ampulla of the antennal pulsatile organ. The neurosecretory axon terminals occurred singly or in small groups rather than closely packed together as in other neurohaemal organs. All axons contained the same type of neurosecretory granule. The granules had varying electron density and a diameter in the range 1000–2500 Å. Some terminals contained small, elliptical, electron-transparent vesicles and the axolemma was apposed to the stroma. Other terminals were large and enveloped by glial tissue and the contents of the terminals exhibited varying degrees of autolytic degeneration. Autolysis was characterized by the occurrence of dense bodies and multilaminate bodies which enclosed mitochondria and neurosecretory granules. It was suggested that the neurosecretory material affects antennal function.     

Ultrastructure of the protocerebral neurosecretory cells of larval Galleria mellonella, in situ and after culture of the brain in vitro

Three major groups of neurosecretory cells are described in the larval brain of Galleria mellonella at two different times during the last larval instar and in larval brains after 72 hr of culture in vitro. The medial group in vivo consists of four distinct neurosecretory cell types, based on characteristic size and morphology, while the posterior and lateral groups each contain a single distinct type of neurosecretory cell. Morphological differences between the same neurosecretory cells at the different times during the last instar are most apparent in the lateral L-1 cells and in the medial M-2 cells, where pleiomorphism is particularly evident in the size, density and accumulations of neurosecretory granules. The only neurosecretory cells in which apparent synthesis of neurosecretory granules is still observed after culture of the brain in vitro are the medial M-2 cells. The other neurosecretory cell types show no accumulation of neurosecretory granules nor new synthesis of neurosecretory material, but are similar to neurosecretory cells in the brain in vivo in all other respects. The morphology of the neurosecretory cells in the larval brain in vivo and in vitro is discussed in relation to their appearance at the light microscopic level and to a known neurohormonal function of the brain which is maintained during 72 hr in vitro.     

Different rate of protein granule formation in the larval fat body of Drosophila melanogaster

The concentration of protein granules was determined cytologically in different regions of the fat body during the latter half of the third larval instar of Drosophila melanogaster. The measurements made at 6 hr intervals from 72–96 hr larvae showed that the concentration of the granules was the highest in the posterior, lowest in the anterior and intermediate in the middle region of the fat body. From these measurements, it was shown that the rate of granule formation was different in each region. Furthermore, there is a strong indication that at any given stage, the rate increases gradually and continuously from the anterior to the posterior region. When the fat body from larvae prior to the time of granule formation was cultured for three days in ecdysterone-containing medium, protein granules were produced in the anterior, middle and posterior regions in the same concentration as that in 90 hr larvae. The same gradient of protein granule formation in vitro is found whether the fat body is cultured as an intact piece or as three separate, dissected regions. The putative adaptive advantage of region-dependent granule formation is discussed.     

Brain neurosecretory cell and ecdysiotropin activity of the non-diapausing,pre-diapausing and diapausing southwestern corn borer, Diatraea grandiosella Dyar

The location and number of brain neurosecretory cells were studied in the larval southwestern corn borer. One posterior, two median and two lateral groups of paraldehyde-fuchsin positive cells were found in each cerebral hemisphere.Implantation of brain parts containing different groups of neurosecretory cells revealed that the median neurosecretory cells contained higher ecdysiotropic activity than the other cell groups. In vitro culture of ecdysial gland with brain or brain-parts extract showed also that the median neurosecretory cells contained much higher ecdysiotropic activity than other neurosecretory cells. To estimate the ecdysiotropic activity of pre-diapausing 6th instar larvae, their brain or brain extract was incubated in culture medium containing an ecdysial gland from a day-4 last-instar non-diapausing larva. Data showed that the ecdysiotropic activity in the pre-diapausing larvae was far lower than in non-diapausing and diapausing larvae.     

Histology, histochemistry, and ultrastructure of neurosecretory cells in the optic lobe of the cockroach, Periplaneta americana

In the region of the distal optic chiasma of each optic lobe of Periplaneta americana, there is a group of about 120 monopolar neurosecretory cells. These cells do not stain with paraldehyde fuchsin but remain acidophilic after oxidation. They stain red or sometimes indigo with the azan technique. Histochemically, the neurosecretory material is positive for protein and the amino acids tryptophan and arginine but negative for 1, 2-glycols and strongly acidic groups. At the ultrastructural level, the cytoplasm of the cells contain many elementary neurosecretory granules 100 to 170 nm in dia. The cells also contain well-developed Golgi bodies and endoplasmic retieulum. The axons from these cells run toward the interior of the optic lobe. In this region, axons containing dense granules (mean diameter 70 nm) and synaptic vesicles synapse onto the axons from the neurosecretory cells. The neurosecretory axons then cross over to the anterior side of the optic lobe and run towards the brain. The function of these neurosecretory cells is unknown, but they may be involved with photoperiodically controlled activity rhythms.     

The role of endocrines in flight-muscle degeneration in Dysdercus cingulatus (Heteroptera: Pyrrhocoridae)

In Dysdercus cingulatus, extirpation of the median neurosecretory cells inhibits flight-muscle breakdown. However, implantation of median neurosecretory cells or corpus allatum into females lacking these neurosecretory cells induces muscle histolysis. Median neurosecretory cells stimulate the corpus allatum to produce juvenile hormone which in turn induces muscle degeneration. Topical application of kinoprene induces muscle breakdown, even in males and starved virgin females. The direct participation of juvenile hormone in muscle degeneration is further confirmed by observations on the effect of precocene II which inhibits muscle histolysis in many individuals. The endocrine basis of vitellogenesis and flight-muscle degeneration could be identical; vitellogenesis however appears to require a higher threshold concentration of juvenile hormone.     

Trypanosoma musculi: In vitro cultivation of blood forms in cell culture media

Blood forms of Trypanosoma musculi were grown at 37°C in media containing adherent peritoneal cells from normal or from T. musculi immune mice. Rat peritoneal cells and Hela cells supported growth equally well which was surprising in view of the strict mouse-host specificity of this parasite in vivo. Cultures showed a 12- to 46-fold increase in numbers over the inoculum during periods up to 23 days whereas control (cell-free) media did not promote growth, the inoculated organisms merely surviving for about 7 days. Sub-culture through several passages was successful and cultured trypanosomes remained fully infective to mice. Foetal calf serum was an essential constituent of the medium.     

Daily changes of neurosecretory type-II cell structure of Pieris larvae entrained by short and long days

The neurosecretory type-II cell (NS-II cell) group of each brain hemisphere consists of three kinds of cells: two small cells, six large ones, and two others having characteristic vacuolated endoplasmic reticulum (ER).Ultrastructural changes of large NS-II cells were observed through the fifth instar and diurnally when short-day and long-day larvae were compared. There were little differences between short-day and long-day larvae in cell structures on corresponding developmental days except for daily changes, but remarkable changes were observed every day through the instar. A secretory cycle through the instar was supposed being based on the ultrastructural changes in NS-II cells: reduced secretory activity on the first day, formation of organelles necessary for the synthesis of secretory materials throughout the instar on the second day, active synthesis and secretion of secretory material during the middle stage (third-fourth day), and reversion to a reduced level of cell activity after the cessation of feeding.In short-day larvae on the third to fourth day, NS-II cells contained large aggregates of secretory granules during the day except for the time of 13 hr after the onset of photophase when a decrease of secretory granules occurred. In long-day larvae, only a small amount of secretory granules was observed at 8 and 13 hr after the onset. Rough ER changed daily paralleling with the quantitative change of the secretory granules.Based on these differences of daily changes in NS-II cell activity between short-day and long-day larvae, it was concluded that photoperiodic time measurement of diapause induction depends on the daily secretory cycle entrained by the photoperiods during the larval stage.