Volume 14, Number 1,March 2021
Pages 11 - 15
Jordan Journal of Biological Sciences
Morphology, Histology and Serotonin Immunoreactivity on
Salivary Glands of Stick Insect, Phobaeticus serratipes
Wan Nurul ‘Ain, W.M.N and Nurul Wahida Othman*
Centre for Insect Systematics, Department of Biology and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan
Malaysia, 43600 Bangi, Selangor, Malaysia.
Received November 11, 2019; Revised Feb16, 2020; Accepted June 1, 2020
The salivary gland plays a significant role in physiological processes in insects including food lubrication, extra-oral
digestion and enzyme secretion. This study was conducted to describe the morphology and histology of salivary glands of
stick insect, Phobaeticus serratipes (Phasmida: Phasmatidae). The observation on gross morphology of salivary glands was
photographed by using DSLR Canon EOS 6D camera attached to a stereo microscope. The histological study of the salivary
glands involves special staining procedures of periodic Schiff’s acid reagent and Alcian blue method. The
immunohistochemical study of the biogenic amine serotonin distribution was observed under fluorescence microscope Zeiss
AxiocamMRm Apotome.2. Results showed that the salivary glands were the acinar type that consists of two cells, parietal
cell and zymogenic cell. The serotonin immunoreactivity of the salivary glands which located on the nerve fibers and might
act as a neurotransmitter.
Keywords: Morphology, histology, serotonin, Phobaeticus serratipes, salivary gland
* Corresponding author e-mail: firstname.lastname@example.org.
Phasmatodea insects are herbivores that use their
bodies’ similarities to twigs, leaves, branches and lichen as
an advantage for camouflaging themselves with vegetation
(Bedford, 1978). Phasmatodea consists of 3000 species
that are divided into 3 Families and 500 Genera (Whiting
et al., 2003) including the largest insect, Phobaeticus
chani Bragg, with the length of the female can reach up to
567 mm (Hennemen and Conle, 2008). They belong to the
monophyletic group within the Orthopteroidea that is
similar with Orthoptera, Blattaria, Dermaptera,
Dictyoptera, Grylloblattodea and Mantophasmatodea
(Flook and Rowel, 1998). The earliest described stick
insects from West Malaysia were Marmessoidea rosea
(Fabricius) in 1793 and Heteropteryx dilatata (Parkinson)
in 1798 (Seow-Choen, 2005). To date, only five families
of stick insects were recorded in Malaysia. They are
Heteronemiidae, Phasmatidae, Aschiphasmatidae,
Bacillidae and Phylliidae.
The salivary gland of insects is the gland associated
with the nutrients intake where secretion is usually
involved in the digestion and lubrication of food (Ali,
1997). There are two main types of the salivary glands of
insects which are acinar as in the locust and cockroaches
and tubular as in blowfly and Lepidoptera (Ali, 1997). The
general function of salivary secretions is digestion but they
may perform additional functions in some insects. For
example, the saliva of locusts and cockroaches contains
digestive enzymes (Gardiner, 1972; Kendall, 1969), the
saliva of mosquitoes contains anticoagulants and irritants
(Gardiner, 1972; Ribeiro, 1992) and labial on Lepidoptera
can produce silk (Kafatos, 1968).
Salivation can be controlled either by direct nervous
innervation or via neurohormone. Biogenic amines are one
of the mediators involved in the control of insect
salivation. They also act as neurotransmitters,
neuromodulators or neurohormones in the nervous system
and various peripheral organs of vertebrates or
invertebrates (Baumann et al., 2003; Blenau and Baumann,
2001; Evans, 1980; Roeder, 1994). Serotonin, dopamine,
octopamine and tyrosine hydroxylase are some of the
biogenic amines. Salivary glands can be innervated from
several sources. Most insects have salivary nerve which
projects from suboesophageal ganglion but some species
like Periplaneta americana and Rhodnius prolixus are also
equipped with a salivary nerve that projects from
stomatogastric nervous system (Baptist, 1941; Davis,
1985). Dopamine and serotonin emulate an important role
in the control of salivary glands in most insects (Baines
and Tyrer, 1989; Berridge, 1970; House, 1973). Salivary
nerves of stick insects receive axonal projection from
salivary nerve 1 (SN1) and salivary nerve 2 (SN2) in the
subesophageal ganglion (Ali and Orchard, 1996).
Immunohistochemistry shows that dopamine presents in
SN1 whereas serotonin presents in SN2.
Not much research has been done on the salivary
glands of stick insects. In this study, we report on the
morphology and histology of the salivary gland of
© 2021 Jordan Journal of Biological Sciences. All rights reserved - Volume 14, Number 1
Phobaeticus serratipes. We present a detailed description
of the salivary gland using stereo microscope and light
microscope. We also present results of the location of
serotonin in the salivary gland. Stick insects are
hypothesized to have an acinar type of salivary glands
based on their feeding behaviour with chewing mouthpart.
Salivary glands of insects have at least two different types
of secretion cells such as parietal cells and zymogenic cells
(Beams and King, 1932). The serotonin might be present
on the nerve of the salivary gland as it is known as a
2. Material and Methods
2.1. Samples preparation
Samples were collected from Langkawi Island, Kedah,
Fraser’s Hill, Pahang and Gunung Ledang, Johor. Overall,
30 fresh samples were used in this study.
2.2. Gross Morphology of Salivary Glands
All stick insects were weighed before dissection.
Solutions of methylene blue were injected at the jointed
segment between the legs and abdomen and also between
the head and thorax of the stick insects. Samples were left
for an hour at room temperature. Dissections were done in
phosphate-buffered saline (PBS) and images of salivary
glands in-situ and ex-situ were captured using a DSLR
Canon EOS 6D camera attached to a stereo microscope.
2.3. Tissue sections
The salivary glands were fixed in the formalin solution
for 2-4 hours. Then, the formalin solution was removed by
washing in 70% ethanol. Next, the glands were dehydrated
through a series of ethanol (50%, 90%, 100%) for an hour
each. Tissue was left in sub-Xylene for an hour, infiltrated
with wax (3x at 58ºC) and embedded. Tissue was
sectioned (3-5µm) using Leica RM2245 microtome. The
slides containing tissues were stained using Alcian blue
staining followed by periodic acid-Schiff's reagent (PAS).
Images of the stained sections were observed under the
light microscope (Zeiss Axio Scope) with iSolutionLite
2.4. Tissue sections immunofluorescence
The serotonin detection was performed in the tissue
sections and whole mounts salivary gland tissue (Wan
Nurul ‘Ain and Nurul Wahida, 2015). Briefly, slides of
tissue sections were rehydrated through a series of
solutions (xylene and ethanol (2x100%, 95%, 70%). Then,
the slides were further rehydrated with phosphate-buffered
saline (PBS) for 10 minutes. The excess wash buffer was
drained. Slides were partially dried after removal from
PBS except for the tissue sections. After that, 2 drops of
pre-blocking agent PBT (PBS of 50ml +0.2% bovine
serum albumin, 25ul +0.1% TritonX-100, 5ul) was used to
cover tissue sections and left for 20 minutes and then
tapped off and wiped away. Tissue sections were covered
with diluted primary antibody (anti serotonin) or negative
control (PBT). The primary antibody was diluted 1 in
1000(1µl in 1ml of PBT + 1% normal goat serum, 100ul)
(PBT+N). Slides were incubated overnight at 4ºC. Slides
were rinsed with PBS to wash off excess serum and
drained. Tissues were then covered with PBT plus normal
goat serum (PBT+N). Then, the tissues were incubated
overnight (4 °C) with secondary antibody conjugated to
Dylight (dilution 1:300). The antibody was washed with
PBT (30 min, 4x) before a complete inversion in PBS and
further washing with series of ethanol (100%, 95%, 70%,
50%) to rehydrate it. The slides were mounted in a mixture
of 50% glycerol and PBS and covered with a coverslip.
The slides were then dried out on a slide warmer overnight
and observed under a fluorescence microscope (Zeiss
AxiocamMRm Apotome.2) with ZenPro2012 software and
Olympus FSX100 microscope.
2.5. Whole mount tissue immunofluorescense
Fresh salivary glands were fixed in 4%
paraformaldehyde in PBS (18 hours at 4ºC). After washing
it with PBS, the tissue was permeabilized by exposing it to
methanol (5 min, 70% MeOH in PBS, 60min, 100%
MeOH and 5min, 70% MeOH in PBS) before washing
with PBS (5min, 2x). The tissues were incubated for
30min in 100 mL of PBT+N (PBT + 5% normal goat
serum) before being processed with 100 mL of diluted
(1:1000) primary antibody serotonin and incubated
overnight at 4ºC. The antibodies were washed off by
multiple rinses with PBS (5min, 3x) and PBT (45min, 2x).
Then, secondary antibody Dylight (1:300) was added to
each vial, and the tissues were incubated at 4ºC overnight.
The tissues were washed in PBT (5min, 3x) before a
complete inversion in PBT for 2 hours. The tissues were
cleared in a mixture of 50% glycerol and 50% PBS
overnight before mounting on slides. The control samples
were processed with similar procedure but with absence of
the primary antibody. The distribution of serotonin was
observed under fluorescence microscope (Zeiss Axiocam
MRm Apotome.2) with ZenPro2012 software and
Olympus FSX100 microscope.
3. Result and Discussion
3.1. General morphology of salivary glands
The salivary glands of Phoebaeticus serratipes consist
of cluster of small globular types of acini. This acinar type
of salivary glands had also been reported in other species
of stick insect such as Carausius morosus (Asimakopoulos
and Orchard, 1998) and other solid feeder insects such as
grasshopper, Gastrimargus musicus (Nurul-Wahida and
Cooper, 2014) and cockroach, Periplaneta americana (Just
and Walz, 1996). Based on the gross morphology of the
salivary glands that had been studied, the salivary glands
of P. serratipes are paired glands that can be found at both
sides of the lateral prothorax and extended to the
© 2021 Jordan Journal of Biological Sciences. All rights reserved - Volume 14, Number 1 13
Figure 1. Image of in-situ (red arrows) along the prothorax to
The size and distribution of salivary glands are
different between the male and female stick insects due to
the difference in the size of their body. The size of female
is bigger than the male stick insect. The process of saliva
secretion occurred at the glands of the globular acinar. The
transparent and fine asinus duct canal acts as the connector
between all the acini. This canal has an outlet from each of
the acinus globules where it channels out the saliva from
the acinus to the lower part of the labium of the mouthpart.
The saliva will be collected in the collecting ducts before it
is secreted. The collecting ducts from both sides of
salivary glands will be fused at the head capsule and
opened to become a salivary cup at the labium (Kendall,
1969), thus forming the main duct (Figure 2). The saliva is
secreted from the main duct.
Figure 2. Ex-situ of acinar salivary glands of P. serratipes. md
(main duct); cd (collecting duct).
Based on ex-situ and in-situ observation, no secretory
gland was detected in P. serratipes. This secretory gland
can be found in other species such as Asceles glaber
(Dossey et al., 2012) and Oreophoetes peruana (Eisner et
3.2. Histology of salivary glands
The acinar glands of P. serratipes consist of two types
of cells, parietal and zymogenic (Figure 3). Each acinus
cell is covered by a basal membrane on the outside. Basal
membrane mould acinus cell a round shape which later
forms acinus globule.
Figure 3. Tissue section of P. serratipes salivary gland. Tissue
was stained with Alcian blue and PAS reagent. A (Acini); Bm
(basal membrane); Pa (parietal cell); z (zymogenic cell).
Parietal cells are cone-shaped and have wide basal
connected with basal membrane cells. They are located
between zymogenic cells and extended towards the centre
of each acinus. The nucleus of the parietal cell is big and
oval-shaped. It is located at the centre of the cell. In
contrast, zymogenic cells have an irregular shape. The
basal of the cell is smaller compared to parietal cells. The
nucleus is small in size and present on the side of each
3.3. Serotonin distributions on salivary glands
The serotonin on the salivary glands of P. serratipes can
be seen clearly at the axons along the ducts to the acini
globules and the nerve fibers in the acini (Figure 4).
Figure 4. The serotonin distributions on the nerve fibers of P.
serratipes salivary glands (yellow arrows).
Moreover, for the cross-section of the tissues, the
serotonin was distributed on both cells in the salivary
glands, parietal cells and zymogen cells (Figure 5). This
result was supported by Nurul-Wahida and Cooper (2014)
who reported the presence of serotonin on both parietal
and zymogenic cells of yellow-winged grasshopper,
Gastrimargus musicus. Serotonin is absent on the salivary
gland of controlled stick insect (Figure 6).
The presence of serotonin will produce saliva with high
protein content (Just and Walz, 1996). Electrical
innervation towards the nerves or glands of the salivary
ducts that superfusion with dopamine and serotonin will
stimulate the secretion of saliva (Just and Walz, 1996).
© 2021 Jordan Journal of Biological Sciences. All rights reserved - Volume 14, Number 1
Liquid secretion rate is controlled by peripheral cell (p-
cell) at the base of each acinar globule and involved in the
transportation of water and electrolytes as in cockroaches,
Periplaneta americana (Kessel and Beams, 1963;
Sutherland and Chillseyzn, 1968). Central cell, also known
as c-cell, will react with serotonergic innervation and
supply the proteinaceous components to the saliva (Just
and Walz, 1994, 1996; Walz et al., 2006). The parietal
cells of stick insects have similar function and morphology
as the peripheral cells or p-cells of the cockroaches,
whereas the zymogenic cells are similar to the central cells
Figures 5-6. (5) Serotonin distributions on the cross section of
salivary glands tissues of P. serratipes (yellow arrows). (6) No
serotonin-like immunoreactive process on salivary glands of
control stick insect.
It can be concluded that the serotonin in P. serratipes
plays a role as a neurotransmitter that is similarly
described in the Periplaneta americana (Ali, 1997; Ali and
Orchard, 1995) due to its presence on the nerve fibers of
the salivary glands. Besides, the distribution of serotonin
on both parietal and zymogenic cells suggests that the
serotonin also innervates the production of proteinaceous
and non-proteinaceous saliva for this species.
The authors would like to thank Universiti Kebangsaan
Malaysia and Ministry of Higher Education of Malaysia
(MOHE) for the facilities and grant provided (Young
Researcher Encouragement Grant GGPM – 2013 – 089
and FRGS/1/2015/WAB13/UKM/02/01), Nazca Scientific
and Olympus from Universiti Putra Malaysia for the
fluorescence microscope facility. We also would like to
express our gratitude to Dr. Azman Sulaiman for helping
us with the samples.
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