Morphology and fine organization of the midgut of Gampsocleis gratiosa (Orthoptera: Tettigoniidae)
Morphology and fine organization of the midgut of Gampsocleis gratiosa (Orthoptera: Tettigoniidae)
Ke Li 0 1
Jin-Hui Zhang 0 1
Yu-Jing Yang 0 1
Wei Han 1
Huan Yin 0 1
0 College of Life Sciences, Shanxi Normal University , Linfen, Shanxi , China , 2 Modern College of Humanities and Sciences, Shanxi Normal University , Linfen, Shanxi , China
1 Editor: Christian Wegener , Biocenter, UniversitaE?t WuE?rzburg , GERMANY
The morphology and ultrastructure of the midgut of Gampsocleis gratiosa (Orthoptera: Tettigoniidae) was examined by light and electron microscopy. The midgut consists of two bulbous gastric caeca and a tubular ventriculus. The general organization of the gastric caeca is similar to that of the ventriculus. They are composed of a peritrophic membrane, an epithelium, a basal lamina and muscle layer from the inside to outside. Three types of cells were identified: regenerative, principal, and endocrine. Regenerative cells occur in groups (called nidi) at the base of principal cells. Principal cells grow from regenerative cells. Rare endocrine cells are scattered throughout the epithelium. Principal cells exhibit intense secretory activity, and regional differences in their ultrastructure were observed along the entire midgut. The microvilli are longer than those in any other region in the posterior region of the midgut. Lysosomes, multivesicular bodies (MVBs), autophagosomes, abundant Golgi apparatuses and lipid droplets primarily occur in the gastric caeca. Three pathways of secretion (merocrine, apocrine and holocrine) occur within the midgut epithelium, and a distinctive type of apocrine bleb was found in the gastric caeca. Therefore, these gastric caeca may be evolving toward a special type of gland.
Data Availability Statement; All relevant data are within the paper
Funding: The undergraduate innovative experiment
project of Shanxi Province of China (2015540)
provided the funding to support this research. The
funder had no role in study design, data collection
and analysis, decision to publish, or preparation of
Competing interests: The authors have declared
that no competing interests exist.
The entire alimentary canal of insects traverses the body from mouth to anus and is generally
separated into three major parts, the foregut, midgut, and hindgut, according to embryological
]. The origin of the foregut and hindgut is the ectoderm, whereas the midgut derives
from the endoderm. The foregut is primarily involved in receiving, transporting and initially
digesting food; the hindgut has important roles in many physical processes of insects, and the
midgut serves as the primary section for enzyme production and secretion, food digestion,
and nutrient absorption [
In most insects, the midgut consists of two parts, the gastric caeca and the ventriculus.
Gastric caeca are midgut projections that are an evolutionary consequence of the feeding habits of
insects. These projections vary among taxa in shape, number, size and site from which they
arise from the midgut [
]. In addition, midgut epithelial cells show regional ultrastructural
differentiation, a characteristic indicating that cells from various regions have different functions
The structure and morphology of the alimentary canal traces the evolution of the digestive
system in Orthoptera [8, 9], an order that is divided into two major suborders: Caelifera and
Ensifera. The latter includes the families Gryllidae and Tettigoniidae. The digestive system of
members of Caelifera is fairly well known [10?12], whereas relatively little information is
available regarding ultrastructural organization in Tettigoniidae. Therefore, we examined the
morphology and structure of the midgut of Gampsocleis gratiosa (Orthoptera: Tettigoniidae) using
microscopy techniques to extend our knowledge and to provide basic data on the functions
and evolution of the insect digestive system.
Materials and methods
Healthy G. gratiosa adults were obtained from the Huo Mountains in Linfen, Shanxi Province,
China. The insects were reared in the laboratory at room temperature (25?C) under a natural
photoperiod and fed carrots.
Twenty specimens were anaesthetized under light CO2, and the entire alimentary canals
were dissected in a physiological saline solution containing (in mM) 130 NaCl, 5 KCl, 2
MgCl2, and 2 CaCl2 at pH = 7.4. The morphology of the midgut was identified under a
stereomicroscope, and the midgut was sectioned into four parts: the gastric caeca, anterior
ventriculus, middle ventriculus and posterior ventriculus. For histological investigations, samples were
fixed in Bouin's liquid for 4 h at 4?C, dehydrated in a graded series of alcohol for 10 min each,
and embedded in paraffin. Sections (7 ?m thick) were stained with hematoxylin-eosin.
For the scanning electron microscope (SEM), samples were fixed in 2.5% glutaraldehyde
for 24 h at 4?C. After rinsing 3 times with phosphate-buffered saline (PBS), the samples were
dehydrated in a graded series of ethanol that transitioned to tertiary butyl alcohol, followed by
freeze-drying using a FD-1-50 vacuum freeze dryer (Boyikang Laboratory Instruments Co.,
Ltd, Beijing, China) at -20?C for 12 hours. Thereafter, the samples were mounted on stubs,
gold coated and observed under a JSM-7500F scanning electron microscope.
Preparation of samples for transmission electron microscopy (TEM) followed that of Li
(2016) . Ultrathin sections (60 nm) were cut by using a glass knife on Reichert and stained
with uranyl acetate and lead citrate, and photographed using an H-7650 transmission electron
No specific permits were required for the described field studies: a) no specific permissions
were required for these locations/activities; b) locations were not privately-owned or protected;
and c) the field studies did not involve endangered or protected species.
The alimentary canal of G. gratiosa is divided into the foregut, midgut, and hindgut (Fig 1).
The midgut consists of two large gastric caeca attached at the anterior end and a tubular
ventriculus (Fig 1). The distal ends of both bulbous gastric caeca are directed forward and embrace
the proventriculus (Fig 1). The dorsal and ventral caeca are similar in size at approximately 6
mm in length and 3.1 mm in width at the widest point. A tubular ventriculus (approx. 2.5 mm
in length) almost uniform in diameter (approx. 2 mm) extends to the hindgut.
The general organization of the gastric caeca and the ventriculus is similar. From the inner
to the outer layer, the peritrophic membrane, epithelium, basal lamina and muscle layer
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Fig 1. Anatomical view of the alimentary canal of G. gratiosa. Scale bar = 5 mm.
constitute the midgut (Fig 2A?2C). The peritrophic membrane was observed to envelop the
food bolus in the entire midgut lumen (Fig 2A and 2B), and a space was observed between the
peritrophic membrane and the epithelium (Fig 2A and 2B). Seven epithelial folds (Fig 2A) of
different lengths pointing toward the lumens of the gastric caeca were only found in the apical
region of the gastric caecum and not in other regions of the midgut.
The epithelium is composed of three cell types: regenerative cells, principal cells and
endocrine cells (Fig 2C). These cells adhere to the basal lamina (Fig 2C), which extends serially
from the base to the apex of the epithelium, with numerous nidi developed (Fig 2A?2C).
Regenerative cells occur in groups at the bottom of the nidi (Fig 2C and 2D) and are
characterized by a large nucleus with heterochromatin as well as scant cytoplasm and few organelles
(Fig 2C and 2D). Principal cells are continually replaced from regenerative cells. According to
the ultrastructural features of principal cells by TEM, these cells are involved in support,
secretion and absorption functions. The apical membrane of principal cells forms numerous
microvilli measuring approximately 4.8 ?m in length in the caecum, 6.2 ?m in the anterior
ventriculus, 10.5 ?m in the middle ventriculus, and approximately 12.6 ?m in the posterior
ventriculus (Fig 3). Many dark secretory granules can be observed among the microvilli (Fig
3). In the middle region of principal cells is an elliptical nucleus with heterochromatin and
euchromatin. Three regions of cytoplasm are found between the microvilli and nucleus. The
apical zone contains filaments (Fig 3A). The middle zone contains abundant polymorphous
mitochondria, a small amount of smooth endoplasmic reticulum (SER) and secretory vesicles
that vary in electron-density and size (Fig 3). Various-sized lysosomes, multivesicular bodies
(MVBs) and a few lipid droplets were primarily observed in this zone of the caeca (Figs 3A, 3B
and 4). Near the nucleus, the basal zone is richly supplied with rough endoplasmic reticulum
(RER) (Fig 5) occurring as groups of elongated cisternae usually arranged in parallel arrays or
in tight whorls. Golgi apparatuses are plentiful among the RER of the caeca and are composed
of a single flattened membrane-bounded cisterna and associated vesicles (Fig 5A). A few
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Fig 2. General organization of the midgut in G. gratiosa. (A) Longitudinal section of the gastric caeca. (B) Transverse section of the
ventriculus. (C) The region delimited by the rectangle in (B). (D) Regenerative cells under TEM. pm: peritrophic membrane; ep: epithelium; bl:
basal lamina; mu: muscle layer; ef: epithelial fold; rc: regenerative cell; pc: principal cell; ec: endocrine cell; lmu: longitudinal muscle; L: lumen.
Scale bars: (A) = 1 mm; (B) = 200 ?m; (C) = 20 ?m; (D) = 1 ?m.
autophagosomes were also observed in this zone (Fig 4D). Extensive infoldings formed by the
basal plasma membrane have an irregular branch-like structure (Fig 6), which together
constitute a basal labyrinth system of channels that could reach almost to the central region of the
cells (Fig 6D). Lipid droplets of variable size occur among the basal labyrinth and are
particularly abundant in that of the caeca (Fig 6A). Mitochondria are abundant and associated with
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Fig 3. The apical region of principal cells from four parts of the midgut of G. gratiosa, as observed by TEM. (A) The gastric caeca. (B) The
anterior ventriculus. (C) The middle ventriculus. (D) The posterior ventriculus. sv: secretory vesicle; mv: microvilli; sg: secretory granule; ld:
lipid droplet; fm: filaments; ls: lysosomes; m: mitochondria; SER: smooth endoplasmic reticulum. Scale bars: (A) = 2 ?m; (B) = 1 ?m; (C) =
2 ?m; (D) = 2 ?m.
PLOS ONE | https://doi.org/10.1371/journal.pone.0200405
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Fig 4. TEM photographs of various-sized lysosomes, multivesicular bodies (MVBs), autophagosomes and a few lipid droplets. (A) MVB
endocytosed by a lysosome. The inset shows the dilation of the MVB in the white rectangle. The MVBs contain dense material, similar to a
secretory vesicle. (B) Lipid droplets hydrolyzed by a lysosome. (C) The MVBs contain dense material, which appears identical to the contents
of secretory vesicles. (D) An autophagosome containing cytosolic components in the middle zone of a principal cell. ls: lysosome; MVB:
multivesicular body; ld: lipid droplet; m: mitochondria; g: Golgi apparatuses; li: lipid; au: autophagosome. Scale bars: (A) = 1 um; (A, inset) =
200 nm; (B-D) = 1 ?m.
basal infoldings in the basal area of the principal cells (Fig 6). The lateral continuous plasma
membranes of adjacent cells form desmosomes and septate junctions with deep
interdigitations (Fig 7A).
The basal lamina is an electron-lucent layer composed of banded fibrils (Fig 6). The muscle
layer consists of an inner substratum of circular muscle and an outer sheath of longitudinal
muscle (Fig 6C), which are embedded inside and out by the basal lamina. Tracheolar cells
were also observed here (Fig 6B). Tracheas ramify into the basal lamina and reach the
tracheolar cells, which form multiple tracheoles that are less than 1 ?m in diameter and penetrate each
cell of the body (Fig 7B and 7C).
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Fig 5. The middle zone of principal cells of the midgut of G. gratiosa, TEM. (A) The gastric caeca. (B) The ventriculus. n: nucleus; RER:
rough endoplasmic reticulum; g: Golgi apparatuses; v: vesicle; trs: tracheoles. Scale bars: (A) = 1 ?m and (B) = 500 nm.
Two secretory devices, merocrine and apocrine, were found in the apical membrane of
principal cells (Fig 8A?8D). The merocrine devices usually bud to form microvilli and release
secretions into the gut lumen (Fig 8C). The apocrine devices have two different shapes: (1) a
bulb-shaped bubble with a smooth surface (Fig 8A and 8B), which is the most common shape
among insects; and (2) a cylindrical-shaped apocrine bubble (Fig 8A) with the membrane
surface developing regular longitudinal ridges (Fig 8A), which primarily occurs in the portion
with the gastric caeca. However, secretion from both merocrine and bulb-shaped apocrine
devices was observed along the entire midgut. In addition, holocrine secretion was observed
by TEM in the gastric caeca of the midgut (Fig 8E).
Gastric caeca occurring in the midgut of many insects vary among taxa with regard to shape,
number, size and position [
]. In Orthoptera, the caeca usually originate at the anterior end of
the midgut. In Gryllidae, the two caeca are bulbous in shape [5, 14], whereas six bi-lobed
finger-shaped caeca occur in Caelifera [
]. The midgut of G. gratiosa consists of two bulbous
gastric caeca and a cylindrical ventriculus. The gastric caeca originate at the anterior end of the
ventriculus, similar to other members of Ensifera, e.g., Melanogryllus desertus (Orthoptera:
Gryllidae) , Mecopoda niponensis (Orthoptera: Mecopodidae) , and Gryllodes sigillatus
(Orthoptera: Gryllidae) . Epithelial folds can increase the area of the midgut and are
commonly observed in the gastric caeca. In G. gratiosa, epithelial folds were observed at the distal
end of both gastric caeca, similar to those in G. sigillatus  and M. niponensis . In
grasshoppers, the epithelium of the caeca is folded into longitudinal ridges [2, 10?12].
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Fig 6. The basal region of principal cells from four parts of the midgut of G. gratiosa, TEM. (A) The gastric caeca. (B) The anterior
ventriculus. (C) The middle ventriculus. (D) The posterior ventriculus. ld: lipid droplet; trs: tracheoles; bl: basal lamina; m: mitochondria; trc:
tracheolar cell; cmu: circular muscle; lmu: longitudinal muscle; tr: tracheas; n: nucleus. Scale bars: (A and D) = 3 ?m; (B) = 2 ?m; (C) = 1 ?m.
The epithelium of most insect midguts contains four cell types: principal, goblet,
regenerative and endocrine [
]. Goblet cells are only found in the midguts of some insects, e.g.,
Ephemeroptera and Lepidoptera . Three midgut epithelial cell types, principal, regenerative and
endocrine cells, were observed in G. gratiosa, similar to previous reports for other Orthoptera
members [5, 12, 14, 16]. Endocrine cells in insect midgut perform important roles by releasing
various peptides in many key physiological functions, and the kind and distribution of
regulatory peptides vary with different species . Thus, further research in this area would be
Principal cells exhibit the cellular structures of classic secretory cells: microvilli, large nuclei,
mitochondria, ribosomes, RER, Golgi complex, and vesicles . Differences in the regional
ultrastructure of principal cells were observed along the midgut of G. gratiosa, a phenomenon
also observed in other insects [12, 19, 20]. Regional differences in the principal cells of G.
gratiosa are highlighted by the length of microvilli and the number and distribution of lipid
droplets, lysosomes, MVBs, autophagosomes and cylindrical-shaped apocrine secretions.
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Fig 7. Cell junctions of principal cells and the distribution of tracheoles in the midgut of G. gratiosa, TEM. (A) Desmosomes and septate
junctions with deep interdigitations of an adjacent cell. (B) Tracheoles in the apical region of a principal cell. (C) Tracheoles in the middle
region of a principal cell. ds: desmosomes; sj: septate junction; m: mitochondria; trs: tracheoles; mv: microvilli; RER: rough endoplasmic
reticulum. Scale bars: (A) = 300 nm and (B and C) = 1 ?m.
Microvilli dramatically increase the apical membrane area of a cell for enzyme secretion
and absorption of digested products, and in G. gratiosa, longer microvilli were found in the
posterior midgut than in the other three regions of the midgut. Therefore, high rates of
absorption likely occur in the posterior midgut.
Lipids play an important role in insects for energy storage for survival, development and
subsequent reproduction [
]. Neutral lipids, digested and absorbed from food, consist
predominantly of triacylglycerols or cholesteryl esters, and the core of lipid droplets is surrounded
by a monolayer of phospholipids and associated proteins [21, 22]. Lipid droplets are present in
various cell types of all organisms, from prokaryotes to eukaryotes [16, 22, 23]. Our study
revealed the highest concentration of lipid droplets in the gastric caeca of G. gratiosa.
Therefore, gastric caeca are likely a vital part of the midgut involved in lipid accumulation, as
observed in Abracris flavolineata (Orthoptera: Acrididae) [
] and Brontocoris tabidus
(Heteroptera: Pentatomidae) .
The intracellular storage and use of lipids are critical for maintaining cellular energy
homeostasis. Lysosomes, a type of membrane-bound organelle , perform multiple
functions in addition to degradation, including energy metabolism, secretion and plasma
membrane repair . MVBs and autophagosomes are closely related to lysosomes in the cellular
energy balance. MVBs are a special type of late endosome that primarily separates and delivers
proteins to lysosomes for degradation (Fig 4A and 4C) [
]. The proteins separated and
delivered by MVBs vary for different cell types; for example, in mammotrophic
hormone-producing cells of the rat anterior pituitary gland, MVBs regulate the secretory process by
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Fig 8. Fine structure of the secretory devices of the midgut of G. gratiosa. (A) Secretory devices in the gastric caeca, SEM.
(B) Secretory devices in the ventriculus, SEM. (C) Merocrine secretion, TEM. (D) Apocrine secretion, TEM. (E) Holocrine
secretion, TEM. bsb: bulb-shaped bubble; csb: cylindrical-shaped bubble; sg: secretory granule; mv: microvilli; n: nucleus.
Scale bars: (A) = 2 ?m; (B) = 10 ?m; (C and D) = 1 ?m; (E) = 3 ?m.
overproducing secretory granules [
]. During autophagy, cellular components are degraded
in lysosomes (Fig 4D), assuring the removal of altered or dysfunctional proteins and organelles
]. Recent studies reveal that multiple mechanisms operate in the delivery of functional
proteins and organelles to lysosomes [
]. One such mechanism is fusion of autophagosomes with
MVBs to generate an amphisome, which fuses with the lysosome to degrade the material
contained within [
]. Additionally, autophagosomes have a key role in lipid metabolism by
shuttling lipid droplets to lysosomes (Fig 4B), where they are hydrolyzed into free fatty acids and
]. MVBs, lysosomes and autophagosomes and lipid droplets were found
together in the principal cells of the gastric caeca in G. gratiosa, similar to the mammotrophic
hormone-producing cells of rat . Therefore, we propose the existence of a regulatory
mechanism of lysosomes over secretory granules, with this mechanism being associated with
intracellular lipid metabolism in G. gratiosa.
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Principal cells, particularly those in the gastric caeca, show intense secretory activity, and
their cytoplasm contains abundant organelles related to secretion, such as RER, Golgi and
secretory vesicles . Three mechanisms release the products of secretion, most likely
including digestive enzymes and other proteins, into the midgut lumen [
]: (a) holocrine, (b)
merocrine and (c) apocrine. During holocrine secretion, all secretory vesicles and the cytoplasm are
released by rupture of the plasma membrane and cellular degradation. In merocrine secretion,
or exocytosis, the products in secretory vesicles reach the lumen via fusion of the limiting
membrane of a vesicle with the apical membrane, without cytoplasm loss. In contrast, the
mechanism for apocrine secretion involves loss of part of the apical cytoplasm as secretory
vesicles are released. In G. gratiosa, a distinctive type of apocrine bleb was found in the gastric
caeca (Fig 8A), with morphology that differed from that of the common apocrine bleb (Fig 8A
and 8B). Further studies are required to ascertain whether these two types of apocrine blebs
have different contents and functions.
In the present study, the morphology and structure of the midgut of G. gratiosa were
examined. The results showed that the midgut of G. gratiosa consists of two bulbous gastric caeca
and a tubular ventriculus. The organizational structure of these two components is similar.
Regional ultrastructural differences along the entire midgut were observed for principal cells,
such as in the gastric caeca, with abundant lysosomes, MVBs, autophagosomes, Golgi, lipid
droplets and a type of distinctive apocrine bleb. These structural differences suggest that the
function of the gastric caeca is more similar to that of a specialized gland. Therefore, we
suggest that the gastric caeca play an essential role in functions of secretion and energy storage,
whereas an absorptive function may be more important for the ventriculus.
Conceptualization: Huan Yin.
Data curation: Huan Yin.
Funding acquisition: Huan Yin.
Methodology: Ke Li, Huan Yin.
Project administration: Huan Yin.
Visualization: Jin-Hui Zhang.
Writing ? original draft: Ke Li.
Writing ? review & editing: Huan Yin.
Investigation: Ke Li, Jin-Hui Zhang, Yu-Jing Yang, Wei Han.
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