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Peptidergic innervation and endocrine cells of insect midgut.

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Archives of Insect Biochemistry and Physiology 22:113-I 32 (1993)
Peptidergic Innervation and Endocrine Cells
of Insect Midgut
DuSan Zitnan, Ivo Sauman, and FrantiSek Sehnal
lnstitute of Ecobiology, Slovak Academy of Sciences, Bratislava (D.Z.), and lnstitute of
EntomoloRy, Czechoslovak Academy of Sciences, CesE Budtjovicc O.S., F.S.),
Czechoslovakia
Antibody against FMRFamide reacts with the stomatogastric innervation and
with the midgut endocrine cells in the representatives of most insect orders.
The innervation was not revealed in Homoptera, Heteroptera, and Hymenoptera, and the endocrine cells were not recognized i n aphids. Other insects
exhibited FMRF-amidepositive endocrine cells of both open and closed types.
The cells are mostly single, rarely grouped, and are distributed unequally in
different midgut regions; some of the cells project cytoplasmic extensions
indicative of a paracrine function. Investigations on Galleria revealed that the
gut innervation persists during midgut reconstruction in the course of metamorphosis. The endocrine cells are sloughed off into the new gut lumen, but
there they maintain their antigenic properties until a new population of
endocrine cells becomes detectable.
Antisera to most mammalian gastroenteropancreatic peptides react specifically
with the innervation and/or the endocrine cells of insect midgut; only antisera to
bombesin, neurotensin, secretin, motilin, and insulin failed to react. All insects
seem to contain antigens that can be detected with antisera to pancreatic
polypeptide, FMRFamide, enkephalins, and vasopressins. Stomatogastric innervation and the endocrine cells of some lepidopterans also possess allatotropinand diuretic hormone-like antigens; stomatogastric ganglia, in particular, a
prothoracicotropic hormone-like antigen. 01993WiIey-~iss,Inc.
Key words: neuropeptides, gut hormones, stomatogastric nervous system, prothoracicotropic hormone, allatotropin, diuretic hormone
Acknowledgments: We express gratitude for the gifts of antisera (Table 2) to Sandoz Crop
Protection (Palo Alto, CA), Dr. H. lshizaki (Nagoya University, Nagoya, Japan), Dr. R. Metz
(Leuven University, Leuven, Belgium), Dr. V. HoiejSi (Institute of Molecular Genetics, Czechoslovak Academy of Sciences, Prague), Dr. M. Nishimura (Shinogi Co., Tokyo, Japan), Dr. N.
Yanaihara (Shizuda College, Shizuda, Japan),Dr. H. Vaudry (Rouen University, Rouen, France),
and Dr. C.J.P. Grimmelikhuijzen (Hamburg University, Hamburg, Germany). Critical reading
of the manuscript by Dr. N.A. Granger of the Universityof North Carolina, Chapel Hill is much
appreciated.
Received December 16,1991; accepted June20,1992.
Address reprint requests to FrantiSek Sehnal, Entomologicallnstitute CSAV, BraniSovska31,370 05
CeskC Bud&jovice,Czechoslovakia.
0 1993 Wiley-Liss, Inc.
114
Zittian et al.
INTRODUCTION
The current boom of research on insect peptidic hormones largely concerns
the neurohormones produced in the central nervous system (CNS) and
adjacent corpora cardiaca. Other sources of peptidic hormones include the
autonomous nervous system and the midgut, as indicated by the finding of
peptidergic neurons in the stomatogastric ganglia [1,2] and of cells with
peptidergic granules in the wall of the midgut [3-51. The stomatogastric
nervous system is diversified within the class of Insecta [ 6 ] ,and it is not clear
whether the occurrence of peptidergic neurons in this system is a general
phenomenon. The midgut endocrine cells have been examined in a relatively
small number of insects and found to contain, similar to the vertebrates, two
types of cells. The apical end of the so-called open type endocrine cells extends
into the gut lumen, whereas cells of the closed type do not have direct contact
with the lumen [7]. The primary aim of our study is (1) to verify whether
visceral innervation and both types of endocrine cells occur in all major insect
taxa, and (2) to determine whether they persist when larval midgut degenerates and a new midgut is formed in the course of metamorphosis,
Numerous immunohistochemical studies support the notion that the peptides of insect stomatogastric nervous system and the midgut endocrine cells
are similar to the ”brain-gut hormones” of the vertebrates [8,9]. In the present
study we review and complement available data and compare insects with
mammals in respect to the localization of comparable peptides in the visceral
nerves vs. the endocrine cells. In addition, since most of the gastroenteropancreatic peptidic hormones also occur in the CNS [8], the digestive tract
of insects has been examined for neurohormones hitherto believed to be found
only in the insect brain. The neurohormones in question include silkworm
bombyxin and PTTH* [ l O , l l ] and tobacco hornworm ATH and DH [12,13].
MATERIALS AND METHODS
Insects and Tissue Preparation
Insect species representing different orders were either collected in the field
or came from laboratory colonies (Table 1). Digestive tracts of adults were
examined in Apterygota and Polyneoptera, and of both larvae and adults in
the remaining insects. At least five specimens were used in each species. The
fate of the gut innervation and of the endocrine cells during metamorphosis
was investigated in the waxmoth, Galleria mellonella, in which the gut is totally
reconstructed between larval and adult stages [14,15]. Gut was examined in
6-12 h intervals between the start of cocoon spinning (day 6 of the last larval
instar) and the pupal ecdysis (day 7.5)’ in 24 h intervals throughout the pupal
instar (total length 7 days), and in freshly ecdysed adults.
Insects selected as midgut donors were immobilized either by carbon
dioxide treatment, by submersion in water, and/or by exposure to 4°C. The
midgut was dissected in PBS (0.16 M NaCl with 0.02 M phosphate buffer, pH
*Abbreviations used: ATH = allatotropic hormone; BrdU = bromdeoxyuridine; CRF = corticotropin
releasing factor; DH = diuretic hormones; HRP = horseradish peroxidase; PBS = phosphatebuffered saline; PP = pancreatic polypeptide; PTTH = prothoracicotropic hormone.
insect Midgut Endocrines
1 15
TABLE 1. Taxonomic Affiliation and Sources of Used Insect Species
Apterygota
Archaeognatha: Lepismachilis notataa
Zygentoma: Thermobiu domesticad and Lepisma saccharin&
Palaeoptera
Ephemeroptera: Cleon ~ p . ~
Odonata: Agrion s p . a (Zygoptera); Sympetrum sp." (Anisoptera)
Polyneoptera
Blattodea: Nuuphoeta cinered and Blabera craniiferd
Mantodea: Mantis religiosaa
Orthoptera-Ensifera: Stenopelmatus uscu6 (Gryllacridoidea);Scudderiujurcutub
(Tettigonioidea);Gryllus bimaculatus (Grylloidea)
Orthoptera-Caelifera: Melanopus biuita tusb and Dissosteira carolinub(Acridoidea)
Phasmatodea: Carausius morosusd
Embioptera: Hoembia ~ p . ~
Paraneoptera
Homoptera: Tibicen canicularia (Cicadoidea);Aphis sp. '(Afhidoidea)
Heteroptera: Pyrrhocoris apterusdand Triatoma sanguisuga
Oligoneoptera
Neuroptera: Chrysopa sp. ';Asculuphus sp.'
Coleoptera: Curubus sp." (Adephaga); Tenebrio molitord (Polyphaga)
Diptera: Tipula sp." (Nematocera).Dosophila melanogastetl' and Calliphora vicinad(Cyclorrhapha)
Lepidoptera: Galleria rnellonellad; Bombyx morid; Lymantria dispar"; Manduca sextad
Hymenoptera: Bomhus s p .' (Aculeata)
f
"Collected around Bratislava, Czechoslovakia;bcollectedaround Burlington, Vermont; 'collected
in Irvine, California; %om laboratory cultures.
7.6), rapidly transferred into a fixative, and left overnight at 4°C. Buffered 4%
or 8%formaldehyde, and occasionallyBouin solution (forvery soft guts), were
used for the whole mounts. The guts designated for sectioning were fixed in
Bouin solutions, dehydrated through an ethanol series and chloroform,
embedded in paraplast, and cut at 7-10 pm.
Immunohistochemistry
Normal goat and horse sera were purchased from Dakopatts (Glostrup,
Denmark) and Vector Laboratories (Burlingame, CA), respectively. Primary
antisera and their suppliers are listed in Table 2. All sera were diluted with
PBS containing 0.05% sodium azide and 0.1% Triton X-100; this solution was
also used for washing fixed tissues and their sections. Biotin-streptavidin-HRP
immunostaining kit (Amersham, Little Chalfont, UK) or the ABC Vectastain
kit (Vector Laboratories) were used routinely to reveal primary antisera.
Activity of most primary antisera against the vertebrate-type peptides was
checked in sections of murine gastroenteropancreatic system. The specificity
of antisera against PTTH, bombyxin, ATH, and DH was verified by immunostaining the nervous system of Galleria mellonella and Manduca sexfa, and
by using antisera saturated with respective antigens.
Deparaffinnized and hydrated sections were treated with 0.1 % hydrogen
peroxide in PBS to block endogenous peroxidase activity and preincubated in
5%normal goat or horse serum for 15min to prevent nonspecific IgG binding.
Unless stated otherwise, the sections were then incubated at room temperature in the following sequence: ( 1)primary antibody overnight at P C , (2) PBS
Twes of antibodies: Gp
=
Guinea pig; R = rabbit; M
=
R(143II)
R
R
R
R
R
R( 117III)
R(1611)
R
R
R
M
M
M(IN-03)
Gp(0002)
R
R
R
R
Gp(1305)
R
R
Type
1:1,000
1:1,000
1:1,000
1:1,000
1:330
1:500
1:500
1:1,000
1:500
1:500
1:500
1:500
1:500
1:500
1:1,000
1:1,000
1:1,000
1:1,000
1:1,000
1:1,000
1:1,000
1:1,000
Dilution
Supplier
Sandoz Crop Protection
Sandoz Crop Protection
Dr. H. Ishizaki
Dr. H. Ishizaki
Dr. V. HoiejSi
Dr. M. Nishimura
Dr. R. Metz
INCSTAR
Dr. N. Yanaihara
Dr. N. Yanaihara
Dr. N. Yanaihara
INCSTAR
INCSTAR
INCSTAR
Dr. R. Metz
Dr. H. Vaudry
Dr. H. Vaudry
Dr. H. Vaudry
Dr. H. Vaudry
Dr. C.J.P. Grimmelikhuijzen
Dr.C.J.P. Grimmelikhuijzen
Dr. C.J.P.Grimmelikhuijzen
mouse, monoclonal; lot identifications are given in parentheses.
Manduca allatotropin
Manduca diuretic hormone
Bombyx PTTH (N-terminal pentadecapeptide),0.5 mg/ml
Bombyx bombyxin I1 (N-terminal decapeptide), 0.5 mg/ml
Insulin, 0.1 mg/ml
Insulin
Glucagon
Vasoactive intestinal peptide (VIP)
Peptide histidine isoleucine (PHI)
Motilin
Gastrin
Cholecystokinin 8
Bombesin
Somatostatin
Neurotensin
a-Melanostimulating hormone (aMSH)
@-Endorphin
Met-enkep halin
Leu-enkephalin
FMRFamide
Bovine pancreatic polypeptide (C-terminal hexapeptide amide)
Arginine vasopressin
Antigen
TABLE 2. List of Prirnarv Antibodies*
Insect Midgut Endocrines
117
wash, (3) secondary biotin-coupled antibody (diluted 1:200), 1 h, (4)PBS
wash, (5) biotin-streptavidin-HRP (1:200) or avidin-HRP (1:200), 1 h, (6)
washing in 0.1 M Tris-HC1, pH 7.6. With the guinea pig primary antisera,
goat antiguinea pig IgG conjugated with HRP (Nordic, Tilburg, The Netherlands) was used as secondary antibody (step 3), followed by washing in
Tris-HC1buffer. Peroxidase activity was revealed by incubating the sections
in 0.01% diaminobenzidine (Sigma, St. Louis, MO) and 0.0003% hydrogen
peroxide in 50 mM Tris-HC1buffer, pH 7.6. The sections were then slightly
counterstained with diluted Mayer's hernatoxylin for 1-2 min, washed in
distilled water, and mounted in glycerin-gelatin (30% and 7% in water).
For the whole mounts, the fixed guts were cut laterally and cleansed of
contents, thoroughly washed in PBS, incubated for 1-2 h in 5% normal goat
or horse serum, and immunostained in the following sequence: (1)primary
antiserum against FMRFamide for 48 h at 4"C with occasional shaking, (2)four
or more PBS washes, 15 min each, (3) secondary biotinylated antiserum
(1:200), overnight at 4"C, (4)four washes in PBS, 15 min each, ( 5 )avidin-HRP
(1:200) or biotin-streptavidin-HRP (1:ZOO) for 4-6 h, (6) three washes in PBS,
and one in 0.1 M Tris-HC1 (pH 7.6), 15 min each. Peroxidase activity was
detected as described above. Guts were washed in PBS-glycerol (l:l),spread
on a slide, and mounted in glycerin-gelatin.
BromdeoxyuridineLabelling and Double Immunohistochemical Staining
BrdU labelling was used to mark cells synthesizing DNA in preparation for
the cell division. Postfeeding waxmoth larvae were water-anaesthetized and
injected (0.5 pl per 50 mg body weight) with 0.33% BrdU (Sigma) in EphrussiBeadle s a h e (prepared freshly from a stock solution of 3.3% BrdU in 30%
ethanol).The midgut was dissected after 2 h, fixed in Bouin's solution, embedded
in paraplast, and cut at 7 pm. Rehydrated sections were pretreated for 30 min
with 2 N HCl in PBS to denature DNA, and after treatmentswith 0.1% hydrogen
peroxide and 10% goat serum, they were double-immunostained according to
the followingprotocol: (1)a mixture of monoclonal anti-BrdU IgG antibody (1:30),
and the rabbit anti-FMRFamide antiserum (1:1,000), overnight at 4"C, (2) three
washes in PBS, 2 min each, (3) a mixture of goat antimouse IgG conjugated to
HRP (1:200), and goat antirabbit IgG conjugated to alkaline phosphatase (1:200),
1h (antibodies from Vector Laboratories), (4)two washes in PBS, one wash in
0.1 M Tris-HC1 (pH 7.6), 2 min each, (5) detection of peroxidase activity as
described above, (6) wash in 0.1 M Tris-HC1, pH 8.5, 5 min, (7) detection of
alkaline phosphatase activity with a mixture containing 15mg naphthol-AS-MXphosphate in 1rnl N,N-dimethylformamide, 50 mg fast blue BB salt, and 24 mg
Levamisole in 70 ml 0.1 M TRIS-HC1buffer, pH 8.5, (8) washing in water and
mounting in glycerol-gelatin.
RESULTS
Innervation and Endocrine Cells in the Midgut of Different Insects
The antiserum against FMRFamide was used to compare the morphology
of midgut innervation and the occurrence of midgut endocrine cells in species
118
Zitnan et al.
listed in Table 1. In the following description, the results are related to insect
subclasses and orders, to which the examined species belong. The data on
innervation concern only that part of the stomatogastric nervous system
which is posterior to the oesophageal nerve.
In Apterygota, either a nonbranching (Archaeognatha) or branching (Zygentoma) oesophageal nerve runs along the dorsal foregut surface to a single
ingluvial ganglion at the foregut/midgut junction. The ganglion contains 4-15
immunopositive cells and sends off either one dorsal and one ventral (Archaeognatha), or several (Zygentoma) nerves that arborize over the midgut
surface into a plexus. Characteristic features of the midgut endocrine cells
include their exclusive localization in the center or close to the center of
regenerative nidi and their occasional arrangement in groups of 2-5. The
morphology and the grouping of cells vary in differentzones of the midgut.
For example, in Lepismachilis, the foremost part of the midgut is devoid of cells
reactive with FMRFamide antibody, whereas a zone comprising about fourfifths of the gut length contains closed-type cells of amoeboid shape; the last
fifth of the midgut accommodates mostly open-type cells, along with small,
round cells of the closed type.
In our study of Palaeoptera, the representatives of mayflies (Ephemeroptera) exhibited very weak immunoreactivity that was detectable only in
the varicosities of gastric nerves. Strongly reacting endocrine cells were seen
at the very beginning of the midgut (mostly closed-type cells) and in the
middle third of its length (mostly long cells of the open type), whereas other
midgut regions seemed devoid of the FMRFarnide-positive endocrine cells. In
contrast, the antibody against FMRFamide revealed a distinct midgut innervation and a widespread occurrence of the endocrine cells in the damseflies
and dragonflies (Odonata). A branching oesophageal nerve terminates in the
ingluvial ganglion that is connected to a pair of proventricular ganglia, which
supply the midgut with a meshwork of anastomosing nerves. The endocrine
cells are singly scattered and of both the open and closed types; the closed
cells often possess cytoplasmic projections and are presumably paracrine. The
density of endocrine cells is highest in the central and in the posterior portions
of the midgut.
Immunoreactivity to FMRFamide antibody was very good in all representatives of Polyneoptera. In the cockroaches (Blattodea), the oesophageal
nerve, which lacks immunoreactive neurons, splits at the frontal crop region
into a dorsal and a ventral ingluvial nerve. The branching point, which was
regarded as a rudimental ingluvial ganglion [6], consists of 1 4 immunoreactive perikarya that are also persent in large numbers along the ingluvial nerves
(Fig. 1A). These send fine branches over the crop and at its posterior margin
they ramify into a number of gastric nerves, providing the meshwork innervation of the midgut (Fig. 1B). The endocrine cells are distributed singly
throughout the midgut. In contrast to some other insects, they lack the
cytoplasmic processes.
In the praying mantis (Mantodea), the walking stick (Phasmodea), and the
embiens (Embioptera), a single dorsal oesophageal nerve with fine side
branches terminates in a distinct ingluvial ganglion containing immunoreactive perikarya. In the walking stick, the perikarya also occur in the vicinity of
Insect Midgut Endocrines
1 19
Fig. 1. Gut innervation and midgut endocrine cells revealed with FMRFamide antibody in
Polyneoptera and Paraneoptera. A: Perikarya (arrows) in the oesophageal nerve (ON) and the
ingluvial nerves (IN)of Nauphoeta. B: Anastomosinggastric nerves on the anterior region of midgut
in Blabera. C: Proventricular ganglion (PG) in Hoernbia. D: A neuron (N) on midgut surface, and
a closed endocrine cell (EC) with fine paracrine processes in Carausius. E: Clusters ( C ) of closed
endocrine cells in midgut nidi, and apparently holocrine immunoreactive secretion (arrows) in the
gut lumen of Gryllus. F: Dominating open-type endocrine cells with a long apical extension in the
anterior region of midgut in Dysdercus. A-D, F, whole mounts, E, section. Bars = 50 prn.
the ingluvial ganglion on the foregut surface, as well as along the gastric
nerves in the proximal midgut region (Fig. 1D). Gastric nerves emanate from
the ingluvial ganglion in the praying mantis and the walking stick, whereas
embiens possess a pair of proventricular ganglia; the gastric nerves start there
120
iitnan et aI.
(Fig. IC). A plexus of anastomosing gastric nerves on the midgut surface is
very obvious. At least some of the midgut endocrine cells are of the closed
type and have an amoeboid shape with cytoplasmic projections; such cells
occur only in the caeca in the praying mantis but in different midgut regions
in Phasmodea and Embioptera. In the walking stick, the endocrine cells of
both closed and open types are of asteroid shape and their paracrine projections point in all directions (Fig. 1D). Although the females of Embioptera
contain both closed and open endocrine cells, only the closed cells are found
in the males.
Paired oesophageal nerves are a common feature of Orthoptera. The two
branches of the nerve begin laterally in the hypocerebral ganglion and run in
a spiral fashion around the crop to a dorsal and a ventral ingluvial ganglion.
In the suborder Ensifera, each of these ganglia contains about 15immunopositive cells and sends off one ingluvial nerve supplying the crop and one caecal
nerve that arborizes over the surface of the two caeca. The caecal nerve splits
into single or paired dorsal and ventral gastric nerves. By contrast, each
ingluvial ganglion of locusts (suborder Caelifera), includes only 2-3 immunopositive cells, whereas additional cells lay along several ingluvial and
two caecal nerves that extend from each ganglion. The caecal nerves split into
numerous gastric nerves that anastomose over the midgut surface. The
distribution and shape of the endocrine cells of both open and closed types
vary along the midgut length. Singly scattered, round cells of the closed type
are most common. In the cricket, however, a midgut zone just behind the
caeca contains also clustered endocrine cells (Fig. 1E). The majority of the
endocrine cells in the caecal region of locusts are not round, but possess
cytoplasmic extensions.
Paraneoptera seem to differ from other insects by the lack of immunoreactivity to FMRFamide antiserum in midgut innervation, indicating that the
gastric nerves of Homoptera and Heteroptera are either reduced or devoid of
FMRFamide-like peptides; a similar observation was made on the bug Rhodnius prolixus [16]. Both open and closed endocrine cells can be visualized with
the FMRFamide antiserum in the cicada and bugs, but no endocrine cells were
discerned in the aphids. In the cicada, some of the cells are paired; some of
the single cells appear paracrine. A characteristic feature of the bugs are very
long, open-type cells in the anterior sack-like region of the midgut (Fig. 1F).
Gut innervation of some Oligoneoptera also reacts poorly with the
FMRFamide antibody. In Neuroptera, there are two oesophageal nerves that
send branches over the crop, and at the end of the crop they dichotomize into
gastric nerves. Only varicosities of these nerves show clear immunoreactivity
in Chrysopa. About 10 perikarya occur at the rear of the foregut in Crysopa,
whereas up to 20 neurons are localized along the gastric nerves in the posterior
quarter of the midgut in Ascalaphus. Endocrine cells are mostly rounded, but
in the posterior half of the midgut in Ascaluphus they are occasionally amoeboid and possess long cytoplasmic processes linking them to one another and
to the innervation. In Chrysopa, the closed-type cells are occasionally paired.
In the Coleoptera, Curubus showed no immunoreactivity in the foregut
region and only weak reaction in four gastric nerves. In Terzebrio, the innervation was clearly stained: a single oesophageal nerve arborizes at the end of
Insect Midgut Endocrines
121
the foregut into a meshwork containing about 20 perikarya and a number of
gastric nerves run from there over the midgut length. Endocrine cells are both
open and closed types, and some of the latter possess cytoplasmic extensions.
In the examined Diptera, a pair of oesophageal nerves terminate in the two
ingluvial ganglia, each of which contains 4 8 immunoreactive perikarya. The
ganglia supply a total of 4 4 gastric nerves that can be traced along the entire
midgut length in Tipula, but only in gastric caeca in Drusophila [17]. Endocrine
cells are large, rounded, and, in Drosophifa, mostly of the open type.
A common feature of Lepidoptera seems to be the single oesophageal nerve
terminating in the ingluvial ganglion, which innervates either a pair of
proventricular ganglia (Galleria) or a loose conglomeration of neurons in the
first quarter of the midgut (Bombyx, Lymantria, Munduca; see Fig. 5F). Eight
gastric nerves begin in the foregut/midgut junction in most species, but in
Lyman tria, numerous gastric nerves appear to extend from individual neurons
located in the anterior region of the midgut (see Fig. 5E). Open bottle-shape
and closed rounded endocrine cells are singly scattered throughout the
midgut epithelium, with increasing density toward the end of midgut.
In the only representative of Hymenoptera, the nerves were not stained,
but about 20 neurons were revealed along the longitudinal muscles in the
foremost part of the midgut. Rounded endocrine cells, mostly of the open
type, occur singly throughout the midgut.
Reconstruction of Gut Endocrines During Metamorphosis
Gut innervation and midgut endocrine cells are found in the larvae, pupae,
and adults of oligoneopterans, including those in which the larval digestive
tract degenerates during metamorphosis and is replaced by a new one. Using
double-immunostaining, we followed the process of midgut replacement and
the fate of endocrine cells in Galleria. The dividing regenerative cells were
marked with BrdU, whereas the endocrine cells were recognized with the
anti-FMRFamide antiserum. No incorporation of BrdU was detected in the
wandering larvae, but it was clear in those that had initiated cocoon spinning.
By comparing a series of insects that were injected with BrdU between
spinning initiation and the completion of apolysis (pharate pupal stage), we
deduced that midgut reconstruction proceeds as portrayed in Figure 2 (to
avoid confusion in the black-and-white photographs, preparations treated
only with the anti-FMRFamideantiserum are shown).
Larval midgut consists of a single layer epithelium containing nidi of
regenerative cells, digestive goblet and columnar cells, and sparsely
scattered endocrine cells (Fig. 2A). The regenerative cells begin to
incorporate BrdU in the middle of the cocoon-spinning period. The
BrdU-labelled cells occur throughout the midgut length, and their number rapidly increases. They remain attached to basal lamina while the
surrounding midgut epithelium detaches and is pushed into the gut
lumen (Fig. 28). The regenerative cells continue to proliferate and in the
postspinning stage, the slowly mobile prepupae form the continuous
epithelium of the pupal midgut (Fig. 2C). At this stage, the new midgut
contains very few FMRFamide-positive cells. The old larval midgut
begins to disintegrate; however, some of the FMRFamide-positive cells
122
iitnan et al.
Fig. 2. Changes in the endocrine cells (arrows) during midgut metamorphosis in Galleria (sections,
antibody to FMRFamide). A: Functional midgut of a feeding larva. 6: Larval gut epithelium (LG)
with endocrine cells, enclosed by newly differentiating pupal gut (PG), in a larva at the end of
cocoon spinning. C: Degenerating larval gut (LG) and pupal gut (PC) in a pharate pupa. D: Midgut
with an imrnunopositive cell 24 h after pupal ecdysis. E: Disintegrating cells of pupal midgut (PG)
with remaining regenerative and endocrine cells in a pharate adult; F: Adult midgut. Bars = 50 prn.
appear to be preserved in the degenerated larval midgut until after pupal
ecdysis. The number of endocrine cells increases during the pupal stage (Fig.
2D), and they remain preserved when the pupal midgut degenerates and the
imaginal midgut is formed (Fig. 2E,F).
Vertebrate-Type Regulatory Peptides in the Innervation and Endocrine Cells
of Insect Midgut
Examples of the immunohistochemical detection of mammalian gastroenteropancreatic peptidic hormones in the gut of insects are provided in Figure
3, and the distribution of these peptides in the innervation and the endocrine
cells of several insects is compared with mammals in Table 3.
Insect Midgut Endocrines
123
In all examined insects, both the gut innervation and the endocrine cells
react with antisera against PP, P-endorphin, enkephalins, FMRFarnide, and
vasopressin; in addition to species listed in Table 3, we detected this reaction
also in the representatives of locusts, crickets, and phasmids (data not shown).
These are the only antisera that yielded a positive reaction in bugs and flies
we examined, but the tse-tse fly was reported not to respond to the PP
Fig. 3. lrnrnunodetection of different regulatory peptides in the endocrine cells of the midgut (sections).
A Group of endocrine cells revealed with antiserum against Arg-vasopressin in lepisma. 6: Reactionwith
antiserum against Met-enkephalin in the nidi of Lepisma. C: Gastrin-irnmunopositive cells in the anterior
region of midgut in Nauphoeta. D: Cholecystokinin-positive cells in the posterior region of midgut in
Nauphoeta. E,F The same midgut, reactions with antisera against Arg-vasopressin and peptide histidine
isoleucine, respectively. C: Endocrinecell revealed with antiserum against FMRFarnide in pyrrhocoris. H:
Endocrine cells containing pancreatic polypeptide-like antigen in Calliphora. Bars = 50 p.
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Lepisma
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+
+
+
-
?
?
+
?
?
Aeschna
N
E
+
+
+
+
+
+
+
+
+
+
+
+
f
f
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Roaches
N
E
+
+
+
+
-
?
?
-
?
+
-
-
-
?
?
?
?
?
-
?
?
?
+
+
-
-
+
+
+
+
-
-
-
+
-
+
-
-
?
?
-
+
+
+
-
?
?
-
?
-
-
+
-
?
?
-
Calliphora
N
E
-
-
?
?
-
Pyrrhocoris
N
E
+
+
+
+
?
?
?
-
-
+
+
+
-
?
?
-
?
-
+
+
+
+
-
+
+
+
+
-
-
Galleria
N
E
-
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
Mammals
N
E
*Listed insects: Silverfish Lqisrna saccharins; dragonfly Aeschna cyunea, data [18,19]; cockroaches: A combination of data on Peripluneta arnericanu
[20,21], Blaberits cmnirfir [22] and Nauphoefa cinerea (our results); bug Fyrrhocuris apterus; fleshfly Calliphora vicina; waxmoth Galleria rnellonella. Data
on peptide localization in the gut innervation in Pyrrhocoris concern the stomodeal nervous system (gastric nerves were not revealed). Distribution of regulatory peptides in the gut of mammals is taken from [2%25]. Abbreviated peptide names: CKF = corticotmpin releasing factor; ACTH
= adrenocorticotropin; MSH = melanization stimulating hormone; + = present; - = absent; ? = not tested; for other abbreviations see
list of Abbreviations used.
Glincentin
Glucagon
VIP
PHI
Gastrins
Cholecystokinin
PP
Somatostatin
Substance P
Neuro tensin
CRF
ACTH
a-MSH
P-Endorphin
Enkephalins
FMRFamide
Vasopressin
Peptide
TABLE 3. Immunohistochemical Identification of Gastroentoropancreatic Peptides in the Gastric Nerves (N)and Midgut Endocrine Cells
(E)of Representative Insects and Occurrence of These Peptides in Mammalian Gut*
Insect Midgut Endocrines
125
antiserum [26]. Most insects appear to contain antigens to additional vertebrate-type regulatory peptides. Antiserum against cholecystokinin reacts in
the gut of dragonflies, locusts, cockroaches, phasmids, lepidopterans, and
beetles (Table 3) [27]. The greatest variety of antigens was found in the gut of
cockroaches.
Insects do not seem to contain neurotensin-like antigens in the gut (Table
3) and possibly also lack peptides related to bombesin, secretin, and rnotilin
(data not shown), which are characteristicfor the gut innervation (bombesin)
and the endocrine cells, respectively, of mammalian gut. We also failed to
detect any specific insulin-like immunoreactivity (see Discussion).
Just as in mammals, some of the regulatory peptides are present both in
the innervation and in the endocrine cells of insect gut, whereas others occur
only in one of these peptidergic sources. Glicentin- and glucagon-like peptides
seem to be confined to the endocrine cells in both mammals and insects, but
gastrins, CRF, and substance P, which are also specific for certain endocrine
cells in mammals, are immunohistochemically detectable both in the endocrine
cells and in the gut innervation in insects (Table 3). Neuropeptide Y, which is
present in mammals exclusively in neurons, was identified both in the nerves
and in the endocrine cells of Locusta migratoria [28]. Finally, the peptide His-Ileu
appears in the gut innervation of mammals, whereas in insects it was immunohistochemically detected in the endocrine cells (Table 3).
The following regulatory peptides, which are believed to be restricted in
vertebrates to the CNS, were immunohistochemicallydetected in both nerves
and endocrine cells of the insect gut: vasopressin-like antigens were revealed
in all examined species (Table3), and antibody to urotensin I reacted in Gryllus
and Periplaneta [29]. Even more surprising, the hypothalamic growth hormone-releasing factor and the luteinizing hormone-releasing factor occur in
the dragonfly A . cyanea and in the cockroach B . craniifer in the endocrine cells
but not in the nerves of the gut [19,21].
Insect Neurohormones in the Innervation and in the Endocrine Cells of the
Midgut
We never found a reaction with bornbyxin antibody, which identified
specific neurons in the CNS [10,30], in the digestive tracts of several lepidopterans. The antibody against PTTH reacted with certain neurons in the
stomodeal nervous system and with gastric nerves, but not with the endocrine
cells (Table 4). Antisera against ATH and DH, however, recognized antigens
both in the innervation and in the endocrine cells of Galleria, Manduca (Table
4), and Lymantria (data not shown). Antisera against PTTH and ATH displayed identical pattern of staining in the neurons and nerves (Fig. 4A,B).
A more detailed study was performed with last instar larvae of Manduca,
the species from which ATH and DH had been isolated [12,13]. Both the
innervation (Fig. 4C,D) and the endocrine cells (Fig. 4G,H) react with these
antisera. DH-like antigen occurs in a higher number of ganglionic neurons
than the ATH-like antigen, but it is wanting in the enteric plexus, in which
ATH-like antigen appears to be present in large amounts (Fig. 4F). A similar
distribution of ATH-immunoreactivity was found in Lymantria (Fig. 4E). In
spite of the lack of DH-like antigen in the enteric plexus, where the gastric
126
Zitnan et al.
TABLE 4. Occurrence of Insect Neuropeptides in Gut Innervation and Midgut Endocrine
Cells
Species and peptides
G. mellonella
P'ITH
ATH
DH
M.sextu
PTTH
ATH
DH
Localizationa
Frontal
ganglion
2
2
2
Frontal
ganglion
2
24
3+4
aNumbers of immunoreactive perikarya;
Ingluvial
ganglion
24
2 4
?
Hypocerebral
ganglion
-
4.4
Proventricular
ganglion
4-6
4
?
Endocrine
cells
-
+
?
Enteric
plexus
Endocrine
cells
+
+
-
-
+
+ = presence, - = absence of reactivity in the nerves.
nerves appear to originate, immunostained axons are found over the entire
midgut surface (Fig. 4H).
DISCUSSION
There is little doubt that peptidergic gut innervation and peptidergic midgut
endocrine cells occur in all insects. With a few exceptions, they can be
visualized with the antisera against FMRFamide. In our study, only the
innervation of foregut in Curubus and Bornbus, the innervation of midgut in
Puruneopteru, and the endocrine cells in aphids failed to respond to FMRFamide antiserum.
The midgut innervation emanates from the stomodeal nervous system,
the arrangement of which is characteristic for insect orders or sub-orders
[ 6 ] .The posterior ganglia (ingluvial and proventricular) are often replaced
by loose neurons; in Manduca, their assembly is called the enteric plexus
[31]. Gastric nerves, which in some species include perikarya, appear to
begin in the posterior ganglia or in the enteric plexus [32], but the distribution of DH-like antigen (Table 4) suggests that they alsocontainaxons
from the frontal ganglion or from the CNS. Gastric nerves form a network
of fine fibers in midgut musculature, and there is evidence that gastric
nerves link the stomodeal nervous system with the proctodeal innervation
[33]. Axons supplying midgut muscles were shown to contain a variety of
neurosecretory granules [34].
As most other animals, the insects contain endodermal endocrine cells of
both open and closed types [35]. The cells are usually distributed singly
through most or all of the midgut, but in the apterygotes, crickets and
Chrysopu, some of them occur in groups [5, and our observations]. Endocrine
cells are derived from the regenerative nidi, which also generate the digestive
cells 136, and our data]. Morphology, size, and abundance of the endocrine
cells are different in different insect taxa, and in various midgut regions of the
same species. Differences in the immunoreactivity of the endocrine cells in
Insect Midgut Endocrines
127
Fig. 4. lmrnunodetection of insect neurohormone-like antigens. Allatotropin-like (A) and PTTH-like
antigens (B) in the frontal ganglion of Galleria larvae (A, end of the penultimate, i.e., 6th instar; 6 ,
beginning of the 6th instar). Allatotropin-like (C) and diuretic hormone-like antigens (D) in the
frontal ganglion of wandering Manduca larvae; immunoreactive neurons (arrows) are present also
in the hypocerebral ganglion (HG). Allatotropin-like antigen in the nervous midgut plexus of fresh
Lymantria pupa (E) and of wandering Manduca larva (F); arrows point to perikarya. Allatotropin-like
(C) and diuretic hormone-like antigens (H)in the fine nerves ( N ) and in the endocrine cells (arrow)
of the feeding (C)
and wandering (H) Manduca larvae. Bars
50 pm.
128
Zitnan et al.
various insect groups are documented in Table 3. Ultrastructural [22,34,37,38],
immunohistochemical [20,28], and immunocytochemical [39] evidence is
available for the diversity of the endocrine cells within the midgut of a single
species.
Widespread occurrence, chemical diversity, and structural complexity,
which are maintained when the gut is rebuilt at metamorphosis, indicate that
the innervation and the endocrine cells of the gut are of great importance. The
information on mammals demonstrates that the two systems are interlinked
and jointly communicate with the CNS [40]. According to Fujita et al. [35],
the endocrine cells function as primary sensors, those of the open type
registering the nutrient contents of the gut, and those of the closed type
perceiving the tension in the gut wall. Upon appropriate sensory stimulation,
the endocrine cells release their secretions primarily into their immediate
vicinity (paracrine secretion). The secretions act mostly locally, affecting gut
movements, production of digestive fluids, rate of replacement of the gut
epithelium, and blood flow to the gut. There is some evidence that the
secretion is released into the gut lumen and acts on the apical sites of the
digestive cells. Paracrine secretions apparently act also on the nerve termini
in the subepithelial nervous reticulum, by which the humoral signal is
transduced into nervous stimuli and eventually causes changes in the nervously controlled functions, including behavior. Finally, some products of the
endocrine gut cells enter the body circulation and exert hormonal effects on
distal targets.
The increasing knowledge of insect peptidic hormones is consistent with
the idea that most of them belong to the same peptide families as the
hormones of vertebrates [41]. Even though the immunohistochemical data
cannot provide a proof of chemical identity and may lead to false conclusions
[42], the consistent demonstration of a number of vertebrate peptides in
various insects and with different antibodies and techniques suggests that
most of the peptidic hormones in insect gut innervation and midgut endocrine
cells are similar to those of the vertebrate gut. The endocrine system of insect
gut parallels that of the vertebrates also in peptide localization in the nerves
vs. the endocrine cells, which seems to be reversed only in the case of peptide
His-Ileu (Table 3). Antisera to neurotensin, secretin, and motilin, which are
the dominant peptides in the gut of mammals, do not react in the insect gut.
A reaction with antiserum against bombesin, another common mammalian
peptide, was reported in a single study of a locust 1391. Secretion of an
insulin-like material in the digestive tract of insects is a subject of dispute.
Whereas radioimmunoassays and metabolic bioassays of midgut extracts have
indicated the presence of insulin-like protein(s) in several insects [43-46], the
immunohistochemical approach has yielded a positive response in one case
only 1391. Differences in the content of midgut hormones among the insect
taxa (Table 3) resemble the situation in the vertebrates [47].
Even in mammals, the physiological effects of gastroenteropancreatic peptides are far from being fully elucidated, and it is becoming clear that in many
cases they are complex. For example, cholecystokinin controls gall bladder
contractions, pancreatic enzyme secretion, and gastric emptying, and via the
latter effectit inhibits food intake [40]. Multiple roles of gut secretions are also
Insect Midgut Endocrines
129
indicated by the data on insects. Involvement in the control of food processing
was proposed by Brown et al. [38], who showed that the number of immunoreactive cells and their content of FRMFamide-like and PP-like material
in the midgut of adult mosquito females decreases after the blood meal,
sugesting depletion of the regulatory peptides. By contrast, in larval corn
earworms, Heliothis zed, feeding evokes an increase in FMRFamide immunoreactivity in the endocrine cells, whereas the blood concentration of the
immunoreactive agent is higher in starved insects [48]. Under in vitro conditions, midgut extracts were shown to stimulate activity of digestive enzymes
[49]. Our present finding of PTTH-, ATH-, and DH-like immunoreactivity in
the midgut (Table 4)indicates that the gut may exert some adenotropic effects
and regulate excretion. The hind gut of certain caterpillars has been shown to
contain PTTH activity [50], and it cannot be excluded that this activity is
derived from gut innervation.
In summary, the secretory products of the gut innervation and of midgut
endocrine cells of insects appear to control a variety of vital functions. They
may also exert important effects on the parasites and pathogens that are
transmitted by insects [51]. Apparent importance of insect gut endocrines
warrants further research with the prospect of developing a new target for
insect manipulations.
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