Rudolph L. Pipa

Studies on the hexapod nervous system: V. The ultrastructure of cockroach gliosomes

Neuroglial cytoplasmic inclusions which correspond to gliosomes have been studied with the electron microscope in the cockroach, Periplaneta americana. The inclusions occur as irregularly-shaped bodies bounded by, or enclosing, variably-elaborated multiple membranes. Gliosomes commonly contain a moderately osmiophilic, homogeneous, finely-granular matrix. The limiting membranes frequently appear to be interrupted. Where this occurs the granular matrix seems continuous with the surrounding glial cytoplasm. The possible cytological significance of these findings is discussed.

Corpus allatum regulation during the metamorphosis of Periplaneta americana: Axon pathways

Abstract The anatomy of the retrocerebral complex was studied after supravital staining with methylene blue, and axonal tracts within the corpora allata (CA) were traced after applying the CoCl2 technique together with Timms sulfide-silver enhancement. Cobalt chloride fills of the nerves to and from the CA revealed two major sources of innervation: the brain and the subesophageal ganglion. Three cell clusters in the brain contribute axons that reach each nervus corporis allati I (NCA I) and, apparently, pass to or beyond the CA. These are: a cluster of 8 to 12 cells in the contralateral pars lateralis, a cluster of 16 to 20 cells in the ipsilateral pars lateralis, and a cluster of 50 to 60 cells in the contralateral pars intercerebralis. PAF-stained sections of other brains revealed a corresponding number of PAF-positive cells in these same regions. The medial and lateral neurons arborize in the neuropile adjacent to the pars intercerebralis, and may associate there. The lateral group also arborizes extensively in the neuropile surrounding the pedunculus of the mushroom body. At least four cell bodies located antero-ventrad in the subesophageal ganglion send axons to the CA via each nervus corporis allati II (NCA II). To determine possible inhibitory pathways to the CA, the NCA I, NCA II, and postallatal nerves of last instar larvae were severed; either singly, or in combination. Additional experiments were performed on last instar larvae to substantiate that superlarvae were a direct result of an enhanced or sustained juvenile hormone titre. These experiments included: implanting two or more CA, extirpating one CA, or applying 100 μg of Altosid topically onto allatectomized larvae. The experiments indicated that only NCA I is an inhibitory pathway and that superlarvae were a direct consequence of CA activation. NCA II does not seem to provide the CA with an essential excitatory innervation; when it and NCA I are severed a supernumerary apolysis will still result. Some of the cells in the brain stainable by the CoCl2 method are most probably identical to those that are PAF-positive. These cells may inhibit the CA in last instar larvae via neurosecretomotor junctions.

Locations and central projections of neurons associated with the retrocerebral neuroendocrine complex of the cockroach Periplaneta americana (L.)

SummaryRetrograde diffusion and precipitation of Co2+ reveals in the ipsilateral pars lateralis (PL) and contralateral pars intercerebralis (PI) of the brain neurons that enter the corpus cardiacum (CC), and, possibly, the corpus allatum (CA) on each side. The PL group consists of 29.6±8.4 somata that fill. Of these, 5.6±0.6 exceed 25 μm in diameter, 14.3±2.7 range from 15–25 μm, and 9.6±7.6 are smaller than 15 μm. After CoCl2 was applied to the right CC-CA of two males, 239 and 265 somata in the left PI stained. Except for 16 ranging from 30–45 μm and chiefly located anteriorly, a majority of these somata measured 10–25 μm.The only somata revealed by staining whole brains with the performic acid-resorcin fuchsin method are neurosecretory cells 10–20 μm in diameter located within the PI. In starved adult males there are 92.4±8.1 on the right, and 93.2±6.9 on the left. The largest somata in the PL group contain numerous granules that stain with paraldehyde fuchsin. These somata also fill with Co2+, and belong to neurosecretory cells that extend into the CC-CA.The cerebral distribution of branches from the PL group, and the relationship of these to the corpora pedunculata, central body, and arborizations from the PI decussation are described.

Supernumerary instars produced by chilled wax moth larvae: Endocrine mechanisms

Young last instar larvae of Galleria mellonella underwent supernumerary ecdyses within 3 to 6 days after being chilled at 0 to 1°C for 30 min. The frequency diminished from 89 ± 9.4% for the survivors of those that were chilled <16 hr after their last ecdysis, to 25 ± 11.2% for those 46 to 88 hr old, and was no longer evident beyond 123 hr. Irrespective of their ages, the larvae never became “superlarvae” unless they had fed after they had been chilled. This was unlike the requirement for metamorphosis, when a feeding period of 40 to 48 hr immediately following ecdysis allowed half the larvae that were subsequently chilled and starved to pupate. The propensity to become superlarvae could be extended by starvation. Chilling signaled the occurrence of the larval moulting program, but its expression was held in abeyance until the larvae had fed. Brains from chilled or unchilled donors were equally effective initiators of supernumerary larval apolyses. The capacity to respond to chilling was abolished following bilateral extirpation of the corpora cardiaca and corpora allata, but not after the corpus cardiacum and corpus allatum of one side were removed. This effect of bilateral cardiacectomy and allatectomy could be remedied by applying Altosid, a juvenile hormone analog. Potentiation of the larval-larval apolysis by chilling and by JH may involve separate mechanisms, for the analog was less effective on unchilled larvae than on those that had been chilled. The results are discussed with reference to the hypothesis that the brains of young larvae produce an “allatotropic hormone”.

Neural influence on corpus allatum activity and egg maturation in starved virgin Periplaneta americana

ABSTRACT. Of sixty‐seven adult female Periplaneta americana (L.) isolated when less than 8h old and kept without food at 25–30°C in LD 12:12, only six produced oothecae during 25–35 days. Growth of the basal oocytes was retarded, and maximal oocyte volume, only one‐third of that in fed virgins, was achieved after about 12 days. By day 14, 60% of the basal oocytes were being resorbed in the starved females, and corpora lutea were usually all that remained beyond day 20. Oocyte growth was potentiated in starved, virgin females by severing either of the two nervi corporis allati 1 (NCA 1), and oothecae were then produced in 45–80% of cases. Sham‐operated controls oviposited in fewer than 27% of the trials. Because oocyte maturation was prevented by extirpating both corpora allata (CA), but not when the glands were replaced, and because the juvenile hormone analogue, ZR 515, was highly effective in causing the starved cockroaches to produce oothecae, the starvation‐induced reproductive failure probably reflects diminished hormone production by the CA. The most likely consequence of severing NCA 1 is de‐repression of juvenile hormone production. The directness of the neural influence was shown by removing the one denervated CA, in which case stimulation of oogenesis was minimal even though the contralateral innervated gland was present. The incidence of ootheca production was not enhanced by transecting the NCA 2, which suggests that the CA of starved cockroaches are not inhibited via this pathway.

Metamorphic shortening of interganglionic connectives of Galleria mellonella (Lepidoptera) in vitro: Stimulation by ecdysone analogues☆

In the absence of other organ systems, β-ecdysone (0·05 to 0·10 μg/ml culture medium) stimulates the shortening of interganglionic connectives of Galleria mellonella that occurs during metamorphosis. There is a direct relations-ship between the amount of β-ecdysone in the medium and the fraction of the sample shortening to at least half the initial length. β-Ecdysone is ∼ 140 × more active than α-ecdysone in eliciting the response. When β-ecdysone and other ecdysone analogues are assayed on this system at uniform dosages (10 μg/ml tissue culture medium), the order of effectiveness (percentage sample shortening to at least half the initial length) is: cyasterone > ponasterone-A = β-ecdysone > inokosterone > α-ecdysone. 22-Iso-α-ecdysone is ineffective in stimulating shortening.

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

SummaryBy 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 ante-roventrally, 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.

Neuroendocrine involvement in the delayed pupation of space-deprived Galleria mellonella (Lepidoptera)☆

Abstract Pupation of last instar larvae of the greater wax moth will be retarded if their free space has been eliminated, or if the dimensions of their free space are unduly restrictive. The response can be abolished by implanting brains, but not by implanting thoracic ganglia. Brains from space-deprived donors are as effective in this regard as brains from donors not space deprived. When brains are implanted into space-deprived last instar larvae at intervals after ecdysis the larvae do not react uniformly; those less than 3 days old undergo additional larval ecdyses, while those older than this pupate. Controls, receiving thoracic ganglia, rarely undergo supernumerary larval ecdyses, and they pupate later than the experimentals. These data support the hypothesis that the juvenile hormone titre is high in the young last instar larva, but diminishes as it approaches pupation. Because the titre declines even though the larvae are space deprived, the possibility that their metamorphosis is inhibited by prolonged corpora allata activity seems unlikely. Also, removal of the corpora allata and corpora cardiaca fails to alleviate the response of last instar larvae to space deprivation. The data suggest that sensory input, presumably via mechanoreceptors, inhibits prothoracotropic hormone secretion by the brains of space-deprived larvae. The neural pathway, however, is unknown.

A cytochemical study of neurosecretory and other neuroplasmic inclusions in Periplaneta americana

Abstract A variety of cytological and cytochemical techniques has been applied to the problem of identifying and classifying neuroplasmic inclusions in the American cockroach. Particular attention has been paid to those found in the supraesophageal ganglion. This has led to the recognition of three types which are believed to exist in addition to mitochondria, ribosomes, and the Golgi complex. To facilitate discussion these are called A-cell NSM, beta-granules, and delta-granules. Colorless vacuoles seen in freshly-teased preparations were identified as bipartite components present in paraffin sections. These occur as sudanophilic crescents or rings (“externa”) of variable thickness which surround less sudanophilic “interna”. Such regions lack cytochemically detectable RNA. Although complete evidence is lacking, it is suggested that the “externa” correspond to the multi-lamellar, nongranular membrane systems (Golgi complex) demonstrable in electron micrographs of nerve cells from a wide variety of different animals. The inability to detect RNA in these zones may simply be a reflection of the nongranular (ribosome-free) nature of the Golgi membranes. The cytochemical staining reactions used indicate that A-cell NSM contains a cystine- or cysteine-rich phospholipoprotein. There was no evidence to support the contention that polysaccharide is present. Yellow-pigmented inclusions occur within the perikarya of freshly-teased neurons. These are well-preserved in Helly-fixed paraffin sections. They are only occasionally found in early instar nymphs, but abound in older nymphs and adults. On the basis of morphological and tinctorial characteristics they are arbitrarily subdivided into two categories, called beta- and delta-granules. Both are intensely PAS-positive and sudanophilic. This seems to be due to the presence of glycolipid. Similarities between these inclusions and lipofuscins described in vertebrate neurons have been pointed out. The relationship between beta- and delta-granules and neurosecretory material remains undetermined.

Insect neurometamorphosis: V. Fine structure of axons and neuroglia in the transforming interganglionic connectives of Galleria mellonella (L.) (Lepidoptera)1

Connectives, some of which shorten 1.4 mm, show a striking reorganization of axons and glia. Axons lose structural uniformity as they come to resemble those which degenerate after being cut from their somata. Some axons contain many close-packed, spindle-shaped vesicles, seemingly originating from neurotubules. Others lack these organelles, and their cytoplasm appears rarefied, or filled with dense bodies, myeloid bodies, autophagic vacuoles, and altered mitochondria. Some axons seem to regress entirely, for their limiting membranes become discontinuous. Larval axons are isolated by glial sheaths, a configuration soon disrupted as extracellular spaces enlarge. During glial metamorphosis microtubules diminish, and rarefaction gives the cytoplasm a polymorphic appearance. Autophagic vacuoles, interrupted cytomembranes, altered mitochondria, and vacuoles also suggest extensive glial degeneration. This may contribute to the adult condition, where numerous unsheathed, contiguous axons occur in irregular bundles delineated by scanty glial cytoplasm. Abundance and size distribution of axons at different stages of development were determined.