Journal of Insect Science, (2019) 19(4): 3; 1–9

doi: 10.1093/jisesa/iez061
Research

The Anatomy and Ultrastructure of the Digestive Tract and

Salivary Glands of Hishimonus lamellatus (Hemiptera:
Cicadellidae)
Lizhen Dai,1 Baodong Yang,1 Jinzhong Wang,1,3 Zhiyong Zhang,1 Rui Yang,1

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Tieqiang Zhang,1 Zhengguang Ren,1 and Caili Lin2
1
Beijing Key Laboratory of New Technology in Agricultural Application, College of Plant Science and Technology, Beijing University
of Agriculture, Beijing 102206, China, 2Institute of Forest Ecology, Environment and Protection, Chinese Academy of Forestry, Beijing
100091, China, 3Corresponding author, e-mail: [email protected]

Subject Editor: Phyllis Weintraub

Received 11 February 2019; Editorial decision 7 May 2019

Abstract
In recent years, we found that Hishimonus lamellatus Cai et Kuoh is a potential vector of jujube witches’-broom
phytoplasma. However, little is known about the anatomy and histology of this leafhopper. Here, we examined
histology and ultrastructure of the digestive system of H. lamellatus, both by dissecting and by semi- and ultrathin
sectioning techniques. We found that the H. lamellatus digestive tract consists of an esophagus, a filter chamber, a
conical midgut and midgut loop, Malpighian tubules, an ileum, and a rectum. Furthermore, both the basal region
of the filter chamber epithelium and the apical surface of the midgut epithelium have developed microvilli. We also
identify the perimicrovillar membrane, which ensheaths the microvilli of midgut loop enterocyte, and the flame-
like luminal membrane, which covers the microvilli of the conical midgut epithelium. In addition, H.  lamellatus
has the principal and accessory salivary glands. Our observations also showed that the endoplasmic reticulum,
mitochondria, and secretory granules were all highly abundant in the secretory cells of the principal salivary glands,
while the accessory glands consist of only one ovate or elbow-like acinus. We also briefly contrast the structure
of the gut of H. lamellatus with those of other leafhopper species. These results intend to offer help for the future
study on the histological and subcellular levels of phytopathogen–leafhopper relationships, including transmission
barriers and the binding sites of pathogens and other microorganisms within their leafhopper vectors.

Key words: leafhopper, digestive system, Malpighian tubule, ultrastructure, histology

Leafhoppers are herbivorous insects belong to the family to the old losses that reach hundreds of millions of dollars annually (Liu et al.
suborder Auchenorrhyncha. More than 25,000 species of leafhoppers 2010). Early studies suggested that the transmission of the JWB
are known worldwide (Dietrich 1997), of which over 1,200 are present phytoplasma occurred mainly by insect vectors such as H. sellatus
in China (Li et al. 2012). Some of the leafhoppers must be mesophyll (La and Woo 1980, Sun et al. 1988). Hishimonus lamellatus was first
feeders and hence do not tap into plant vasculature to suck plant sap, reported in Huolu County, Hebei Province, China (Cai et al. 1995).
thereby directly harming plants. Moreover, many leafhoppers are re- Both leafhopper species were found to be carrying JWB phytoplasma
sponsible for the transmission of plant pathogens—including viruses in vivo, but vector competence of H. lamellatus was not empirically
and bacteria—thus causing serious losses of production in the agricul- tested. (Hao et al. 2015).
ture and forestry industries (Nault and Ammar 1989, Bové et al. 2003). Phytoplasmas, the single-celled prokaryotes, are parasitic on
To date, 118 species of leafhopper are known vectors (Weintraub and the phloem of plants. Insects that feed on phloem can acquire and
Wilson 2010). In East Asia, the pathogen of rice dwarf disease is spread transmit phytoplasmas (Lee et  al. 2000, Weintraub and Beanland
by leafhoppers, which is the reason for the sharp decline in rice produc- 2006), and it is generally believed that phytoplasmas circulating in
tion every year (Ruan et al. 1985, Sivamani et al. 1999). Furthermore, insects need to pass through the midgut and salivary glands before
mulberry dwarf disease phytoplasma, which is transmitted by transmission to healthy plants via interface and salivary secretions
Hishimonus sellatus (Hemiptera: Cicadellidae), has caused a severe de- during feeding (Weintraub et al. 2004, Weintraub and Beanland 2006,
cline of mulberry tree (Kawakita et al. 2000, Mitsuhashi et al. 2002). Ammar et al. 2011).
Jujube witches’-broom (JWB) phytoplasma is known to be trans- To date, few researchers have conducted detailed analyses of the
mitted by leafhoppers, and the resulting disease results in economic digestive systems of leafhopper vectors. Gil-Fernandez and Black

© The Author(s) 2019. Published by Oxford University Press on behalf of Entomological Society of America.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/
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2 Journal of Insect Science, 2019, Vol. 19, No. 4

(1965) observed the general structure of the digestive tract of Agallia Semi-Thin Section Sample Preparation for Light
constricta Van Duzee  (Hemiptera: Cicadellidae) (Gil-Fernandez Microscopy
and Black 1965). In addition, Lindsay and Marshall (1980) de- The selected adult H.  lamellatus individuals, anesthetized with
scribed the morphology and ultrastructure of the Eurymela distincta ether, were placed on single concave slides. We then added 2.5%
Signoret  (Hemiptera: Eurymelidae) filter chamber (Lindsay and glutaraldehyde fixative and quickly dissected the digestive tract of the
Marshall 1980), Cheung and Purcell (1993) revealed the ultrastruc- leafhopper using tweezers. Next, the digestive tract was then placed
ture of the digestive system of Euscelidius variegatus (Kirschbaum) in a centrifuge tube containing 1.5 ml of glutaraldehyde fixative. The
(Hemiptera: Cicadellidae) (Cheung and Purcell 1993), and Wayadande digestive tract was fixed for 48  h in the dark at room temperature.
et al. (1997) compared the general morphology of the digestive tracts Next, fixed tracts were washed with phosphate-buffered saline (PBS)
of Circulifer tenellus (Hemiptera: Cicadellidae) and Dalbulus maidis for 3 h. Afterward, we added 1% osmium tetroxide to the digestive
(Hemiptera: Cicadellidae)  (Wayadande et  al. 1997). More recently, tracts for 90 min before rinsing with phosphate buffer. Then, the sam-
Zhang et al. (2012) observed the ultrastructure of the digestive tract ples were washed with PBS for 30 min. The samples were dehydrated

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of Psammotettix striatus (Linnaeus) (Hemiptera: Cicadellidae) (Zhang in a series of ethanol concentrations of 30, 50, 70, 80, 90, and 100%
et  al. 2012), and Utiyama et  al. (2016) studied similar features in for 6 min, respectively, and again in that of 100% ethanol for 30 min.
Bucephalogonia xanthophis (Hemiptera: Cicadellidae) (Utiyama et al. Samples were then infiltrated with a 1:2 mixture of ethanol and epoxy
2016). Taken together, the results of these analyses suggest that there resin 618 for 2  h and pure epoxy resin 618 for 12  h. The samples
are important differences in the morphology and ultrastructure of were transferred to plastic flat embedding mold (Electron Microscopy
the digestive tracts and salivary glands of different leafhopper species Sciences) and polymerized for 24 h at 37°C, 12 h at 45°C, and 48 h
(Sōgawa 1965, Wayadande et al. 1997). Globally, there are 41 species of at 60°C. The embedded block was trimmed to the appropriate size
Hishimonus, of which there are 17 Hishimonus in China (Li and Wang and the sample was cut into 2-μm-ultrathin sections with a glass knife
2004). However, little information is currently available regarding the of Leica UC6 ultrathin slicer. The sections were placed in a saturated
structural characteristics of the digestive systems of Hishimonus in- solution of NaOH in absolute ethanol for 3 min. The samples were
sects. Actually, there are few research on the histology and ultrastruc- infiltrated in a series of ethanol concentrations of 100, 95, 80, 70, 50,
ture of the digestive tract and salivary glands of H. lamellatus. and 35% for 5 min. Slides were immersed in a staining jar containing
Therefore, the purpose of this study was to characterize the 2% hydrochloric acid for 5 min, and then in a staining jar containing
structure of the digestive system of H. lamellatus using optical and distilled water for 2 min. The samples were stained in Harris’s hema-
transmission electron microscopy (TEM), and to understand the toxylin for 20 min, and washed in tap water. Slides were immersed in a
ultrastructural characteristics of the digestive tract, the Malpighian staining jar containing acid alcohol (0.5:99 v/v) differentiation solution
tubule (MT) system, and the salivary glands. for 30 s, and then in a staining jar containing tap water for 15 min, and
counterstained in 1% aqueous eosin for 2 min. Slides were immersed
in a staining jar containing tap water for 5 min. The samples were de-
Materials and Methods
hydrated in a series of ethanol concentrations of 80 and 95% for 1 min
Leafhopper Rearing and twice in 100% for 1 min. The samples were sealed with a drop
Hishimonus lamellatus individuals were raised in the insect of neutral balsam, capped with a cover slip, and then kept in drying
breeding room of the Beijing Key Laboratory of New Technology oven. The treated samples then were observed with a Zeiss microscope
in Agricultural Application. Leafhoppers were originally collected Imager A1 (Aparicio and Marsden 1969).
in Liucun, Changping District, Beijing in August 2010. A  labora-
tory population was established in the insect breeding room and Sample Preparation for TEM
identified by Professor Cai Ping. The H. lamellatus population was To prepare samples for analysis using TEM, we used the same
reared on jujube (Ziziphus jujuba Mill (Rhamnales: Rhamnaceae)) method as described in ‘Semi-Thin Section Sample Preparation for
seedlings and placed in a 40-cm-high cylindrical transparent plastic Light Microscopy’. At least five embedded blocks were made for
worm cage, which was sealed with 40 × 40 mesh gauze. The feeding each sample to be observed. The ultrathin resin sections (60  nm)
temperature was maintained at 25  ± 1°C, the relative humidity were cut with an ultramicrotome (Leica UC6) using a glass knife,
ranged between 50 and 70%, and the photoperiod was 16 L:8 D transferred to copper grids, and stained with 2% (w/v) uranyl
(Hao et al. 2015). acetate for 15  min (in dark), and then washed in distilled water.
The sections were stained with lead citrate for 20  min, and then
Sample Preparation for Light Microscopy washed in distilled water again. The sections were kept in a des-
Hishimonus lamellatus samples were chilled at −20°C for 20  min iccator (Ghanim et  al. 2016, Ammar et  al. 2017). Then ultrathin
and then each sample was then placed on a grooved glass slide (SAIL sections were examined at 80 kV using an Hitachi H-7500 trans-
BRAND, 7103 Single Concave). Under a stereoscopic microscope mission electron microscope at the electron microscope laboratory
(Motic, K Series), the wings and feet of leafhoppers were removed with of the Institute of Food Science and Technology, Chinese Academy
forceps (Dumont, 0208-5-po), and the side line of the abdomen stalk of Agricultural Sciences. About 30 leafhopper individuals were
was cut with scissors. A drop of phosphate buffer solution was added examined by TEM.
dropwise to this cut, and an anatomical needle was used to extract the
digestive tract from abdominal segments 1–7. The salivary glands, lo-
Results
cated at the top of the head, were gently separated using forceps. The
salivary glands were dissected and stained with toluidine blue solution Histology and Morphology Features of the
and photographed using a stereomicroscope (Zeiss SteREO Discovery. Digestive Tract
V20) (Gil-Fernandez and Black 1965, Sōgawa 1965). About 80 leaf- The foregut, midgut, and hindgut (Fig. 1) are the three major parts
hopper individuals were examined by light microscopy. of the digestive tract of adult H. lamellatus.
Journal of Insect Science, 2019, Vol. 19, No. 4 3

Foregut Malpighian tubule: Hishimonus lamellatus has four MTs that extend

The esophagus is a narrow, slender tube. The esophagus is approxi- out of the same side of the ileum as the FC. Each of the malpighian
mately 50 μm in diameter and is translucent or pale white in color tubes is divided into four parts. The first part is connected to the FC,
(Fig. 1). which is short and has a smooth surface. The second part is wavy,
smooth, translucent, and is not easily stained with toluidine blue. The
diameter of this stage is about 60  μm. The third part is larger in
Filter Chamber
diameter (about 100  μm) and is thicker than the first segment. Its
The filter chamber (FC) has a half-moon shape, a diameter of 300–
surface is rough. The fourth part is a short translucent tube with a
400 μm, and is light milky white in color. The FC is composed of the
smooth surface that is also not easily stained with toluidine blue. Its
anterior segment of the midgut, the posterior segment of the midgut,
diameter is about 50 μm, and it merges with the rectum to form a
the base of the Malpighian tube, and the basal hindgut. It is also con-
complex (Fig. 1).
nected to the conical midgut (CM).

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Midgut Hindgut
The midgut is divided into a CM and a midgut loop (ML). The The hindgut consists of an elongated tubular ileum (Fig. 1) and
spherical or long vesicular CM is about 1,200  μm long and an enlarged rectum (RE; see Fig. 1). The ileum is connected to the
700  μm wide. The surface of the CM is translucent and some- rectum through four MTs (Fig. 1). Its surface is smooth, translucent,
times contains white particulate matter. The apical of the CM, and is not easily stained with toluidine blue; its diameter is approxi-
which has a thick basement membrane, rapidly collapses into the mately 100 microns. The ileal apical swells into the rectum, and the
ML. The ML has a diameter of about 400–600  μm and is only end of the ileum sac is open to the anal tube.
slightly smaller than the diameter of the rest of the midgut (Fig.
1). Its surface is rough and its color is yellowish. The basement Ultrastructure of the Digestive Tract
membrane is relatively thin, and the columnar epithelium cells Filter Chamber and Conical Midgut
(CECs) found there are large and are located near the basement The FC has a thin membrane known as the peritoneal membrane
membrane (Fig. 2). (PM), which is adjacent to the longitudinal muscles (LMU). Cells of
these tissues include large numbers of mitochondria, which distrib-
uted around developed muscle fibers (Fig. 3C). The circular muscles
(CMU) lie on the inside of the LMU surround the basement mem-
brane. The basement membrane has channels, formed by infolding
(IF), which gives it a network-like and highly developed appearance.
The intima is highly specialized into microvilli (MV) which are dense
and regularly arranged. Many mitochondria were also observed in the
MV-containing cells. Furthermore, many well-developed MV extend
into the lumen (L) (Fig. 3D). Moreover, there are also a large number
of secretory vesicles (SVs) in the cytoplasm of many of these cells in
these tissues (Fig. 3A and B), and many cells also contain a highly de-
veloped rough endoplasmic reticulum (RER) connecting the SVs and
the basement membrane (Fig. 3B). In many cases, there are also dis-
tinct septal desmosomes (SDs) between the cells (Fig. 3D).
The basement membrane of the CM was found to be thinner
than that of the FC, and we also found that the basement membrane
has one or more IFs. Mitochondria are present between the base-
ment membrane and the IF (Fig. 3E). The intima is highly specialized
into dense and regularly arranged MV (Fig. 3E and F). In addition,
its uniform and well-aligned flame-like luminal membrane (FLM) is
Fig. 1.  Light micrograph of the alimentary canal of H. lamellatus. CM, conical covered in MV (Fig. 3).
midgut; FC, filter chamber; IL, ileum; ML, midgut loop; MT, Malpighian
tubule; OE, esophagus; RE, rectum.
Midgut Loop
The midgut’s PM is relatively thick (Fig. 4A and B) and is wrapped
with developed LMU (Fig. 4B). There is a tracheole (TR) distributed
between the PM and the LMU (Fig. 4B), and the LMU are found
adjacent to the CMU (Fig. 4A). The basement membrane has chan-
nels formed by IF (Fig. 4A), and these are network-like and highly
developed; in addition, there are a large number of mitochondria dis-
tributed therein (Fig. 4A). Many well-developed MV extend into the
lumen of the midgut (Fig. 4A, C, and D), and these MV are generally
covered with a perimicrovillar membrane (PMM) (Fig. 4A and C).
Moreover, many mitochondria were observed near the developing
Fig. 2.  Semi-thin section of the digestive tract of H. lamellatus. BM, basement MV (Fig. 4D). SVs in the cytoplasm (Fig. 4A) were found to differ in
membrane; CEC, columnar epithelial cell; CM, conical midgut; ML, midgut size and shape. We also found unknown inclusions present in the SVs
loop; N, nucleus. (Fig. 4C), and there were SDs between the cells (Fig. 4A). In addition,
4 Journal of Insect Science, 2019, Vol. 19, No. 4

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Fig. 4.  Transmission electron microscopy (TEM) photographs of the midgut
loop of H. lamellatus. (A) Midgut loop (partial) cross-section. (B) Longitudinal
muscle at the peritoneal membrane. (C) Lysosome in the cytoplasm. (D)
Microvilli at the lumen of the midgut. CMU, circular muscle; L, lumen;
LMU, longitudinal muscle; LY, lysosome; MIT, mitochondria; MV, microvilli;
PM, peritoneal membrane; PMM, perimicrovillar membrane; SD, septate
desmosomes; SV, secretory vesicle; TR, tracheole.

with low electron density were scattered around the nuclei (Fig. 6A).
Sparse MV were observed on the outside of the intima (Fig. 6D).
Fig. 3. Transmission electron microscopy (TEM) photographs of the filter
chamber and conical midgut of H.  lamellatus. (A) Cross-section of the filter Morphology of Salivary Glands
chamber (partial). (B) Secretory vesicles in the cells are amplified. (C) Basement
The salivary glands of H. lamellatus are located in the head cavity.
membrane infolding in the filter chamber cells. (D) Microvilli developing in
The salivary gland complex consists of pairs of principal glands
the basement membrane. (E) Cross-section of the conical segment (partial).
(F) Conical midgut cells are attached to a flame-like luminal membrane (black (PGs) and accessory glands (AGs) (Fig. 7).
arrow). CMU, circular muscle; FLM, flame-like luminal membrane; IF, infolding;
L, lumen; LMU, longitudinal muscles; MIT, mitochondria; MV, microvilli; N, Principal Gland
nucleus; PM, peritoneal membrane; RER, rough endoplasmic reticulum; SD,
The vesicular PG is a major part of the salivary glands (Fig. 7A).
septate desmosomes; SV, secretory vesicle.
The PG includes both an anterior lobe (AL) and posterior lobe (PL)
(Fig. 7B). The distal and proximal regions of the AL consist of small,
many lysosomes (LY) were observed in the cytoplasm of ML cells.
closely packed acini that have a smooth surface. In addition, we
These cells also show internal organelle fragments (Fig. 4C).
identified five larger acini in the middle region that are loosely ar-
ranged. The PL consists of about 10 acini that are divided into two
Ileum types: the first type is a group of about five relatively small acini
The nucleus of ileal cells is relatively large. N1 has a distinct hetero- that are closely arranged in a petal shape and have a rough surface.
chromatin, whereas N2 is not obvious (Fig. 5A). The ileum PM is The second type refers to about five larger acini present in a loose
thin, and is adjacent to the developed circular muscle (Fig. 5B and pattern around the periphery of the petal-like acinus (Fig. 7B). The
C). The cytoplasm of the cells of the ileum PM is filled with SVs acini of the PL are slightly larger than the acini of the middle region
and mitochondria that are circular, oval, or clavate. We also identi- of the AL. Finally, the PGs are connected via the lateral salivary
fied a developed RER around mitochondria (Fig. 5C). An elliptical ducts which converge to form the common salivary duct (Fig. 7B).
bacterial-like structure is present in the ileum cytoplasm of 50%
leafhopper individuals (Fig. 5C).
Accessory Glands
The AGs (Fig. 7A) found in the salivary gland complex were rod- or
Malpighian Tubule elliptically-shaped. These are connected to the PG by a short acces-
The cells of the MT contained many SVs (Fig. 6A) in which a large sory duct. AGs are simply constructed and have only one acinus,
number of brochosomes (BRs) were found. The cell showed the wide- which is similar in structure to the acini of the PL. AGs are free on
spread RER (Fig. 6B). Mitochondria were located near SVs (Fig. 6C). both sides of PG.
The outer wall of each BR was honeycomb-shaped. BRs were ob-
served: Somes had high central electron density and appeared dark
(Fig. 6C; white arrow); while other BRs were centrally transparent Ultrastructure of the PG
(BR2), embedded (BR1), or showed multiple small cavities (BR3) (Fig. The nucleus of secretory cells contained a variety of heterochromatin
6C). This may be mitochondria at different developmental stages. SVs (Fig. 8A), and was generally located in the basement membrane
Journal of Insect Science, 2019, Vol. 19, No. 4 5

Fig. 5.  Transmission electron microscopy (TEM) photographs of the ileum of H.  lamellatus. (A) Ileum cross-section. (B) A  section of panel A  is enlarged to
show an abundance of mitochondria. (C) Microorganisms (black arrows) are present in ileum cells. N1 and N2, nucleus; CMU, circular muscle; L, lumen; MIT,
mitochondria; RER, rough endoplasmic reticulum; SV, secretory vesicle.

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edge, and the core of SG2 is opaque and has no filaments (Fig. 9A).
We also identified dispersed SVs that have MV-like protrusions along
the inner edge of the core (Fig. 9B). In the salivary gland cells, sig-
nificant muscles were observed (Fig. 9C). We observed the presence
of rod-shaped bacteria-like structures in the cytoplasm of salivary
gland cells (Fig. 9D).

Discussion
The Ultrastructure and Function of the Tubular
Midgut in H. lamellatus
The ML of H. lamellatus is divided into two parts. In the front is
the cone, which is connected to the FC. The tubular midgut is also
connected to the FC, thus forming the ‘midgut ring’. The presence of
this structure is consistent with previous reports of other leafhoppers
(Tsai and Perrier 1996, Zhong et  al. 2014). Our results also con-
firmed that the epithelial cells of the tubular midgut of the digestive
tract of H.  lamellatus were present in the shape of a column, and
that the endoplasmic reticulum and SVs were abundant in the cells.
We found many well-organized and developed brush-like borders of
Fig. 6.  Transmission electron microscopy (TEM) photograph of a Malpighian MV, which have a specialized function during digestion (Silva et al.
tubule of H. lamellatus. (A) Cross-section of the cell tip region. (B) A developed 1995). As observed in the midguts of other leafhoppers (Zhong et al.
rough endoplasmic reticulum near the secretory vesicles. (C) A  section of
2014), these MV play an important role in the absorption of digested
panel A  is enlarged to show different forms of brochosomes. Also visible
nutrients including carbohydrates, amino acids, and water.
is an inclusion in the central cavity of BR1, the fact that the cavity of BR2
is transparent, and that the center of BR3 is a plurality of small cavities. The midguts of most insects have a peritrophic matrix, which is
Brochosomes in the opaque center (white arrow). (D) Sparse microvilli. BR, mainly used to protect midgut cells from pathogens and food par-
brochosomes; BV, brochosomes vesicles; MIT, mitochondria; MV, microvilli; ticles, and has a compartmentalization effect on the midgut (Terra
RER, rough endoplasmic reticulum; SV, secretory vesicle. 1990). However, there is no peritrophic membrane in the midgut of
Hemiptera insects. Instead, a layer of lipoprotein membranes covered
with MV tips is present, termed the PMM (Terra 1988). This mem-
brane is derived from the inner membrane of a double-membrane ves-
icle, which are produced by budding from the Golgi region (Werner
et al. 1991, Silva et al. 1995). Here, we identified an obvious PMM
in the tubular midgut of H. lamellatus. Its morphology is similar to
that found in the midgut cells of Dysdercus peruvianus (Hemiptera:
Pyrrhocoridae), Mahanarva posticata (Hemiptera: Cercopidae), and
Cicadella viridis (Hemiptera: Cicadellidae)  (Fonseca et  al. 2010,
Zhong et al. 2014). The PMM and MV form a closed space, i.e., the
perimicrovillar space that mediates the digestion and absorption of
Fig. 7.  Light microscopy photograph of the salivary glands of H. lamellatus.
nutrients in the midgut (Terra 1990).
(A) Overall morphology of the salivary gland. (B) Unilateral salivary gland
morphology. AD, accessory duct; AG, accessory gland; AL, anterior lobe; PD, Doi et al. (1967) first discovered phytoplasmas in plants infected
principal duct; PG, principal gland; PL, posterior lobe. with yellow-type diseases (e.g., aster yellows disease), and similar
pathogens were found to be present in the insect digestive tract (Doi
region, as were the TRs (Fig. 8B). Unknown inclusions were also et al. 1967, Maramorosch et al. 1968, Granados 1969). Later, Raine
present in the salivary duct (Fig. 8C, see asterisk [*]). The atrium is and Forbes (1969) also found that phytoplasmas were present in
lined with a cuticle, which is surrounded by IFs of the apical plasma salivary sheaths (i.e., in saliva) secreted by leafhoppers (Raine and
membrane (Fig. 8C). Forbes 1969). Wayadande et  al. (1997) showed that the pathogen
We observed the presence of many secretory granules (SGs) must pass through the basal layer, the basal plasma membrane,
within the cells (Fig. 9A). There are differences between the SGs, and the apical plasma membrane before they can be ejected with
SG1 has a transparent core and filaments are attached to its inner the saliva (Wayadande et al. 1997). In general, when phytoplasma
6 Journal of Insect Science, 2019, Vol. 19, No. 4

Fig. 8.  Transmission electron microscopy (TEM) photograph of the principal gland of H. lamellatus. (A) Salivary gland cells are highly enlarged to show the
multicellular nucleus. (B) The tracheole, present near the basement membrane. (C) A cross-section of the salivary duct. AMP, apical plasma membrane; CU,
cuticle; MIT, mitochondria; N, nucleus; RER, rough endoplasmic reticulum; SD, salivary duct; SG, secretory granule; SV, secretory vesicle; TR, tracheole; *The
contents of the lumen.

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principal and AGs are significantly different (Ammar et  al. 1985).
Our observations revealed that the salivary glands of H. lamellatus,
including both the principal and AGs, are lightly cream-colored or
translucent. The former is bulky, has a more complex structure,
and consists of an aggregate body containing many acini. In con-
trast, the latter is a short rod-shaped tubular gland with a struc-
ture similar to that of salivary gland of H. sellatus (Sōgawa 1965).
However, the salivary glands of H. lamellatus are morphologically
distinct from those found in other leafhoppers, such as P.  striatus
(L.), where the AGs are more slender than those of H.  lamellatus
(Zhang et al. 2012). Moreover, the AGs of C. tenellus and D. maidis
are both relatively large (Wayadande et  al. 1997), and the PGs of
Nephotettix cincticeps (Hemiptera: Cicadellidae)  and Chlorita
flavescens (Hemiptera: Cicadellidae)  have a small number of acini
and a relatively simple structure (Sōgawa 1965)
We also observed that the PG is composed of two parts: an AL
and a PL. The AL is itself divided in two parts, i.e., the head and tail
portions. The ALs of H. sellatus are quite different from those found
in Graminella nigrifrons (Hemiptera: Cicadellidae)  or D.  maidis
(Sōgawa 1965, Tsai and Perrier 1993). Moreover, as mentioned by
Fig. 9. Transmission electron microscopy (TEM) photograph of the Sōgawa (1965), the size, number, and shape of the ALs vary from
principal gland of H. lamellatus. (A) Secretory granules are placed at higher species to species in the Cicadellidae. The PL consists of approxi-
magnification. (B) Secretory vesicles are placed at higher magnifications. (C)
mately 13 acini arranged in a multilayered petal-like shape similar
Muscle tissue in salivary glands. (D) Microorganisms (black arrows) in the
to the shape and structure of the principal salivary glands of the sub-
host cells. MU, muscle; RER, rough endoplasmic reticulum; SG1 and SG2,
secretory granule; SV, secretory vesicle. family Deltocephalinae (Sōgawa 1965). The principal salivary gland
is a complex gland that contains at least two secretory systems, one
that secretes precursors of the salivary sheath and another that pro-
circulate through the digestive tract and salivary glands of insects, duces water saliva that contains several enzymes (Sōgawa 1965). The
they must adhere to the intestinal epithelial cells of the insect vec- AGs secrete mucus and phenolic enzymes which, together with pro-
tors. Recently, several variable membrane proteins were  used by teins secreted by the principal salivary gland, constitute the salivary
the phytoplasma to bind to the PMM—which covers the MV of sheath (Miles 1964, 1965). In general, the salivary sheath of the leaf-
leafhopper gut cells have been identified. These membrane pro- hoppers contains lipids and neutral mucus substances (Sōgawa 1965,
teins may promote the degradation of PMM protein components, 1967). Saliva secreted by leafhoppers can damage host plants due
so that phytoplasma reaching the MV apical membrane region of to the presence of toxins and anticoagulants, and saliva containing
the midgut epithelium adhere and can colonize gut cells (Arricau- pathogenic microorganisms may transmit them during mouthpart
Bouvery et al. 2018). This binding transport mechanism was present penetration (Raine and Forbes 1969, Sauer 1977). A  variety of
in Trypanosoma cruzi (Trypanosomatina: Trypanosomatidae), whi plant pathogens have been reported in the salivary glands of the
ch adheres to the PMM of the Chagas disease vector bug Rhodnius leafhoppers (Ghanim and Medina 2007, Ammar et  al. 2009), and
prolixus (Hemiptera:  Reduviidae)  (Gutiérrez-Cabrera et  al. 2016). the phytoplasma associated with mulberry dwarf disease has been
Taken together, we believe that our data improve our understanding observed in the salivary glands of H.  sellatus and Hishimonoides
of the cellular and subcellular structure of the H.  lamellatus gut, sellatiformis (Hemiptera: Cicadellidae)  (Kawakita et  al. 2000). In
which is important because it is vital to improve our understanding addition, we used molecular data to show that the JWB phytoplasma
of the nature of pathogenic microbial interaction with the cells of was present in some leafhoppers (Hao et  al. 2015). Moreover, re-
insect vectors to mitigate the impact of plant diseases. cent studies of the relationship between microorganisms and their
insect vectors have suggested that the primary glands are beneficial
The Morphology and Function of Salivary Glands in to the survival of microorganisms, since they are very sensitive to
Leafhoppers nutritional quality in the intracellular environment (Crotti et  al.
The salivary glands of Cicadellidae insects are composed of the PG, 2009, 2010). Kwaik (1996) found that the vesicular RER and its SVs
the AG, and its salivary duct. Importantly, the morphology of the may provide a rich environment replete with essential nutrients for
Journal of Insect Science, 2019, Vol. 19, No. 4 7

microorganisms. This is thought to be due to the fact that there is a with mitochondria and openings of the basal lamina. In addition,
lot of RER, mitochondria, and numerous SGs within leafhopper PG the RER around the mitochondria is clearly visible, as is the apical
cells (Kwaik 1996). Here, we found rod-shaped or oval microorgan- membrane of the epithelial cells, which is tightly arranged into MV.
isms in the PGs of the leafhoppers, although these glands are known The mitochondria at the base of the MV of enterocytes provide a
to contain phytopathogens in other leafhopper species (Gonzalez large amount of energy for transport, and we also identified a large
2016). Although to date there has been no detailed investigation of number of SVs in the cells, which play an important role in the ab-
the saliva of H. lamellatus, our histological and ultrastructure results sorption, storage, and secretion of metabolites (Silva et  al. 2004).
suggest that the salivary glands of H. lamellatus play an important Thus, the absorption of nutrients and ions occurs in the CM and
role in the transmission of plant pathogens (Reis et al. 2003). the anterior midgut (Cheung and Marshall 1973). In addition, the
MV are covered with a filamentous membrane complex similar to
The Structure of the Hindgut of the Leafhopper and the FLM found in the CM of the leafhopper B.  xanthophis. This
Its Microorganisms membrane complex originates from intestinal epithelial cells, and

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does not secrete product directly. The MV tips of the enterocytes are
Ultrastructural observations showed that cells of the ileum (hindgut)
often constricted, thereby forming a membrane that projects into
of H. lamellatus possessed apical IFs associated with SVs as well as
the midgut lumen and remains associated with MV (Utiyama et al.
an abnormal abundance of mitochondria. These IFs were delicately
2016). However, unlike the PMM, they may be anchored during in-
formed by the invaginations of apical plasma membrane. Similar IFs
testinal digestion. Enzymes such as cathepsin and a-glucosidase pre-
have been described in the intestines of the leafhoppers P. striatus (L.)
vent excretion and binding to amino acids, thereby constricting and
and Cic. viridis (Zhang et al. 2012, Zhong et al. 2014), and the IFs
concentrating them at the absorption site and enhance to effect of
were found to increase the contact surface with food in the luminal
absorption (Cristofoletti et al. 2003). In the end, further studies are
space. In addition, we identified oval and short rod-shaped micro-
needed to explore the relationship between this FLM complex and
organisms embedded within the epithelial cells of the hindgut wall
intestinal microorganisms.
in H. lamellatus. These microorganisms are likely to be prokaryotic
microbes (i.e., bacteria), and resemble those found in the intestines
of other leafhopper vectors including Cic. viridis and A.  constricta Acknowledgments
(Van Duzee) (Gil-Fernandez and Black 1965, Zhong et al. 2014). The
We sincerely appreciate Prof. P. Cai (Soochow University, China) for his work
microbes present in insect intestines and gut cells are probably in-
identifying leafhopper species. We thank Dr. Zhang Qing and Yang Liu at
volved in digestion processes (Caetano et  al. 2009), and may par-
Beijing Key Laboratory of New Technology in Agricultural Application for
ticipate in many different metabolic pathways. Thus, the presence
their help with photomicrography. This research was supported by grants
of microorganisms is thought to be generally beneficial to host in- from the Beijing Municipal Natural Science Foundation, and the Beijing
sects (Ishikawa 2003). These microorganisms were observed to be Municipal Education Commission Science and Technology Plan Key Pro-
surrounded by the endoplasmic reticulum, mitochondria, and cyto- ject (KZ201810020026), the Beijing Municipal Natural Science Foundation
plasm of the host cells, which again is likely beneficial for leafhoppers (6182002), the National Key R&D Program of China (2017YFD020030703),
digestion and essential nutrient uptake (Ishikawa 2003, Salehi et al. the Beijing Municipal Science & Technology Commission (Z15100002115030-
2007). Because the leafhoppers feed on phloem sap, which contains 2), the National Natural Science Foundation of China (31272099, 31170602),
few amino acids, they are likely to rely on microbes to supply certain and the Beijing Agricultural Commissioner Project. We acknowledge TopEdit
LLC for linguistic editing and proofreading during the preparation of this
nutrients that are lacking in their food. However, more research is
manuscript.
needed to explore the identity and characteristics of these microbes.

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