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: jinzhw9276@163.com

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

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2
(1965) observed the general structure of the digestive tract of Agallia
constricta Van Duzee  (Hemiptera: Cicadellidae) (Gil-Fernandez
and Black 1965). In addition, Lindsay and Marshall (1980) de-
scribed the morphology and ultrastructure of the Eurymela distincta
Signoret  (Hemiptera: Eurymelidae) filter chamber (Lindsay and
Marshall 1980), Cheung and Purcell (1993) revealed the ultrastruc-
ture of the digestive system of Euscelidius variegatus (Kirschbaum)
(Hemiptera: Cicadellidae) (Cheung and Purcell 1993), and Wayadande
et al. (1997) compared the general morphology of the digestive tracts
of Circulifer tenellus (Hemiptera: Cicadellidae) and Dalbulus maidis
(Hemiptera: Cicadellidae)  (Wayadande et  al. 1997). More recently,
Zhang et al. (2012) observed the ultrastructure of the digestive tract
of Psammotettix striatus (Linnaeus) (Hemiptera: Cicadellidae) (Zhang
et  al. 2012), and Utiyama et  al. (2016) studied similar features in
Bucephalogonia xanthophis (Hemiptera: Cicadellidae) (Utiyama et al.
2016). Taken together, the results of these analyses suggest that there
are important differences in the morphology and ultrastructure of
the digestive tracts and salivary glands of different leafhopper species
(Sōgawa 1965, Wayadande et al. 1997). Globally, there are 41 species of
Hishimonus, of which there are 17 Hishimonus in China (Li and Wang
2004). However, little information is currently available regarding the
structural characteristics of the digestive systems of Hishimonus in-
sects. Actually, there are few research on the histology and ultrastruc-
ture of the digestive tract and salivary glands of H. lamellatus.
Therefore, the purpose of this study was to characterize the
structure of the digestive system of H. lamellatus using optical and
transmission electron microscopy (TEM), and to understand the
ultrastructural characteristics of the digestive tract, the Malpighian
tubule (MT) system, and the salivary glands.
Materials and Methods
Leafhopper Rearing
Hishimonus lamellatus individuals were raised in the insect
breeding room of the Beijing Key Laboratory of New Technology
in Agricultural Application. Leafhoppers were originally collected
in Liucun, Changping District, Beijing in August 2010. A  labora-
tory population was established in the insect breeding room and
identified by Professor Cai Ping. The H. lamellatus population was
reared on jujube (Ziziphus jujuba Mill (Rhamnales: Rhamnaceae))
seedlings and placed in a 40-cm-high cylindrical transparent plastic
worm cage, which was sealed with 40 × 40 mesh gauze. The feeding
temperature was maintained at 25  ± 1°C, the relative humidity
ranged between 50 and 70%, and the photoperiod was 16 L:8 D
(Hao et al. 2015).
Sample Preparation for Light Microscopy
Hishimonus lamellatus samples were chilled at −20°C for 20  min
and then each sample was then placed on a grooved glass slide (SAIL
BRAND, 7103 Single Concave). Under a stereoscopic microscope
(Motic, K Series), the wings and feet of leafhoppers were removed with
forceps (Dumont, 0208-5-po), and the side line of the abdomen stalk
was cut with scissors. A drop of phosphate buffer solution was added
dropwise to this cut, and an anatomical needle was used to extract the
digestive tract from abdominal segments 1–7. The salivary glands, lo-
cated at the top of the head, were gently separated using forceps. The
salivary glands were dissected and stained with toluidine blue solution
and photographed using a stereomicroscope (Zeiss SteREO Discovery.
V20) (Gil-Fernandez and Black 1965, Sōgawa 1965). About 80 leaf-
hopper individuals were examined by light microscopy.
Journal of Insect Science, 2019, Vol. 19, No. 4
Semi-Thin Section Sample Preparation for Light
Microscopy
The selected adult H.  lamellatus individuals, anesthetized with
ether, were placed on single concave slides. We then added 2.5%
glutaraldehyde fixative and quickly dissected the digestive tract of the
leafhopper using tweezers. Next, the digestive tract was then placed
in a centrifuge tube containing 1.5 ml of glutaraldehyde fixative. The
digestive tract was fixed for 48  h in the dark at room temperature.
Next, fixed tracts were washed with phosphate-buffered saline (PBS)
for 3 h. Afterward, we added 1% osmium tetroxide to the digestive
tracts for 90 min before rinsing with phosphate buffer. Then, the sam-
ples were washed with PBS for 30 min. The samples were dehydrated
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in a series of ethanol concentrations of 30, 50, 70, 80, 90, and 100%
for 6 min, respectively, and again in that of 100% ethanol for 30 min.
Samples were then infiltrated with a 1:2 mixture of ethanol and epoxy
resin 618 for 2  h and pure epoxy resin 618 for 12  h. The samples
were transferred to plastic flat embedding mold (Electron Microscopy
Sciences) and polymerized for 24 h at 37°C, 12 h at 45°C, and 48 h
at 60°C. The embedded block was trimmed to the appropriate size
and the sample was cut into 2-μm-ultrathin sections with a glass knife
of Leica UC6 ultrathin slicer. The sections were placed in a saturated
solution of NaOH in absolute ethanol for 3 min. The samples were
infiltrated in a series of ethanol concentrations of 100, 95, 80, 70, 50,
and 35% for 5 min. Slides were immersed in a staining jar containing
2% hydrochloric acid for 5 min, and then in a staining jar containing
distilled water for 2 min. The samples were stained in Harris’s hema-
toxylin for 20 min, and washed in tap water. Slides were immersed in a
staining jar containing acid alcohol (0.5:99 v/v) differentiation solution
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-
hydrated in a series of ethanol concentrations of 80 and 95% for 1 min
and twice in 100% for 1 min. The samples were sealed with a drop
of neutral balsam, capped with a cover slip, and then kept in drying
oven. The treated samples then were observed with a Zeiss microscope
Imager A1 (Aparicio and Marsden 1969).
Sample Preparation for TEM
To prepare samples for analysis using TEM, we used the same
method as described in ‘Semi-Thin Section Sample Preparation for
Light Microscopy’. At least five embedded blocks were made for
each sample to be observed. The ultrathin resin sections (60  nm)
were cut with an ultramicrotome (Leica UC6) using a glass knife,
transferred to copper grids, and stained with 2% (w/v) uranyl
acetate for 15  min (in dark), and then washed in distilled water.
The sections were stained with lead citrate for 20  min, and then
washed in distilled water again. The sections were kept in a des-
iccator (Ghanim et  al. 2016, Ammar et  al. 2017). Then ultrathin
sections were examined at 80 kV using an Hitachi H-7500 trans-
mission electron microscope at the electron microscope laboratory
of the Institute of Food Science and Technology, Chinese Academy
of Agricultural Sciences. About 30 leafhopper individuals were
examined by TEM.
Results
Histology and Morphology Features of the
Digestive Tract
The foregut, midgut, and hindgut (Fig. 1) are the three major parts
of the digestive tract of adult H. lamellatus.
Journal of Insect Science, 2019, Vol. 19, No. 4
Foregut
The esophagus is a narrow, slender tube. The esophagus is approxi-
mately 50 μm in diameter and is translucent or pale white in color
(Fig. 1).
Filter Chamber
The filter chamber (FC) has a half-moon shape, a diameter of 300–
400 μm, and is light milky white in color. The FC is composed of the
anterior segment of the midgut, the posterior segment of the midgut,
the base of the Malpighian tube, and the basal hindgut. It is also con-
nected to the conical midgut (CM).
Midgut
The midgut is divided into a CM and a midgut loop (ML). The
spherical or long vesicular CM is about 1,200  μm long and
700  μm wide. The surface of the CM is translucent and some-
times contains white particulate matter. The apical of the CM,
which has a thick basement membrane, rapidly collapses into the
ML. The ML has a diameter of about 400–600  μm and is only
slightly smaller than the diameter of the rest of the midgut (Fig.
1). Its surface is rough and its color is yellowish. The basement
membrane is relatively thin, and the columnar epithelium cells
(CECs) found there are large and are located near the basement
membrane (Fig. 2).
Fig. 1.  Light micrograph of the alimentary canal of H. lamellatus. CM, conical
midgut; FC, filter chamber; IL, ileum; ML, midgut loop; MT, Malpighian
tubule; OE, esophagus; RE, rectum.
Fig. 2.  Semi-thin section of the digestive tract of H. lamellatus. BM, basement
membrane; CEC, columnar epithelial cell; CM, conical midgut; ML, midgut
loop; N, nucleus.
3
Malpighian tubule: Hishimonus lamellatus has four MTs that extend
out of the same side of the ileum as the FC. Each of the malpighian
tubes is divided into four parts. The first part is connected to the FC,
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
diameter (about 100  μm) and is thicker than the first segment. Its
surface is rough. The fourth part is a short translucent tube with a
smooth surface that is also not easily stained with toluidine blue. Its
diameter is about 50 μm, and it merges with the rectum to form a
complex (Fig. 1).
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Hindgut
The hindgut consists of an elongated tubular ileum (Fig. 1) and
an enlarged rectum (RE; see Fig. 1). The ileum is connected to the
rectum through four MTs (Fig. 1). Its surface is smooth, translucent,
and is not easily stained with toluidine blue; its diameter is approxi-
mately 100 microns. The ileal apical swells into the rectum, and the
end of the ileum sac is open to the anal tube.
Ultrastructure of the Digestive Tract
Filter Chamber and Conical Midgut
The FC has a thin membrane known as the peritoneal membrane
(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
covered in MV (Fig. 3).
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
MV (Fig. 4D). SVs in the cytoplasm (Fig. 4A) were found to differ in
size and shape. We also found unknown inclusions present in the SVs
(Fig. 4C), and there were SDs between the cells (Fig. 4A). In addition,
4
Fig. 3. Transmission electron microscopy (TEM) photographs of the filter
chamber and conical midgut of H.  lamellatus. (A) Cross-section of the filter
chamber (partial). (B) Secretory vesicles in the cells are amplified. (C) Basement
membrane infolding in the filter chamber cells. (D) Microvilli developing in
the basement membrane. (E) Cross-section of the conical segment (partial).
(F) Conical midgut cells are attached to a flame-like luminal membrane (black
arrow). CMU, circular muscle; FLM, flame-like luminal membrane; IF, infolding;
L, lumen; LMU, longitudinal muscles; MIT, mitochondria; MV, microvilli; N,
nucleus; PM, peritoneal membrane; RER, rough endoplasmic reticulum; SD,
septate desmosomes; SV, secretory vesicle.
many lysosomes (LY) were observed in the cytoplasm of ML cells.
These cells also show internal organelle fragments (Fig. 4C).
Ileum
The nucleus of ileal cells is relatively large. N1 has a distinct hetero-
chromatin, whereas N2 is not obvious (Fig. 5A). The ileum PM is
thin, and is adjacent to the developed circular muscle (Fig. 5B and
C). The cytoplasm of the cells of the ileum PM is filled with SVs
and mitochondria that are circular, oval, or clavate. We also identi-
fied a developed RER around mitochondria (Fig. 5C). An elliptical
bacterial-like structure is present in the ileum cytoplasm of 50%
leafhopper individuals (Fig. 5C).
Malpighian Tubule
The cells of the MT contained many SVs (Fig. 6A) in which a large
number of brochosomes (BRs) were found. The cell showed the wide-
spread RER (Fig. 6B). Mitochondria were located near SVs (Fig. 6C).
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
(BR2), embedded (BR1), or showed multiple small cavities (BR3) (Fig.
6C). This may be mitochondria at different developmental stages. SVs
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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).
Morphology of Salivary Glands
The salivary glands of H. lamellatus are located in the head cavity.
The salivary gland complex consists of pairs of principal glands
(PGs) and accessory glands (AGs) (Fig. 7).
Principal Gland
The vesicular PG is a major part of the salivary glands (Fig. 7A).
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,
closely packed acini that have a smooth surface. In addition, we
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
types: the first type is a group of about five relatively small acini
that are closely arranged in a petal shape and have a rough surface.
The second type refers to about five larger acini present in a loose
pattern around the periphery of the petal-like acinus (Fig. 7B). The
acini of the PL are slightly larger than the acini of the middle region
of the AL. Finally, the PGs are connected via the lateral salivary
ducts which converge to form the common salivary duct (Fig. 7B).
Accessory Glands
The AGs (Fig. 7A) found in the salivary gland complex were rod- or
elliptically-shaped. These are connected to the PG by a short acces-
sory duct. AGs are simply constructed and have only one acinus,
which is similar in structure to the acini of the PL. AGs are free on
both sides of PG.
Ultrastructure of the PG
The nucleus of secretory cells contained a variety of heterochromatin
(Fig. 8A), and was generally located in the basement membrane
Journal of Insect Science, 2019, Vol. 19, No. 4
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.
Fig. 6.  Transmission electron microscopy (TEM) photograph of a Malpighian
tubule of H. lamellatus. (A) Cross-section of the cell tip region. (B) A developed
rough endoplasmic reticulum near the secretory vesicles. (C) A  section of
panel A  is enlarged to show different forms of brochosomes. Also visible
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.
Brochosomes in the opaque center (white arrow). (D) Sparse microvilli. BR,
brochosomes; BV, brochosomes vesicles; MIT, mitochondria; MV, microvilli;
RER, rough endoplasmic reticulum; SV, secretory vesicle.
Fig. 7.  Light microscopy photograph of the salivary glands of H. lamellatus.
(A) Overall morphology of the salivary gland. (B) Unilateral salivary gland
morphology. AD, accessory duct; AG, accessory gland; AL, anterior lobe; PD,
principal duct; PG, principal gland; PL, posterior lobe.
region, as were the TRs (Fig. 8B). Unknown inclusions were also
present in the salivary duct (Fig. 8C, see asterisk [*]). The atrium is
lined with a cuticle, which is surrounded by IFs of the apical plasma
membrane (Fig. 8C).
We observed the presence of many secretory granules (SGs)
within the cells (Fig. 9A). There are differences between the SGs,
SG1 has a transparent core and filaments are attached to its inner
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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
MV, which have a specialized function during digestion (Silva et al.
1995). As observed in the midguts of other leafhoppers (Zhong et al.
2014), these MV play an important role in the absorption of digested
nutrients including carbohydrates, amino acids, and water.
The midguts of most insects have a peritrophic matrix, which is
mainly used to protect midgut cells from pathogens and food par-
ticles, and has a compartmentalization effect on the midgut (Terra
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
nutrients in the midgut (Terra 1990).
Doi et al. (1967) first discovered phytoplasmas in plants infected
with yellow-type diseases (e.g., aster yellows disease), and similar
pathogens were found to be present in the insect digestive tract (Doi
et al. 1967, Maramorosch et al. 1968, Granados 1969). Later, Raine
and Forbes (1969) also found that phytoplasmas were present in
salivary sheaths (i.e., in saliva) secreted by leafhoppers (Raine and
Forbes 1969). Wayadande et  al. (1997) showed that the pathogen
must pass through the basal layer, the basal plasma membrane,
and the apical plasma membrane before they can be ejected with
the saliva (Wayadande et al. 1997). In general, when phytoplasma
6
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.
Fig. 9. Transmission electron microscopy (TEM) photograph of the
principal gland of H. lamellatus. (A) Secretory granules are placed at higher
magnification. (B) Secretory vesicles are placed at higher magnifications. (C)
Muscle tissue in salivary glands. (D) Microorganisms (black arrows) in the
host cells. MU, muscle; RER, rough endoplasmic reticulum; SG1 and SG2,
secretory granule; SV, secretory vesicle.
circulate through the digestive tract and salivary glands of insects,
they must adhere to the intestinal epithelial cells of the insect vec-
tors. Recently, several variable membrane proteins were  used by
the phytoplasma to bind to the PMM—which covers the MV of
leafhopper gut cells have been identified. These membrane pro-
teins may promote the degradation of PMM protein components,
so that phytoplasma reaching the MV apical membrane region of
the midgut epithelium adhere and can colonize gut cells (Arricau-
Bouvery et al. 2018). This binding transport mechanism was present
in Trypanosoma cruzi (Trypanosomatina: Trypanosomatidae), whi
ch adheres to the PMM of the Chagas disease vector bug Rhodnius
prolixus (Hemiptera:  Reduviidae)  (Gutiérrez-Cabrera et  al. 2016).
Taken together, we believe that our data improve our understanding
of the cellular and subcellular structure of the H.  lamellatus gut,
which is important because it is vital to improve our understanding
of the nature of pathogenic microbial interaction with the cells of
insect vectors to mitigate the impact of plant diseases.
The Morphology and Function of Salivary Glands in
Leafhoppers
The salivary glands of Cicadellidae insects are composed of the PG,
the AG, and its salivary duct. Importantly, the morphology of the
Journal of Insect Science, 2019, Vol. 19, No. 4
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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
Sōgawa (1965), the size, number, and shape of the ALs vary from
species to species in the Cicadellidae. The PL consists of approxi-
mately 13 acini arranged in a multilayered petal-like shape similar
to the shape and structure of the principal salivary glands of the sub-
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-
duces water saliva that contains several enzymes (Sōgawa 1965). The
AGs secrete mucus and phenolic enzymes which, together with pro-
teins secreted by the principal salivary gland, constitute the salivary
sheath (Miles 1964, 1965). In general, the salivary sheath of the leaf-
hoppers contains lipids and neutral mucus substances (Sōgawa 1965,
1967). Saliva secreted by leafhoppers can damage host plants due
to the presence of toxins and anticoagulants, and saliva containing
pathogenic microorganisms may transmit them during mouthpart
penetration (Raine and Forbes 1969, Sauer 1977). A  variety of
plant pathogens have been reported in the salivary glands of the
leafhoppers (Ghanim and Medina 2007, Ammar et  al. 2009), and
the phytoplasma associated with mulberry dwarf disease has been
observed in the salivary glands of H.  sellatus and Hishimonoides
sellatiformis (Hemiptera: Cicadellidae)  (Kawakita et  al. 2000). In
addition, we used molecular data to show that the JWB phytoplasma
was present in some leafhoppers (Hao et  al. 2015). Moreover, re-
cent studies of the relationship between microorganisms and their
insect vectors have suggested that the primary glands are beneficial
to the survival of microorganisms, since they are very sensitive to
nutritional quality in the intracellular environment (Crotti et  al.
2009, 2010). Kwaik (1996) found that the vesicular RER and its SVs
may provide a rich environment replete with essential nutrients for
Journal of Insect Science, 2019, Vol. 19, No. 4
microorganisms. This is thought to be due to the fact that there is a
lot of RER, mitochondria, and numerous SGs within leafhopper PG
cells (Kwaik 1996). Here, we found rod-shaped or oval microorgan-
isms in the PGs of the leafhoppers, although these glands are known
to contain phytopathogens in other leafhopper species (Gonzalez
2016). Although to date there has been no detailed investigation of
the saliva of H. lamellatus, our histological and ultrastructure results
suggest that the salivary glands of H. lamellatus play an important
role in the transmission of plant pathogens (Reis et al. 2003).
The Structure of the Hindgut of the Leafhopper and
Its Microorganisms
Ultrastructural observations showed that cells of the ileum (hindgut)
of H. lamellatus possessed apical IFs associated with SVs as well as
an abnormal abundance of mitochondria. These IFs were delicately
formed by the invaginations of apical plasma membrane. Similar IFs
have been described in the intestines of the leafhoppers P. striatus (L.)
and Cic. viridis (Zhang et al. 2012, Zhong et al. 2014), and the IFs
were found to increase the contact surface with food in the luminal
space. In addition, we identified oval and short rod-shaped micro-
organisms embedded within the epithelial cells of the hindgut wall
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
(Van Duzee) (Gil-Fernandez and Black 1965, Zhong et al. 2014). The
microbes present in insect intestines and gut cells are probably in-
volved in digestion processes (Caetano et  al. 2009), and may par-
ticipate in many different metabolic pathways. Thus, the presence
of microorganisms is thought to be generally beneficial to host in-
sects (Ishikawa 2003). These microorganisms were observed to be
surrounded by the endoplasmic reticulum, mitochondria, and cyto-
plasm of the host cells, which again is likely beneficial for leafhoppers
digestion and essential nutrient uptake (Ishikawa 2003, Salehi et al.
2007). Because the leafhoppers feed on phloem sap, which contains
few amino acids, they are likely to rely on microbes to supply certain
nutrients that are lacking in their food. However, more research is
needed to explore the identity and characteristics of these microbes.
Structure and Function of FC and Cone Segment
Our results also showed that the muscles of the FC of H. lamellatus
were well developed. The MV of the epithelial cells of the FC were
tubular, uniform in length, and regularly arranged. Many insects in
the Cicadomorpha that feed on plant juices have developed LMU,
which power the contraction of the FC, allowing a large amount of
water to move directly from the anterior midgut into the rear end of
the midgut and the MT. This in turn concentrates sap in the xylem
and phloem before absorption, and the FC then acts as a waterway
(Cheung and Marshall 1973). Studies on the fine structure of the FC
in the leafhopper E. distincta showed that the cells in the anterior
midgut had a storage and secretion function, while the cells in the
posterior midgut had an ion-secretion function, and the cells in the
FC were similar to the cells in the anterior midgut. There is only
one type of enterocyte in the inner tubule, and this is related to the
passive diffusion and active transport of ions from the hemolymph
to the lumen; the inner ileum thereby facilitates the absorption of
water (Lindsay and Marshall 1980).
We observed that the anatomy of the CM of the H. lamellatus
is different from that of the leafhopper P.  striatus (L.) (Zhang
et al. 2012), which itself is similar to the gross morphology of the
xylem-feeding leafhopper Cic. viridis (Zhong et  al. 2014). The
basement membrane is deeply deepened and forms IFs associated
7
with mitochondria and openings of the basal lamina. In addition,
the RER around the mitochondria is clearly visible, as is the apical
membrane of the epithelial cells, which is tightly arranged into MV.
The mitochondria at the base of the MV of enterocytes provide a
large amount of energy for transport, and we also identified a large
number of SVs in the cells, which play an important role in the ab-
sorption, storage, and secretion of metabolites (Silva et  al. 2004).
Thus, the absorption of nutrients and ions occurs in the CM and
the anterior midgut (Cheung and Marshall 1973). In addition, the
MV are covered with a filamentous membrane complex similar to
the FLM found in the CM of the leafhopper B.  xanthophis. This
membrane complex originates from intestinal epithelial cells, and
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does not secrete product directly. The MV tips of the enterocytes are
often constricted, thereby forming a membrane that projects into
the midgut lumen and remains associated with MV (Utiyama et al.
2016). However, unlike the PMM, they may be anchored during in-
testinal digestion. Enzymes such as cathepsin and a-glucosidase pre-
vent excretion and binding to amino acids, thereby constricting and
concentrating them at the absorption site and enhance to effect of
absorption (Cristofoletti et al. 2003). In the end, further studies are
needed to explore the relationship between this FLM complex and
intestinal microorganisms.
Acknowledgments
We sincerely appreciate Prof. P. Cai (Soochow University, China) for his work
identifying leafhopper species. We thank Dr. Zhang Qing and Yang Liu at
Beijing Key Laboratory of New Technology in Agricultural Application for
their help with photomicrography. This research was supported by grants
from the Beijing Municipal Natural Science Foundation, and the Beijing
Municipal Education Commission Science and Technology Plan Key Pro-
ject (KZ201810020026), the Beijing Municipal Natural Science Foundation
(6182002), the National Key R&D Program of China (2017YFD020030703),
the Beijing Municipal Science & Technology Commission (Z15100002115030-
2), the National Natural Science Foundation of China (31272099, 31170602),
and the Beijing Agricultural Commissioner Project. We acknowledge TopEdit
LLC for linguistic editing and proofreading during the preparation of this
manuscript.
References Cited
Ammar, E. D., L. R. Nault, and J. G. Rodriguez. 1985. Internal morphology
and ultrastructure of leafhoppers and planthopper, pp. 127–162. In L. R.
Nault and J. G. Rodriguez (eds.), Leafhoppers & planthoppers. John Wiley
& Sons, New York.
Ammar, e. l.-.D., D. Gargani, J. M. Lett, and M. Peterschmitt. 2009. Large
accumulations of maize streak virus in the filter chamber and midgut
cells of the leafhopper vector Cicadulina mbila. Arch. Virol. 154:
255–262.
Ammar, E., R. G. Shatters, and D. G. Hall. 2011. Localization of Candidatus
Liberibacter asiaticus, associated with citrus huanglongbing disease, in its
psyllid vector using fluorescence in situ hybridization. J. Phytopathol. 159:
726–734.
Ammar, E. D., D. G. Hall, and R. G. Shatters, Jr. 2017. Ultrastructure of the
salivary glands, alimentary canal and bacteria-like organisms in the Asian
citrus psyllid, vector of citrus huanglongbing disease bacteria. J. Microsc.
Ultrastruct. 5: 9–20.
Aparicio,  S.  R., and P.  Marsden. 1969. Application of standard micro-
anatomical staining methods to epoxy resin-embedded sections. J. Clin.
Pathol. 22: 589–592.
Arricau-Bouvery, N., S. Duret, M. Dubrana, B. Batailler, D. Desqué, L. Beven,
J.  Danet, M.  Monticone, D.  Bosco, S.  Malembic-Maher, et  al. 2018.
Variable membrane protein a of flavescence dorée phytoplasma binds the
midgut perimicrovillar membrane of Euscelidius variegatus and promotes
adhesion to its epithelial cells. Appl. Environ. Mocrob. 84: e02487-17.
8
Bové,  J.  M.,  J. Renaudin,  C. Saillard,  X. Foissac, and M.  Garnier. 2003.
Spiroplasma citri, a plant pathogenic molligute: relationships with its two
hosts, the plant and the leafhopper vector. Annu. Rev. Phytopathol. 41:
483–500.
Caetano, F. H., M. L. Bution, and F. J. Zara. 2009. First report of endocytobionts
in the digestive tract of ponerine ants. Micron. 40: 194–197.
Cai, P., S. Y. Cui, and Z. L. Ge. 1995. A new species of Hishimonus injurious
to Ziziphus jujuba (Homoptera: Cicadellidae, Euscelidae). Acta Entomol.
Sin. 38: 217–219.
Cheung, W. W., and A. T. Marshall. 1973. Studies on water and ion transport
in homopteran insects: ultrastructure and cytochemistry of the cicadoid
and cercopoid midgut. Tissue Cell 5: 651–669.
Cheung,  W.  W.  K., and A.  H.  Purcell. 1993. Ultrastructure of the digestive
system of the Le Fhopper Euscelidius variegatus Kirshbaum (Homoptera:
Cicadellidae), with and without congenital bacterial infections. Int.
J. Insect Morphol. Embryol. 22: 49–61.
Cristofoletti, P. T., A. F. Ribeiro, C. Deraison, Y. Rahbé, and W. R. Terra. 2003.
Midgut adaptation and digestive enzyme distribution in a phloem feeding
insect, the pea aphid Acyrthosiphon pisum. J. Insect Physiol. 49: 11–24.
Crotti, E., C. Damiani, M. Pajoro, E. Gonella, A. Rizzi, I. Ricci, I. Negri, P.
Scuppa,  P. Rossi,  P. Ballarini, et  al. 2009. Asaia, a versatile acetic acid
bacterial symbiont, capable of cross-colonizing insects of phylogenetically
distant genera and orders. Environ. Microbiol. 11: 3252–3264.
Crotti,  E.,  A. Rizzi,  B. Chouaia,  I. Ricci,  G. Favia,  A. Alma,  L. Sacchi,  K.
Bourtzis, M. Mandrioli, A. Cherif, et al. 2010. Acetic acid bacteria, newly
emerging symbionts of insects. Appl. Environ. Microbiol. 76: 6963–6970.
Dietrich,  C.  H. 1997. The role of grasslands in the diversification of leaf-
hoppers (Homoptera: Cicadellidae): a phylogenetic perspective. In
C.  Warwick, (ed.), Fifteenth North American Prairie Conference, Bend,
Oregon. http://digicoll.library.wisc.edu/cgi-bin/EcoNatRes/EcoNatRes-
idx?id=EcoNatRes.NAPC15.
Doi,  Y., M.  Teranaka, K.  Yora, and H.  Asuyama. 1967. Mycoplasma- or
PLT group-like microorganisms found in the phloem elements of plants
infected with mulberry dwarf, potato witches’ broom, aster yellows, or
paulownia witches’ broom. Japan. J. Phytopathol. 33: 259–266.
Fonseca,  F.  V.,  J.  R. Silva,  R.  I. Samuels,  R.  A. DaMatta,  W.  R. Terra, and
C.  P.  Silva. 2010. Purification and partial characterization of a midgut
membrane-bound alpha-glucosidase from Quesada gigas (Hemiptera:
Cicadidae). Comp. Biochem. Physiol. B. Biochem. Mol. Biol. 155: 20–25.
Ghanim,  M., and V.  Medina. 2007. Localization of tomato yellow leaf curl
virus in its whitefly vector Bemisia tabaci. In H.  Czosnek (ed.), Tomato
yellow leaf curl virus disease. Springer, Dordrecht, the Netherlands.
Ghanim,  M.,  S. Fattah-Hosseini,  A. Levy, and M.  Cilia. 2016. Morphological
abnormalities and cell death in the Asian citrus psyllid (Diaphorina citri)
midgut associated with Candidatus Liberibacter asiaticus. Sci. Rep. 6: 33418.
Gil-Fernandez,  C., and L.  M.  Black. 1965. Some aspects of the internal
anatomy of the leafhopper Agallia constricta (Homoptera: Cicadellidae).
Ann. Entomol. Soc. Am. 58: 275–284.
Gonzalez,  J.  G. 2016. Interactions of maize bushy stunt phytoplasma with
the leafhopper vector, Dalbulus maidis (Delong and Wolcott) (Hemiptera:
Cicadellidae) and associated microbiota. University of São Paulo, Piracicaba.
Granados, R. R. 1969. Electron microscopy of plants and insect vectors in-
fected with the corn stunt disease agent. Contr. Boyce Thompson Inst. PI.
Res. 24: 173–188.
Gutiérrez-Cabrera,  A.  E., A.  Córdoba-Aguilar, E.  Zenteno, C.  Lowenberger,
and B. Espinoza. 2016. Origin, evolution and function of the hemipteran
perimicrovillar membrane with emphasis on Reduviidae that transmit
Chagas disease. Bull. Entomol. Res. 106: 279–291.
Hao,  S.  D., Y.  Q.  Chen, J.  Z.  Wang, H.  Wang, W.  Q.  Tao, Z.  Y.  Zhang,
X.  Y.  Shi, and S.  Zhou. 2015. Multiplex-PCR for identification of two
Hishimonus species (Hemiptera: Cicadellidae) in jujube orchards and de-
tection of jujube witches’ broom JWB phytoplasma in their bodies. Acta
Entomol. Sin. 58: 264–270.
Ishikawa, H. 2003. Insect symbiosis: an introduction, pp. 1–22. In K. Bourtzis
and T. A. Miller (eds.), Insect symbiosis. CRC Press, Boca Raton, FL.
Kawakita,  H.,  T. Saiki,  W. Wei,  W. Mitsuhashi,  K. Watanabe, and M.  Sato.
2000. Identification of mulberry dwarf phytoplasmas in the genital organs
Journal of Insect Science, 2019, Vol. 19, No. 4
and eggs of leafhopper Hishimonoides sellatiformis. Phytopathology 90:
909–914.
Kwaik,  Y.  A. 1996. The phagosome containing Legionella pneumophila
within the protozoan Hartmannella vermiformis is surrounded by
the rough endoplasmic reticulum. Appl. Environ. Microbiol. 62:
2022–2028.
La, Y. J., and K. S. Woo. 1980. Transmission of jujube witches’ broom myco-
plasma by the leaf hopper Hishimonus sellatus Uhler. J. Korean For. Soc.
48: 29–39.
Lee,  I.  M.,  R.  E. Davis, and D.  E.  Gundersen-Rindal. 2000. Phytoplasma:
phytopathogenic mollicutes. Annu. Rev. Microbiol. 54: 221–255.
Li, Z. Z., and L. M. Wang. 2004. Notes on Chinese species of Hishimonus with
descriptions of two new species (Homoptera, Cicadellidae, Euscelinae).
Acta Zootaxon. Sin. 29: 486–490.
Downloaded from https://academic.oup.com/jinsectscience/article-abstract/19/4/3/5527870 by guest on 09 July 2019
Li, H., R. H. Dai, and Z. Z. Li. 2012. DNA barcoding technique and its ap-
plication in Cicadellidae (Insecta, Hemiptera, Cicadelloidae) research. J.
Mount. Agricult. Biol. 31: 432–438.
Lindsay, K. L., and A. T. Marshall. 1980. Ultrastructure of the filter chamber
complex the alimentary canal of Eurymela distincta Signoret (Homoptera,
Eurymelidae). Int. J. Insect Morphol. Embryol. 9: 179–198.
Liu,  M.  J., J.  Zhao, and J.  Y.  Zhou. 2010. Jujube witches’ broom disease.
China Agriculture Press, Beijing, China.
Maramorosch,  K., E.  Shikata, and R.  R.  Granados. 1968. Structures resem-
bling mycoplasma in diseased plants and in insect vectors. Transact. NY
Acad. Sci. 30: 841–855.
Miles, P. W. 1964. Studies on the salivary physiology of plant bugs: the chem-
istry of formation of the sheath material. J. Insect Physiol. 10: 147–160.
Miles, P. W. 1965. Studies on the salivary physiology of plant-bugs: the sal-
ivary secretions of aphids. J. Insect Physiol. 11: 1261–1268.
Mitsuhashi, W., T. Saiki, W. Wei, H. Kawakita, and M. Sato. 2002. Two novel
strains of Wolbachia coexisting in both species of mulberry leafhoppers.
Insect Mol. Biol. 11: 577–584.
Nault, L. R., and E. D. Ammar. 1989. Leafhopper and planthopper transmis-
sion of plant viruses. Ann. Rev. Entomol. 34: 503–529.
Raine, J., and A. R. Forbes. 1969. Mycoplasma-like bodies in the saliva of the
leafhopper Macrosteles fascifrons (Stål) (Homoptera: Cicadellidae). Can.
J. Microbiol. 15: 1105–1107.
Reis, M. M., R. M. Meirelles, and M. J. Soares. 2003. Fine structure of the sal-
ivary glands of Triatoma infestans (Hemiptera: Reduviidae). Tissue Cell.
35: 393–400.
Ruan, Y. L., S. X. Chen, and D. D. Jin. 1985. Dynamics and chemical control
of the leafhopper vector: Nephotettix cincticeps, which spread rice virus
disease. Insect Knowledge 3: 54–57.
Salehi, M., K. Izadpanah, M. Siampour, A. Bagheri, and S. M. Faghihi. 2007.
Transmission of ‘Candidatus Phytoplasma aurantifolia’ to Bakraee (Citrus
reticulata hybrid) by feral Hishimonus phycitis leafhoppers in Iran. Plant
Dis. 91: 466.
Sauer, J. R. 1977. Acarine salivary glands–physiological relationships. J. Med.
Entomol. 14: 1–9.
Silva, C. P., A. F. Ribeiro, S. Gulbenkian, and W. R. Terra. 1995. Organization,
origin and function of the outer microvillar (perimicrovillar) membranes
of Dysdercus peruvianus (Hemiptera) midgut cells. J. Insect Physiol. 41:
1093–1103.
Silva, C. P., J. R. Silva, F. F. Vasconcelos, M. D. Petretski, R. A. Damatta, A. F.
Ribeiro, and W. R. Terra. 2004. Occurrence of midgut perimicrovillar
membranes in paraneopteran insect orders with comments on their
function and evolutionary significance. Arthropod Struct. Dev. 33:
139–148.
Sivamani,  E., H.  Huet, P.  Shen, C.  A.  Ong, A.  D.  Kochko, C.  Fauquet, and
R. N. Beachy. 1999. Rice plant (Oryza sativa L.) containing rice tungro
spherical virus (RTSV) coat protein transgenes are resistant to virus infec-
tion. Mol. Breed. 5: 177–185.
Sōgawa,  K. 1965. Studies on the salivary glands of rice plant leafhop-
pers. I.  Morphology and histology. Japan. Soc. Appl. Entomol. Zool. 9:
275–290.
Sōgawa, K. 1967. Chemical nature of the sheath materials secreted by leafhop-
pers (Homoptera). Appl. Entomol. Zoolog. 2: 13–21.
Journal of Insect Science, 2019, Vol. 19, No. 4
Sun, S. M., F. W. Zhang, and X. D. Tian. 1988. Studies on the biology and
control of Hishimonus sellatus Uhler: a vector of jujube witches’-broom
disease. Acta Phytophyl. Sin. 15: 173–177.
Terra, W. R. 1988. Physiology and biochemistry of insect digestion: an evolu-
tionary perspective. Braz. J. Med. Biol. Res. 21: 675–734.
Terra,  W.  R. 1990. Evolution of digestive systems of insects. Ann. Rev.
Entomol. 35: 181–200.
Tsai,  J.  H., and J.  L.  Perrier. 1996. Morphology of the digestive and re-
productive systems of Dalbulus maidis and Graminella nigrifrons
(Homoptera: Cicadellidae). Fla. Entomol. 79: 563–578.
Tsai,  J.  H., and J.  L.  Perrier. 1993. Morphology of the digestive and repro-
ductive systems of Peregrinus maidis (Homoptera: Delphacidae). Fla.
Entomol. 76: 428–436.
Utiyama,  A.  H., W.  R.  Terra, and A.  F.  Ribeiro. 2016. The digestive system
of the leafhopper Bucephalogonia xanthophis (Hemiptera, Cicadellidae):
the organization of the luminal membrane complex. J. Entomol. Res. 40:
339–346.
Weintraub,  P.  G., and L.  Beanland. 2006. Insect vectors of phytoplasmas.
Annu. Rev. Entomol. 51: 91–111.
9
Weintraub, P. G., and M. R. Wilson. 2010. Control of phytoplasma diseases and
vectors, pp. 233–249. In P. G. Weintraub and P. Jones (eds.), Phytoplasmas:
genomes, plant hosts and vectors. CABI, Wallingford, United Kingdom.
Weintraub, P. G., S. Pivonia, A. Rosner, and A. Gera. 2004. A new disease in
Limonium hybrids. II. Insect vectors. HortScience 39: 1060–61
Wayadande,  A.  C., G.  R.  Baker, and J.  Fletcher. 1997. Comparative ultra-
structure of the salivary glands of two phytopathogen vectors, the beet
leafhopper, Circulifer tenellus (Baker), and the corn leafhopper, Dalbulus
maidis DeLong and Wolcott (Homoptera: Cicadellidae). Int. J.  Insect
Morphol. Embryol. 26: 113–120.
Werner,  K., K.  Moutairou, and G.  Werner. 1991. Formation and structure
of the surface coat in the midgut of a waterstrider, Gerris najas Deg.
(Heteroptera: Gerridae). Int. J. Insect Morphol. Embryol. 20: 69–77.
Zhang, F. M., C. N. Zhang, W. Dai, and Y. L. Zhang. 2012. Morphology and
Downloaded from https://academic.oup.com/jinsectscience/article-abstract/19/4/3/5527870 by guest on 09 July 2019
histology of the digestive system of the vector leafhopper Psammotettix
striatus (L.) (Hemiptera: Cicadellidae). Micron 43: 725–738.
Zhong, H. Y., Y. L. Zhang, and C. Wei. 2014. Morphology of the alimentary
canal of the leafhopper Cicadella viridis (Hemiptera: Cicadellidae). Ann.
Entomol. Soc. Am. 108: 57–69.