STUDY OF NEUROSECRETORY CELLS OF ORDER DIPTERA

1. INTRODUCTION

The neurosecretory cell (NSCs) plays a central part in the life of the insect. They receive a
variety of stimuli from the external and internal environments. Environmental signals are
processed by the nervous system to alter motor programs controlling behaviour, or to regulate
release of hormones controlling development and other physiological phenomena as
neurosecretion plays an important role as a link between the nervous and endocrine systems
(Orchard,1985; Kloot,1960). The possibility to identify NSCs has greatly facilitated
physiological studies since it enabled scientists to perform surgical ablations of cell clusters or
dissections of single cells for bioassays, and intracellular recording and staining (Agui,1979;
Kostron ,1996). In the larval brain of dipteran insects, there are two medial and three lateral
groups of neurons innervating the ring gland. One lateral group extends fibers to the corpus
allatum. After metamorphosis, a large cluster of the medial group in the pars intercerebralis and
two lateral groups in the pars lateralis innervate the retrocerebral complex and some neurons
from the lateral group and a few from the medial group extend fibers to the corpus allatum in
the adults ( shiga S.,2003).

In insects the nervous system consists of a chain of central ganglia connected with sensory and
effector organs via afferent and efferent nerves. Each ganglion is composed essentially of
amplifying neuroglial cells having supporting and nutritive functions and the more distinctive
numerous neurons which are specialized for nervous functions and have prolongations and
branches (Richards, 1977) . Neurons in central nervous systems of insects are characteristically
monopolar, their prolongations and branches are processes that are especially adapted for
neural transmission and can relatively rapidly generate and conduct electrochemical nervous
impulses ( Shankland, 1985).

Neurosecretory cells in the brain of the housefly, previously described by (Ramade 1969)
were:-

Type I Neuron: These are relatively small neurons situated near the calyces of the corpora
pedunculata in the protocerebrum and often referred to as globuli cells.

Type II Neuron: They are the smallest neurons in the brain measuring about 3.5-5 U in diameter
and occur in the optic lobes.

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Type III Neuron: These are medium-sized neurons approximately 9-12 U in diameter and are
distributed throughout the brain. The chromatin is predominantly diffused with only occasion
dense patches.

Type IV Neuron: These neurons are located at the base of the ocellar stalk in the anteromedian
region of the protocerebrum and are about 5-6 U in diameter. Their chief distinguishing
characteristics are that they are bipolar. The nucleus occupies a large proportion of the cell
body.

Type V Neuron: In this category are the giant neurons about 22-30 U in diameter which are
situated in the anteroventral region of the protocerebrum and in the anterodorsal region of the
deutocerebrum. The rounded nucleus contains a single eccentrically located nucleolus.

However, the present report is concerned with the structural organization of the neural sheath,
neurons and glial cells in the brain, ganglion connected with sensory organs, and many more
related to the adult housefly, Musca domestica, drosophila, mosquitoes which belongs to the
order diptera.

1.1. NEUROSECRETORY CELLS IN MUSCA DOMESTICA

1.1.1 BRAIN
The head and anterior part of the thorax was stain with hematoxylin staining method result
found that - The brain lies just above the oesophagous between the abdomen of the tentorium.
It is the dorsal ganglionic center of the head. Histologically, the two main divisions of the brain
are cortex and medulla or neuropile. Cortex is the cellular peripheral part possessing three main
types of cells i.e. Neurons, Glial or supporting cells and Neurosecretory cells (Firdausia ,1996).
Five distinct types of conventional nerve cells were identified in different regions of the brain.
The major differences concern size, nuclear-cytoplasmic ratio, and relative development and
distribution of cell organelles. The stomata are surrounded by one or more layers of
glycoplasm. The neuropile is the central part of brain and is traversed by axons of different
diameter, are arranged in definite bundles and form distinct fiber tracts (Horridge, 1965). The
responses of certain classes of interneurons in the optic lobes and brain to visual stimuli. After
suitable amplification, response and stimulus signals were sent to an analogueto-digital
conversion system and from there to an IBM 360/44 computer digitized data were stored.

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A report describing the use of this system in detail results in more than 40 distinct classes of
neurons (each several times) in the medulla, lobula-lobular plate, and brain of dipterans (Dill,
1969).

Cholesterol is taken up by the brain tissue of larvae of Musca domestica in preference to -

sitosterol. The uptake reaches a maximum value when both sterols are available to the insect
each as 0.01% of the diet. These results are obtained with low levels of choline and B-
methylcholine in the housefly. These studies suggested a preferential uptake of choline into
the nervous system, where it was accumulated in the form of phosphatidylcholine. They are
unable to synthesize either cholesterol, tissues should be dependent upon their relative
concentration in the diet, absorption from the gut, efficiency of transport and their specific
requirements for the development of the insect ( Dwivedy, 1982).

1.1.2 NEUROSECRETORY CELLS

Neurosecretory cells are predominantly present in the parts Intracerebral region of the
protocerebrum where they are present in groups. All these cells have rounded or elliptical nuclei
with a conspicuous nucleolus and centrally concentrated chromatin material. Their nuclei range
from 7.5-15μm in length and 5-7.5μm in width. The cell size ranges from 12-20μm in length
and 9-20μm in width (Cook,1972; Richards,1977). The staining intensity of the median
neurosecretory cells (MNC) in Musca domestica increased as oogenesis progressed from stages
2 to 10. The amount of neurosecretory material within the MNC was dependent upon the
presence of ovaries with developing or mature follicles. Eight type of median neurosecretory
cells (MNCA) are located near the median cleft of the pars intercerebralis anterior to the
insertion of the ocellar nerve (Adams,1974).

1.1.3 NEURAL SHEATH

The neural sheath consists of an outer acellular fibrous layer and a subjacent cellular layer
which have been termed the neural lamella and the perineurium respectively ( ashhurst, 1939).
In Musca, the neural lamella consists of an external lamina, approximately 600 A in thickness,
which is composed of finely granular and fibrillar material similar to that constituting basement
membranes. Beneath the external lamina lie striated fibrils presumably collagenous in nature
which are embedded in an amorphous matrix (Ashhurst, 1968). These fibrils are about 80-100
A thick; their maximum length recorded in the sectioned material is about 3U. Thin processes
of glial cells form perineuronal sheaths. Three distinct types of glial cells are recognized and

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are arbitrarily designated as Type I, Type II and Type III. Based on the evidence obtained from
histochemistry (Ashhurst,1961), biochemical analysis (Harper et al.,1967) electron microscopy
(Hess,1958; Wigglesworth,1959; Ashhurst,1961). It is now well established that the neural
Lamella is composed of collagen fibrils embedded in a matrix of mucopolysaecharides. These
extracellular structures appear to be secreted by the perineurial cells (Ashhurst, 1964; Pipa,
1965); however the involvement of hemocytes has also been suggested (Wiggtesworth, 1956;
boschek ,1971).

1.1.4 NERVOUS SYSTEM

Supra-vital staining of the nerves of larva in 1 : 1000 solution of Methylene Blue shows the
nervous system of the larva is greatly modified due to the concentration and fusion of the
cephalic, the thoracic and the abdominal ganglia. This fused ganglionic mass is situated in the
IV and V segments of the body, except the presence of paired segmental nerves arising from it.
It is pierced through by the oesophageal tube into two parts: a dorsal portion formed by the
two Cerebral Ganglia situated above the oesophagus and a large, unpaired, Ventral Ganglionic
mass (VG) situated below the oesophagus. The cerebral ganglia are smooth, globular and
bilobed joined to the anterior part of the ventral ganglionic mass, representing the sub-
oespohageal ganglion (Ranade, 1966)

Comparative study of certain parts of the central nervous system between the larval and the
adult stage of the house flies observed During the metamorphosis, the central nervous system
of the house flies are not only changed in size and shape, but also the whole structure. In the
larval stage, the two brain hemispheres are connected mind ventrally, where is perforated by
the oesophagus and the ventral ganglionic mass is anteriorly connected to the ventral point of
fusion of the brain hemispheres and consists of the suboesophageal ganglion located in the
forth segment and reach to about the end of the fifth segment. In the adult house flies, the
suboesophageal ganglion is fused with the brain and hardly distinguished externally (Hewitt
,1914)

The first visual ganglion of Musca is built of sub- units called cartridges, each of which is
composed of six retinular cell axons synapsing on to five different neurons: some of which are
second order neurons, some of higher order. One of these five neurons, "L", has two long
collaterals at the base of the lamina, which run from the cartridge to the two neighboring
cartridges in the hexagonal array (Debbge,1975). The peripheral retina and laminal ganglion
of Musca domestica have been investigated electron microscopically. Laminal ganglion are of
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- Four monopolar neuron somata have been found distal to each optical cartridge in the lamina.
The four fibers of these neurons form a bundle and enter the crown of photoreceptor cell axon
terminals. Two large diameter fibers which are derived from two type of somatic form the
central second order neurons, L 1 and L2 in each cartridge (Boschek,1971).

2.1 NEUROSECRETORY CELLS IN MOSQUITO

2.1.1 BRAIN
The head of mosquito larvae is large and sclerotized. The shape of the head may be elongate
or broad (Aedes and Culex). The head bears two eyes, two antennae and brush- or comb-like
mouthparts. The eyes are generally small, simple (not compound) and are found on either side
of the head. The antennae are quite variable. The mouthparts are composed of articulating
appendages of the mandible and maxilla. Setae of the head are numerous and variable in length
and form. The arrangement, length, branching and shape of head setae are used in the
identification of larvae (Cadena,2013). The brain and other ganglia consist of an outer layer of
neuronal cell bodies (perikaryal rind) and inner masses ofaxons (neuropil), which together are
enclosed within a sheath, the neurolemma. The brain and subesoesophageal ganglion, joined
by two thick circum oesophageal connections, form a globular mass, with a narrow passage
between them for the oesophagus. The supra oesophageal ganglion is divided into the
protocerebrum, deutocerebrum, and tritocerebrum (Childress, 1984).

2.1.2 NEUROSECRETORY CELLS

Distribution of neurosecretory cells in the pupal brain of mosquitoes. Highmann reported their
presence in pupa of Mimes. They were fixed in Carnoy's solution. They appear at the time of
moulting of pupal cuticle when they become conspicuous by their large size which is three or
four times the size of the ordinary nerve cells. The neurosecretory cells are located not less than
six groups in each half of the brain (Banerjee,1958). Mosquitoes have several sensory
appendages important for sensing their environment and executing behaviors. The wing margin
may also contain chemosensory neurons. the total number of neurons in the mosquito antenna
could indicate a maximum number of olfactory neurons in this tissue. However, the number
of neurons that innervate Anopheles mosquito appendages is unknown (Joanna, 2000).

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 2.1.3 NERVOUS SYSTEM
The nervous system is ectodermal in origin; in the adult mosquito, it has three divisions: the
central, visceral, and peripheral (Christophers,1960). The central division includes: (a) the
supraesophageal ganglion and optic lobes, which we will refer to as the brain; (b) the
subesophageal ganglion; (c) four fused ganglia in the thorax (prothoracic, mesathoracic,
metathoracic, and first abdominal ganglia); (d) the ganglia in the second to sixth abdominal
segments; (e) the fused terminal ganglia in the seventh abdominal segment (Pratt, et al., 1986).

The stomatogastric system is made up of the frontal ganglion, the recurrent nerve, the
hypocerebral ganglion, the paired ventricular ganglia, and nerves to the mouthparts and
digestive tract. The ventral and caudal visceral nervous systems innervate the respiratory,
reproductive, and digestive organs. The peripheral division consists of motor nerves,
originating in the central ganglia, and sensory nerves associated with the cuticle, muscles,
viscera, and sense organs ( Brown,et al.,1989).

Adult male and female C. salinarius used in the ultrastructural study were reared. Procedures
used were described by (Meola et al., 1998). Result observed that each antenna of a mosquito
contains 13 flagellar segments, innervated by a pair of antennal nerves in which the sensory
nerves of the antennae extend to the antennal lobe of the deutocerebrum. The axons of these
antennal neurosecretory cells (ANC) could be traced within the antennal nerve (AN) to their
terminals on glomeruli (G) of the antennal lobe where the ANC form neurotransmitter terminals
on processes of the internuncial neurons comprising the neuropile of the glomeruli (Shirlee ,
Bhatkar,1999).

3.1 NEUROSECRETORY CELLS IN DORSOPHILLA

3.1.1 BRAIN
On the basis of 1200 Golgi-impregnated brains the structure of the central complex of
Drosophila melanogaster was analyzed at the cellular level. The four substructures of the
central complex — the ellipsoid body, the fan shaped body, the noduli, and the protocerebral
bridge — are composed of (a) columnar small-field elements linking different substructures
or regions in the same substructure ( b) tangential large-field neurons forming strata
perpendicular to the columns (Hanesch, et al., 1989). The deutocerebral/tritocerebral boundary
region in the embryonic Drosophila brain displays developmental genetic features similar to

6
those observed for the midbrain/hindbrain boundary region in vertebrate brain development.
This suggests that a tripartite organization of the embryonic brain was already established in
the last common urbilaterian ancestor of protostomes and deuterostomes (Hirth, et al.,2003 ).

In the larval brain and subesophageal ganglion there are about 35 neurons in each hemisphere
expressing in situ signals, in addition to numerous Kenyon cells .These neurons are located
dorsally and ventrally in the protocerebrum and in the subesophageal ganglion, neurons in each
protocerebrum have larger cell bodies and are likely to be neurosecretory cells In the ventral
nerve cord there are segmentally distributed neurons both in thoracic and abdominal
neuromeres. In each side of the thoracic and abdominal neuromeres there are three to four
lateral cell bodies and one dorso-median cell body (Nässel, et al., 2008). In the adult Drosophila
central brain, we identify five different types of glia based on its location, morphology, marker
expression, and development. Perineurial and subperineurial glia reside in two separate single-
cell layers on the brain surface, cortex glia form a glial mesh in the brain cortex where neuronal
cell bodies reside, while ensheathing and astrocyte-like glia enwrap and infiltrate into
neuropils, respectively ( Awasaki, et al., 2008 ).

3.1.2 NEUROSECRETORY CELLS

Automatic Neuron Type Identification ,Localization in the Drosophila Medulla the testing data
contain 379 neurons that were manually categorized into 89 types. category Among the 89
manually defined types, 33 types have only one sample, there by leaving 346 neurons for
testing classification accuracy ( Ting Zhao and Stephen, 2014).

Glial cells exist throughout the nervous system, and play essential roles in various aspects of
neural development and function. Distinct types of glia may govern diverse glial functions as
glia requires systematic characterization of glia diversity and development (Lee,2008 ). The
neuropile of the Drosophila brain is subdivided into anatomically discrete compartments.
Compartments are rich in terminal neurite branching and synapses; neuropile domains in which
signal processing takes place. Compartment boundaries are defined by more or less dense
layers of glial cells as well as long neurite fascicles. These fascicles are formed during the
larval period, when the approximately 100 neuronal lineages that constitute the Drosophila
central brain differentiate. Each lineage forms an axon tract with a characteristic trajectory in
the neuropile (Pereanu, et al.,2010).

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 The cellular organization of the fru circuit suggests how multiple distinct sensory cues are
integrated in the fly's brain to drive sex-specific courtship behavior. sensory processing and
motor control are mediated through circuits that are largely similar in males and females. Sex-
specific behavior may instead arise through dimorphic circuits in the brain and nerve cord that
differentially couple sensory input to motor output. Some 1500 neurons in the fly's nervous
system express the sex-specific transcripts of the fru gene (Jai, et al., 2010 ). The
neurosecretory cells seen in the brain of, thoracic and abdominal ganglia of the fruit fly
Chatodacus cucurbita Coq. (Trypetida: Diptera). A general description of the neurosecretory
cells appeared to be characteristic distribution in the thoracic ganglion, forming a more
conspicuous cluster than in the brain. Result observed that the neurosecretory cells Situated in
the pars intercerebralis, is a cluster of about a dozen cells which show well-defined secretory
activity (Naya,1954).

3.1.3 NERVOUS SYSTEM

The fact that the Drosophila nervous system is composed of structural modules that are defined
by discrete lineages offers the exciting possibility of getting closer at the link between genes
and behavior. In a developmental sense, lineages represent “units of gene expression”. The
expression pattern of more than fifty transcription factors in specific embryonic neuroblasts
has been described (Urbach and Technau, 2003).

In the embryo, a given transcription factor becomes active in one, or a small number of,
neuroblasts; a particular neuroblast there by acquires a “genetic address”, consisting of specific
sets of transcription factors which involved in shaping the morphology and function of that
lineage. Lineages also represent to some extent structural modules of the brain. a lineage with
its highly restricted dendritic arborization and axonal pathway, will be involved in a single or
a limited number of behaviors, “behavioral subroutines” (Larsen , Spindler, 2009).

The neuroendocrine system and nervous system of the adult black blow fly, Phormia regina
(Meigen), were observed using phase contrast, Nomarski light and electron microscopy. thus,
it is non ganglionic. Neurosecretory granules (NSG) are found in some axons of the junction.
Two types of neurosecretory cells can be identified in the pars intercerebralis of the brain based
one size and electron opacity of their NSG, and their staining reaction with the paraldehyde
fuchsin (PAF) method, the nervi corporis cardiaci one from each cerebral hemisphere approach
and join with the recurrent nerve to form a larger nerve bundle, the cardiac-recurrent nerve (
Dai, Stoffolano,1987).

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4.1 CONCLUSION

Diptera is an order of single pair winged insects commonly known as flies/true flies. About
150,000 species are described in 150 families are also use as bioindicator for environment, and
biological control agents, some Diptera produce useful products, they are foods for other
animals, they participate as pollinators, dispersal of seeds, and symbionts. The nervous system
of Diptera can be divided into a brain and a ventral nerve cord. The head capsule is made up of
6 fused segments. The first 3 pairs of ganglia are fused into the brain, while the three following
pairs are fused into a structure of 3 pairs of ganglia under the insect’s oesophagus, called the
subesophageal ganglion. Musca domestica, have all the body ganglia fused into a single large
thoracic ganglion. At least a few insects have nociceptors, cells that detect and transmit
sensations of pain.

During the metamorphosis of holometabolous insects, the central nervous system changes
from the larval form into the adult form. The head of mosquito larvae is large and sclerotized.
The shape of the head may be elongate or broad.The Drosophila Brain. The brain consists of
supraesophageal and subesophageal ganglia. The supraesophageal ganglia is made from three
neuromeres, or neural segments. These neuromeres are formed during neurogenesis, coincident
with the formation of the rest of the central nervous system.

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