Comparative distribution of leucokinin and functionallyrelated peptides in the nervous system of several insects

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Comparative distribution of leucokinin and functionally related peptides in the nervous system of several insects

Chen, Yuetian, M.S.

The University of Arizona, 1993

U M I 300 N. ZeebRd. Ann Arbor, MI 48106

Comparative distribution of leucokinin and functionally related peptides in the nervous system of several insects

by

Yuetian Chen

A thesis Submitted to the Faculty of the

DEPARTMENT OF ENTOMOLOGY

In Partial Fulfillment of the Requirements

For the Degree of

MASTER OF SCIENCE

WITH A MAJOR IN ENTOMOLOGY

In the Graduate College

THE UNIVERSITY OF ARIZONA

1993

2

STATEMENT BY AUTHOR

This thesis has been submitted in partial fulfillment of requirements for an advanced degree at the University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library.

Brief quotations from this thesis are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his or her judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author.

SIGNED

APPROVAL BY THESIS DIRECTOR

This thesis has been approved on the date shown below:

Henry'H. Hagedorn Date Professor of Entomology

3

Acknowledgment

I would like to express my sincere appreciation to Dr. Henry H.

Hagedorn for serving as my major adviser and for his guidance and funding

during my graduate studies. Drs. Jan Veenstra and Norman T. Davis had a

great effect on my research and I am grateful for their time and valuable

advice.

I would also like to thank Drs. Reginald F. Chapman and Eddie W.

Cupp for serving as members of my graduate committee and giving advice

during my studies, and Drs. Diana E. Wheeler and Martin Taylor for

reviewing this thesis.

I wish to thank my husband Shi Niu for giving me support during my

graduate studies.

4

TABLE OF CONTENTS

I. LIST OF ILLUSTRATIONS 7

II. LIST OF TABLES 9

III. ABSTRACT 10

IV. INTRODUCTION 11

The neuroendocrine system 11

Physiological approaches to neuroendocrinology 12

Molecular approaches to neuroendocrinology 14

Immunological study of neuropeptides 16

Interspecific comparisons 16

Identifying synthesis and releasing sites 17

Monitoring hemolymph titers 18

Diuretic neuropeptides in insects 19

Leucokinins: myotropic neuropeptides possibly involved in diuresis 20

V. MATERIALS AND METHODS 27

Experimental animals 27

Antiserum preparation 28

Conjugate preparation 28

Rabbit injection and bleeding 28

Antisera purification 29

Antisera labeling 29

Tissue section staining 30

Peroxidase-antiperoxidase section staining 31

5

Fluorescent labeled section staining 32

Whole mount staining 32

Enzyme-linked immunosorbent assay (ELISA) of leucokinins 33

Testing the conjugate 34

Establishing a standard curve for leucokinins 35

HPLC fractionation of N. cinerea neuropeptides 36

Tissue preparation 36

HPLC 36

VI. Results 38

Specificity of antisera 38

Leucokinin homogeneity in N. cinerea 40

Distribution of leucokinin and vasopressin immunoreactive patterns in N. cinerea 43

Distribution of leucokinin and vasopressin immunoreactive patterns in A. domesticus 52

Distribution of leucokinin immunoreactive pattern in S. americana 60

Distribution of leucokinin immunoreactive pattern in A. aegypti 68

Distribution of leucokinin immunoreactive pattern in A. mellifera 69

Distribution of leucokinin and diuretic hormone immunoreactive neurons in M. Sexta 76

VII. DISCUSSION 82

Conservation of the leucokinin system 82

Interspecific comparisons of the anatomy of leucokinin secretion, transport and release 85

Significance of leucokinin and vasopressin co-localization 87

6

Leucokinin and diuretic hormone neurosecretion in M. sexta 89

Comparison of the distribution pattern of leucokinin-like peptides with

FMRFamide-like peptides in insects 90

VIII. REFERENCES 92

7

LIST OF ILLUSTRATIONS

FIGURE 1. Anti-leucokinin IV affinities to leucokinin IV, HPLC immunoreactivity peak fraction from corpora cardiaca of Nauphoeta cinerea, allatotropin and corazonin 39

FIGURE 2. Leucokinin IV immunoreactivity of HPLC fractions from brains, corpora cardiaca and abdominal ganglia of adult Nauphoeta cinerea 41

FIGURE 3. Anti-leucokinin IV affinities to immunoreactive peaks in 35 min from brains, abdominal ganglia and corpora cardiaca 42

FIGURE 4. Distribution of leucokinin positive cells and processes in Nauphoeta cinerea 47

FIGURE 5. Leucokinin positive neurosecretory cells and processes in Nauphoeta cinerea 49

FIGURE 6. Distribution of leucokinin positive cells and processes of abdominal ganglia and their neurohemal release sites of Nauphoeta cinerea 51

FIGURE 7. Distribution of leucokinin positive neurosecretory cells and processes in Acheta domesticus 55

FIGURE 8. Leucokinin and vasopressin positive neurosecretory cells and processes in Acheta domesticus 57

FIGURE 9. Distribution of leucokinin positive neurosecretory cells in abdominal ganglia and their neurohemal release sites of Acheta domesticus 59

FIGURE 10. Distribution of leucokinin positive neurons and processes in Schistocerca americana 63

FIGURE 11. Leucokinin positive cells and processes of Schistocerca americana 65

FIGURE 12. Distribution of anti-leucokinin reactive neurosecretory cells in abdominal ganglia and their neurohemal release sites of Schistocerca americana 67

8

FIGURE 13. Distribution of leucokinin positive cells and processes in Aedes aegypti 71

FIGURE 14. Leucokinin positive cells and processes in Aedes aegypti and Apis mellifera 73

FIGURE 15. Distribution of leucokinin positive cells and processes in abdominal ganglia of Apis mellifera 75

FIGURE 16. Leucokinin and Manduca diuretic hormone immunoreactive neurons of Manduca sexta 79

FIGURE 17. Distribution of leucokinin positive neurosecretory cells in abdominal ganglia and their neurohemal release sites of Manduca sexta larva 81

FIGURE 18. Major similarities in the locations of leucokinin positive cells in the six species examined 84

9

LIST OF TABLES

TABLE 1. Known amino acid sequence of diuretic peptides 25

TABLE 2. Known amino acid sequence of leucokinins and related neuropeptides 26

10

Abstract

Antisera against leucokinin IV were used to test for the presence of

leucokinin-like peptides in the central nervous systems of Nauphoeta

cinerea, Acheta domesticus, Schistocerca americana, Aedes aegypti, Manduca

sexta and Apis mellifera. Leucokinin immunoreactive neurosecretory cells

were found in the pars intercerebralis and pars lateralis of the brains of N.

cinerea, A. domesticus, A. aegypti, but not in the brains of S. americana, M.

sexta and A. mellifera. The neurohemal release sites were also very different

among species. In contrast, the distribution patterns of leucokinin

immunoreactive neurosecretory cells were very similar among abdominal

ganglia of all six species. The identity of the leucokinin immunoreactive

material in the brain, corpora cardiaca and abdominal ganglia of N. cinerea

was shown by HPLC combined with ELISA. In N. cinerea and A. domesticus,

leucokinin and vasopressin were found to co-localize in the same

neurosecretory cells. In M. sexta, leucokinin and diuretic hormone co-

localized in the same neurosecretory cells in abdominal ganglia, but not in

the brain.

INTRODUCTION

11

The neuroendocrine system

The neuroendocrine system of animals serves as a mode of internal

communication between the nervous system and the endocrine system. This

system consists of neurosecretory cells secreting neuropeptides and other

biogenic amines in the central and peripheral nervous systems. The same

neuropeptide may work either as a neurotransmitter or neuromodulator

affecting only neighboring neurons, or as a neurohormone, sending signals to the

entire body for regulation of different physiological processes.

In vertebrates, neurosecretory cells (i.e., the cells in the hypothalamus)

primarily communicate between the nervous and endocrine systems, as is the

case in the anterior pituitary (Scharrer, 1967). A few neurohormones, such as

vasopressin and oxytocin, originate from neurosecretory cells in the

hypothalamus and are released from the posterior pituitary to act directly on

non-endocrine target tissues. In contrast, in invertebrates, most neurosecretory

products appear to act directly on non-endocrine target tissues (Gainer et al.

1977).

Neuropeptides in insects have been shown to control many processes

including diapause (Williams 1946,1947, Hasegawa 1957, Takeda and Girardie,

1985), molt initiation (Williams 1947,1952), heart rate (Gersch 1958, Veenstra

1989), color change (Dupont-Raabe 1951, Hashiguchi et al., 1965, Suzuki et al,

1976, Matsumoto et al., 1985,1986) and metabolism (Mayer & Candy, 1969).

These peptides are synthesized by neurosecretory cells that are distributed in the

12

central nervous system (brain and segmental ganglia), corpus cardiaca-corpus

allata system and peripheral nervous system of insects. Comparison between the

corpus cardiaca-corpus allata system of the insect brain and the hypothalamic-

hypophysial system of vertebrate brain reveals that these neuroendocrine

systems have striking structural and functional similarities. The hypothalamic

nuclei of the vertebrates seem to be equivalent to the pars intercerebralis of the

insects. Neurosecretory cells are found in both centers, which send nerve fibers

to endocrine organs: the pituitary gland and the corpora cardiaca-corpora allata

respectively (Scharrer & Scharrer 1944). In addition to these similarities between

vertebrate and insects, the neurosecretory cells in insects are present throughout

the nervous system and are, therefore, more decentralized. The products of these

cells play important roles in controlling the development, metabolism and

reproduction of insects.

Physiological approaches to neuroendocrinology

Morphological and cytophysiological investigations have provided

indirect evidence for the role of neurosecretory products in the regulation of

developmental, reproductive and metabolic processes. Secretory activity of

nerve cells was first reported in the teleost fish, Phoxinus laevis (Scharrer 1928).

Later, the Scharrers examined a wide variety of vertebrate and invertebrate

groups and confirmed the apparently universal occurrence of neurosecretory

cells (Scharrer 1977). With the development of the electron microscope, major

sites of synthesis and release of neurosecretory products could be identified, as

secretions were packaged into membrane-bound, electron-dense granules

(Bargmann & Scharrer 1951). Additional experiments established that

13

neurosecretory cells are functionally similar to other protein-secreting cells in

that the neurosecretory products are packaged at the Golgi apparatus and

released by exocytosis (Scharrer & Brown 1961). Since the products of

neurosecretory cells appeared to be released in the blood, these neuropeptides

were initially considered to function as hormones. The idea that these

neuropeptides could also function as neurotransmitter was introduced with the

discovery of peptidergic neurons by Bargmann (1967). The peptidergic neurons

include neurons showing the classical synaptic pattern and others that are

neither local (synaptic) or truly neurohormonal but have a relatively narrow zone

of extracellular stroma between the terminal and effect cells. Still other

peptidergic neurons have synaptic-like contacts in which the two neurons are in

close contact, thereby providing for a reciprocal exchange of neurotransmitter. It

was demonstrated that the peptide factor extracted from molluscan ganglia could

modulate bursting pacemaker activity, suggesting that neuropeptides could

function also as neuromodulators (Ifshin et al. 1975). Buijs and Swaab (1979)

demonstrated vasopressin and oxytocin synapses in the rat limbic system by

immuno-electron microscopy and suggested that these peptides function as

neurotransmitters. Thus it became clear that neuropeptides could function in

variety of roles, i.e. as neurotransmitters, neuromodulators and neurohormones,

and therefore that the same peptide could have multiple roles.

Different techniques such as organ culture, transplantation experiments

(Nikitovitch-Winer & Everett 1958, Oksche et al. 1964), electrical stimulation, and

lesion experiments of specific hypothalamic centers (Szentagothai et al. 1962)

provided further evidence for the physiological functions of various

neurosecretory cells. On the basis of histological evidence, Fukuda (1967)

14

identified a single pair of large neurosecretory cells in the subesophageal

ganglion in Bombyx mori as the site of diapause hormone release. However,

extracts of pupal brains also showed diapause hormone activity (Sonobe & Keino

1975). Pars intercerebralis and lateral neurosecretory cells were shown to

produce prothoracicotropic hormone (PTTH) in the locust (Girardie 1974,

Girardie & De Reggi 1978), using electrical stimulation followed by

determination of hemolymph ecdysteroid titers. But the experiment was not

sufficiently precise to define the location of the PTTH neurosecretory cells.

Synthesis sites of diuretic hormone were identified in Rhodnius by testing the

diuretic activity of isolated neurons (Berlind & Maddrell 1979); but it was

unproved that these were the only neurons that synthesized diuretic hormone.

In general, these early studies were limited by lack of knowledge of the nature of

the compounds released, whether they are composed of peptides only, or

whether they are several different compounds. In addition, the exact sites of

release and location of receptors remained to be determined.

Molecular approaches to neuroendocrinology

As a result of advances in biochemistry and molecular biology, the exact

structure and chemical character of the neurosecretory products and their

physiological function could be better studied. To date, more than 100

neuropeptides have been isolated and identified. Proctolin was the first insect

neuropeptide to be structurally characterized (Sterratt & Brown 1975). Since

then, over 60 additional insect neuropeptides have been sequenced including the

adipokinetic hormone/hyperglycemic hormone (AKH/HrGH) family (Stone et

al. 1976), the bombyxin family (Nagasawa et al. 1979,1984) and the leucokinin

15

family (Holman et al. 1986,1987). Also, some of the specific genes which codes

for certain peptides have been investigated.

Immunological techniques have greatly enhanced the further

characterization and purification of neuropeptides from species with well-

characterized peptides. An example is the use of radioimmunoassay to

characterize and purify the thyrotropin-releasing factor from several species

(Grimm-J0rgenser et al. 1975, Berson & Yalow 1973). Antisera raised against a

number of mammalian neuropeptides were found to be reactive against nervous

systems of diverse phyla, including several invertebrates (Remy et al. 1977,

Strambi et al. 1979, Veenstra et al. 1984), indicating that vertebrate-like peptides

also exist in invertebrates. It remains to be seen whether they fulfill the same

functions. An antiserum against vertebrate Arg-vasopressin was used to purify

related oligo-peptides from the locust (Proux et al. 1987). FMRF amide peptides,

with the structure (Phe-Met-Arg-Phe-NH2), were also purified from Drosophila

using similar techniques (Nambu et al. 1988). The cardio-accelerator peptide

corazonin was first isolated from the American cockroach (Veenstra 1989). With

this purified peptide, antisera against corazonin were used in a very sensitive

enzyme-linked immunosorbent assay (ELISA) to quantify the peptide, and

isolate it from Schistocerca americana, Nauphoeta cinerea and Manduca sexta

(Veenstra 1991).

The structural and functional similarities in the neuroendocrine systems of

insects and vertebrates have counterparts in the chemical similarity of peptide

messenger substances used by the nervous system (Scharrer 1987). Some of these

peptides share very high levels of similarity of their amino acid sequence

16

(Bodenmiiller & Schaller 1981, Nachman et al. 1986, Thorpe & Duve 1988). A

better understanding of the evolutionary relationships between these peptides

was obtained when genes of the peptides were isolated and described. cDNA-

clones coding for insulin-related peptides have been identified in Locusta

migratoria (Lagueuxef al. 1990) and Bombyx mori (Adachi et al. 1989, Iwami et al.

1989). A cDNA-clone coding for FMRF-amide was identified from both

Drosophila virilis and D. melanogaster (Schneider & Taghert 1988,1990, Nambu et

al. 1988). Structures of the precursors of AKHI and AKHII have also been

elucidated (Schulz-Aellen 1989). Results from biochemical and molecular

biological investigations have indicated that the primary and secondary structure

of insulin and insulin-related peptides appear quite conserved and thus have

long evolutionary histories (Steiner & Chan 1988), whereas others such as

eclosion hormones have no known vertebrate homologues (Nagasawa et al.

1987).

Immunological study of neuropeptides

Antibodies, which are natural defense molecules produced by vertebrate

immune systems in response to specific foreign antigens, can be used to purify

neuropeptides, and study their distribution and physiological roles. Specific uses

of neuropeptide antisera are:

Interspecific comparisons. Antisera against purified neuropeptides can be

used to screen related peptides in other species. Antisera against insulin in

vertebrates were used to screen insulin-related peptides in invertebrates (Tager et

al. 1976, Duve et al. 1979, El-Salhy et al. 1984, Hansen et al. 1990). Proctolin was

originally isolated from the American cockroach. Proctolin immunoreactivity

17

has been demonstrated in members of the Crustacea, Annelida, and Mammalia

(Adams 1990). Crustacean cardioactive peptide (CCAP) is a peptide that was

found to be a cardioacceleratory substance in extracts from the pericardial organs

of Carcinus maenas (Price & Greenberg 1977) and has been biochemically

characterized (Stangier et al. 1987). Radioimmunological studies demonstrated

that peptides related to CCAP are also present in Locust migratoria (Stangier et al.

1989). Using immunocytochemical methods, CCAP immunoreactive neurons

were also found in Tenebrio molitor (Breidbach & Dircksin 1991). FMRF-amide is

a cardioaccelerator from molluscan heart which belongs to a large "RF" peptide

family (for cholecystokinin, gastric and pancreatic polypeptide, the last two

amino acids are Arg-Phe-NH2). Different orders of insects have been found to

produce FMRF-amide-related peptides using appropriate antisera (Boer et al.

1980, Veenstra 1984, Remy et al. 1988, Tsang et al. 1991, Homberg et al. 1991,

Eichmuller et al. 1991)

Identifying synthesis and releasing sites. Using immunocytochemical

staining, the neurosecretory cells and nerve pathway of specific peptides can be

mapped in both vertebrates and invertebrates. In vertebrates, the synthetic sites

of vasopressin have been investigated and their receptors localized by

immunocytochemistry (Leclerc & Pelletier 1974). The neurosecretory cells

producing vasopressin-related peptides in insects and their release sites were

also investigated in different insects (Remy & Girardie, 1980, Veenstra, 1984,

Nassel et al. 1989, Davis et al. 1992). Many other insect neurosecretory cells which

produce different neuropeptides were also mapped in more than one insect

nervous system such as FMRF-amide-related peptides, CCAP peptide,

vasopressin-like peptides, and proctolin peptides.

18

Monitoring hemolymph titers. Using an antibody against a specific peptide,

the developmental fluctuations of this peptide can be monitored which may help

to understand the physiological function of the peptide. Recently, an

radioimmunoassay was developed to test the titer of bombyxin in the

hemolymph of Silkmoth, Bombyx mori, during larval-larval, larval pupal and

pupal-adult development using monoclonal antibody against this peptide

(Saegusa flZ. 1992).

The distribution, site of synthesis and titer changes in the hemolymph of

neuropeptides are very important to understanding the neuroendocrine system.

Some groups of peptides are distributed in different insects such as AKH, PTTH

and vasopressin. PTTH shows variation in the sites of synthesis among Locusta,

(median neurosecretory cells and lateral neurosecretory cells; Girardie 1967,1974,

Girardie & De Reggi 1978), Rhodnius (median neurosecretory cells;

Wigglesworth, 1940), Bombyx (median neurosecretory cells; Mizoguchi et al. 1987)

and Manduca (lateral neurosecretory cells only; Agui et al. 1979). In contrast, For

vasopressin the distribution patterns of neurosecretory cells were very similar

among different insects (Davis 1992, Davis, unpublished data, Veenstra, 1984). It

is possible that vasopressin has a longer evolutionary history or a more

conserved function in insects. By comparing the distribution of the same group

of peptides within the nervous system of different insects, the evolutionary

history of the peptides, and their underlying communication systems, can be

partially revealed. A complication in understanding evolution is that one

physiological process may be controlled by more than one peptide, and so the

whole suite of controlling peptides may have to be studied to understand the

evolutionary history of the dependent physiological processes.

19

Diuretic neuropeptides in insects

Insect diuresis can be divided into two parts, secretion by Malpighian

tubules and reabsorption by the rectum. The Malpighian tubules absorb a liquid

from the hemolymph and the rectum reabsorbs various amounts of water and

amino acids, sugars, lipids and certain ions to the hemolymph. The volume of

fluid excreted by Malpighian tubules, and selective re-absorption by rectum

varies depending on physiological need, and appears to be regulated by the

neuroendocrine systems in insects. The change of diuretic activity has been

determined by measuring the amount of urine secreted (Maddrell, 1963),

modifications in the transepithelial voltage (Maddrell & Klunsuwan 1973,

Williams & Beyenbanch 1983,1984), and the intracellular level of cAMP (Morgan

& Mordue 1985). When Malpighian tubules are stimulated by diuretic

neuropeptides, the fluid secretion can be accelerated more than a thousand times

(Maddrell et al. 1992).

In earlier studies, the investigation of insect diuretic peptides was based

on histological evidence. A relationship between neurosecretion in the type A

neurosecretory cells in the nervous system and osmoregulation in the female

blow fly (Calliphora erythrocephala) was established in histophysiological studies

(Thomsen 1952). These type A cells also produce anti-diuretic hormones as later

demonstrated by immunocytochemistry (Tamarelle & Girardie 1989).

Histological studies of Oryctes rhinoceros (Kannan & Prabhu 1985), Locusta

migratoria (Girardie 1970), Aeshna cyanea (Charlet 1974) and Leucophaea maderae

(De Besse 1978) indicated that storage of type A neurosecretory cell products in

the brain and corpora cardiaca was variable but could be correlated with

20

humidity. Differences in the amount of products in neurosecretory cells of

ventral ganglia of R. prolixus were also correlated with environmental humidity

(Baudry 1968,1969). These histophysiological studies indicated that diuresis is

controlled by neurosecretory cells and that these neurosecretory cells are present

in brain, corpora cardiaca and abdominal ganglia.

Bioassay methods more directly demonstrated the presence of a diuretic

hormone in the brain and abdominal ganglia of several insects. By sectioning

nerves of S. gregaria (Mordue & Goldsworthy 1969) and activating different parts

of the brain of the stick insect, Carausius morosus, (Pilcher 1970), the central region

of the protocerebrum has been shown to control diuretic activity. By selectively

removing the pars intercerebralis of the brain, the regions, in which a diuretic

hormone occurs in the dragonfly A. cyanea (Charlet 1974) and in L. migratoria

(Proux 1979) were localized. Diuretic hormone was also found in the abdominal

ganglia of R. prolixus by incubating isolated neurons with Malpighian tubules

and measuring secretory activity (Berlind & Maddrell 1979).

Distinct diuretic and antidiuretic factors have been isolated from the

storage and glandular lobes of the corpora cardiaca of various insects (Morgan &

Mordue 1983, Proux et al. 1984, Herault et al. 1985). Several factors that control

the diuresis of the mosquito A. aegypti have been isolated from head extracts

(Petzel et al. 1985). However, the structures of some of these diuretic

neurohormones in different insects has only been recently determined (see Table

2). Vasopressin-like factors with diuretic activity were isolated from the corpora

cardiaca of L. migratoria (Morgan 1987). Several diuretic hormones that resemble

mammalian corticotropin releasing factor (CRF) have been sequenced from L.

21

migratoria (Kay et al. 1991, Lehmberg et al. 1991), M. sexta (Kataoka et al. 1989,

Blackburn et al. 1991), A.domesticus (Kay 1991) and P.americana (Kay et al 1992)

and these CRF-related diuretic peptides have 30-46 amino acid residues (Table 1).

A cDNA clone encoding a precursor of the M. sexta diuretic hormone has been

isolated (Digan et al. 1992). In addition to diuretic hormone, some of the

myotropic peptides such as leucokinins (Holman et al. 1986), achetakinins (Coast

et al. 1990) and culekinin depolarizing peptides isolated from mosquito Culex

salinarius (Hayes, unpublished data) also have diuretic effects (Hayes et al. 1989,

Coast et al. 1990). Thus diuresis appears to be controlled by more than one

peptide.

The mechanism by which these peptides control diuresis in insects is not

well understood. It was demonstrated that diuretic hormone can affect cAMP

levels and membrane potential of the Malpighian tubules and rectum. However,

the patterns of the effects were different among these peptides during bioassays.

Some peptides stimulate fluid secretion by influencing membrane potential but

not changing the levels of cAMP (Coast et al. 1991, Hayes et al. 1989). In contrast,

some peptides stimulate fluid secretion by changing cAMP levels (Morgan &

Mordue 1985, Rafaeli et al. 1987, Lehmberg et al. 1991, Kay et al, 1991,1992).

Spring and Clark (1990) have developed a model to account for the actions of

these diuretic peptides I, II and an antidiuretic peptide in the cricket. The model

suggests that diuretic peptide-I could elevate intracellular cAMP levels,

presumably by activating a G-protein, which would in turn activate cAMP-

dependent protein kinases. The result of this cascade would be the opening of

sodium channels in the basolateral membrane, increasing intracellular sodium

concentration and activating the apical cation pump. Diuretic peptide-II could

22

have multiple effects. First, it could activate an inhibitory G-protein, blocking

adenylate cyclase, and activate a phosphodiesterase to lower the cAMP

concentrations. Second, it could act to lower intracellular calcium concentrations,

closing the potassium-channel and increasing potassium transport by the apical

cation pump. Antidiuretic hormone, on the other hand, may act by elevating

intracellular calcium concentrations, possibly via the phosphatidyl inositol (PI3)

pathway, opening the potassium channel that would hyperpolarize the

basolateral membrane and decrease the potassium available to the cation pump.

However, how diuretic peptides control diuresis in insects still needs to be

investigated.

Finding the synthesis and releasing sites of diuretic neuropeptides is

another approach to understanding physiological function. The synthesis sites of

a diuretic hormone in M. sexta have been localized (Veenstra & Hagedorn 1990).

Intra and interspecific comparisons of the distribution of various diuretic

peptides can help us further understand the mechanism of diuretic regulation.

Leucokinins: myotropic neuropeptides possibly involved in diuresis

Leucokinins are a class of myotropic neuropeptides isolated from head

extracts of the cockroach, Leucophaea maderae (Holman et al. 1986a, 1986b, 1987a,

1987b). They consist of a group of eight neuropeptides, all of which contain a

similar C-terminal core sequence of 5 amino acids [Phe-X-Ser-Trp-Gly-NH2]

(Table 2), which has been demonstrated to be essential for maintenance of their

physiological effects (Hayes et al. 1989). The physiological roles of these

peptides are not well understood. The other series of five myotropic peptides,

the achetakinins, have been isolated and structurally characterized from head

23

extracts of the cricket, A. domesticus, using a purification system similar to the

method applied to the leucokinins (Holman, et al. 1990).

The structures of these five myotropic peptides also contain a C-terminal

core that can stimulate hindgut contraction of L. maderae, and which has a very

similar sequence to that of the leucokinins (Table 2). In addition to the

stimulation of hindgut contraction of the cockroach, achetakinins can stimulate

fluid secretion by the Malpighian tubules of A. domesticus (Coast et al. 1991).

Leucokinins and achetakinins clearly belong to the same neuropeptide family.

Recently, a related peptide was isolated from L. migratoria that had the same C-

terminal sequence as that of the leucokinins (Schoofs et al. 1992; Table 2). Later,

two leucokinin peptides which were called culekinin depolarizing peptides were

isolated from the mosquitoes, Culex salinarius (Hayes et al. 1992, from Clements,

1992, Table 2). Using immunocytochemical staining, leucokinin-like peptides

have been found in the blow fly, Phormia terraenovae (Nassel 1991) and turnip

moth, Agrotis segetum (Cartera 1992). Experiments examining the effect of

leucokinins on the Malpighian tubules of the mosquito, A. aegypti, indicated that

leucokinins can stimulate fluid secretion and depolarize transepithelial

membrane potential even in these distantly related insects. These results were

very similar to the effects of the factors extracted from the mosquitoes

themselves. Thus it seems that leucokinins are a class of neuropeptides that exist

in all insects and are important to diuretic activity.

In this thesis I report an immunocytochemical study of the distribution of

leucokinin-like peptides in the nervous systems of Nauphoeta cinerea and five

other species representing four different orders of insects: Schistocerca americana,

24

Manduca sexta, A. domesticus, A. aegypti and Apis mellifera. The presence of

leucokinins in the nervous system of N. cinerea was confirmed by separation of

the peptides from the nervous system with HPLC and testing the

immunoreactivity to anti-leucokinin antiserum using a ELISA. The distribution

of leucokinins in the nervous systems of the insects examined was compared

with the distributions of Lys-vasopressin immunoreactive cells in N. cinerea and

A. domesticus, and the distribution of diuretic hormone in M. sexta. The results

indicated that Lys-vasopressin immunoreactive cells, which may also be

involved in diuresis in some insects, co-localized in the same neurosecretory cells

in the abdominal ganglia with the leucokinins in N. cinerea, A. domesticus and M.

sexta. M. sexta diuretic hormone co-localized with leucokinin in the same

neurosecretory cells in the abdominal ganglia of M. sexta.

in 04 Table 1. Known amino acid sequence of diuretic peptides

Peptides Sequences

P-DP T G S G P R L R I JNPLD V T. R 0 R L L L E I A R R R M R n R 0 D 0 I O A N R E T T. 0 T I-NHo

L-DP M £ M G P R T. R T V N P M D V T, R o R L L L E I A R R R T. R n A K E 0 I R A N R D F T. 0 0 I-NHO

A-DP T G A 0 R T. R T V A P L D V T. R 0 R L M N E L N R R R N R K T. 0 G R R I 0 0 N R 0 T, T. T R I-NHT

M-DP I R M P S L S I D L P M S V L R 0 K L S L E K E R K. _V H A L R A A A N R N F L N D I-NH2

m-dph S F S V N P A V D I L Q H R Y M E K_ _V A Q N N F L N R I-NH2.

* A-DP Acheta diuretic peptide. L-DP Locusta diuretic peptide. M-DP Manduca diuretic peptide. P-DP

leucophaea diuretic peptide. Underline indicate the identical amino acids sequence.

26

Table 2. Known amino acid sequence of leucokinins and related neuro­

peptides

Peptides * Sequence

Lk-I AsD-Pro-Ala-Phe-Asn-Ser-TrD-GlvNHo

Lk-II Aso-Pro-Glv-Phe-Ser-Ser-Tr-D-GlvNHo

Lk-III AsD-Gln-Glv-Phe-Asn-Ser-TrD-GlvNHo

Lk-IV AsD-Ala-Ser-Phe-His-Ser-TrD-GlvNHo

Lk-V Glv-Ser-Glv-Phe-Ser-Ser-Trn-GlvNH2

Lk-VI PGlu-Ser-Ser-Phe-His-Ser-Tro-GlvMHo

Lk-VII AsD-Pro-Ala-Phe-Ser-Ser-Tm-GlvNHo

Lk-VIII Glv-Ala-AsD-Phe-Tvr-Ser-TrD-GlvNHo

A-I Ser-Glv-Ala-Aso-Phe-Tvr-Pro-Tro-GlvNHo

A-II Ala-Tvr-Phe-Ser-Pro-Tro-GlvNHo

A-III Asn-Phe-Lvs-Phe-Asn-Pro-TrD-GlvNHo

Lc-I Ala-Phe-Ser-Ser-Tro-GlvNHo

CDP-I Asn-Pro-Phe-His-Ser-Tm-GlvNH2

CDP-II Asn-Asn-Ala-Asn-Val-Phe-Tur-Pro-TrD-GlvNH2

* Lk=leucokinin; A=achetakinin; Lc=locustakinin; CDP=Culekinin

depolarizing peptide. See text for references.

27

MATERIALS AND METHODS

Experimental animals

Since leucokinins were originally isolated from a cockroach (L. maderae),

another cockroach (N. cinerea) was tested for the presence of leucokinin-like

peptides. In order to see whether the leucokinin IV which is the peptide has high

effect on diuresis antiserum would recognize leucokinin-related peptides in other

insect species and how widely the leucokinin-like peptides were distributed in

the insects, five additional insect species representing several orders were also

investigated: Acheta domesticus (Orthoptera), Schistocerca americana (Orthoptera),

Manduca sexta (Lepidoptera), Aedes aegypti (Diptera) and Apis mellifera

(Hymenoptera)

Cockroaches (N. cinerea) were reared on dog chow and water in the

laboratory at room temperature. Mosquitoes (A. aegypti of the Rock strain) were

reared in the laboratory according to Shapiro and Hagedorn (1982). Locusts (S.

americana), reared on wheat leaves were obtained from Dr. E. A. Bernays

laboratory (University of Arizona, Tucson). Tobacco hornworm (M. sexta) were

obtained from the laboratory in Division of Neurobiology (University of Arizona,

Tucson). Adult honey bees (A. mellifera) were obtained from the Carl Hayden

Bee Research Center in Tucson. Crickets {A. domesticus) were obtained from

Flukers Cricket Farm, Baton Rouge, LA.

28

Antiserum preparation

Conjugate preparation. Since leucokinin is a very short peptide (only 8

amino acids), it is too small to induce antibody production. Such peptides need

to be conjugated to a carrier protein that is immunogenic in order to generate

antisera. Using glutaraldehyde (Harrow and Lane 1990), leucokinin IV

(Peninsula Laboratories, Belmont, CA. USA) was conjugated to bovine

thyroglobulin (St. Louis, MO. USA) as the carrier protein. Thyroglobulin (3.5

mg) was dissolved in 1 ml phosphate buffered saline (PBS: 0.15M, pH 7.5).

Leucokinin IV was dissolved in 50 jxl H2O and added to the thyroglobulin

solution. Glutaraldehyde (2 ml of 0.25%) was then slowly added into the

solution containing thyroglobulin and leucokinin TV. After one hour at room

temperature, 10 ml of 1 M glycine in PBS (0.15M, pH 7.5) were added in to stop

the reaction. After 1 hour, the conjugate mixture was dialyzed against PBS

(0.15M, pH 7.2) for 2 days at 4°C with 4 solution changes. The conjugate was

stored at -20°C.

Rabbit injection and bleeding. Two rabbits (New Zealand strain, female)

were used to raise the antisera. Before injection, 220|i.g of conjugate (1 ml

conjugate solution) was mixed with 1 ml PBS and added to a pre-warmed (37° C

water bath for 10 min.) vial of RIBI adjuvant (Hamilton, MN, USA) containing 0.5

mg of monophosphoryl lipid A, 0.5 mg synthetic trehalose dicorynomycolate, 0.5

mg cell wall skeleton, 0.04 ml squalene and 0.004 ml monooleate (Tween 80) and

mixed. RIBI function as Freund's complete but it is not harmful to the rabbits.

Each rabbit was injected with 1 ml of the mixture at multiple sites along the back

bone, representing 150-200 |xl per injection site. Six weeks later, 130|xg (about

29

600|il) of conjugate were mixed with 1 ml of Freund's incomplete adjuvant

(lacking killed mycobacterium) and 0.4 ml of PBS and mixed. Each rabbit was

injected with 1 ml of the homogenate.

The first bleeding was done one week after the second injection. The

blood was taken from the central ear vein with a cannulated needle and a 50 ml

plastic syringe. A light bulb was used to increase the temperature of the ear to

stimulate the blood circulation. Booster shots were given four weeks after each

bleeding.

Antisera purification. About 25-30 ml of blood was obtained from the first

rabbit and 10 ml of blood was obtained from the second rabbit after each

bleeding. Blood was allowed to coagulate at room temperature for about 5

hours. After centrifugation at 10,000 rpm/min., the serum was collected, divided

into aliquots and stored at -70°C. A total of eight bleedings was obtained from

one rabbit, and six from the other rabbit.

Antisera labeling. Two kinds of labels were used in our experiment, a

peroxidase-antiperoxidase (PAP) label and a fluorescent label. In order to test

whether two different peptides (leucokinin and vasopressin or leucokinin and

diuretic hormone of M. sexta) co-localize in the same neurosecretory cells, the

peptides were labeled differently. In double labeling experiments, one peptide

was labeled indirectly [the peptide was first recognized by its antiserum (raised

in rabbits) and the IgG of the antiserum was recognized by anti-rabbit

immunoglobulins IgG labeled with fluorescent rhodamine]. The second peptide

was recognized directly by labeling the IgG with fluorescein isothiocyanate.

Anti-leucokinin IgG and anti-C-terminal diuretic hormone IgG were partially

30

purified and conjugated with fluorescein isothiocyanate (FITC) using a kit from

Boehringer Mannheim (Indianapolis, IN, USA) and kindly provided by Jan

Veenstra. The FITC to anti-leucokinin IgG protein ratio was 2.56 and to anti­

diuretic hormone IgG protein ratio is 2.43.

Tissue section staining

The central nervous system was dissected from the insects under saline

(0.9% NaCl) and fixed in GPA (1 ml 25% glutaraldehyde, 40 (J.1 glacial acetic acid,

3 ml saturated aqueous picric acid) or Bouin's mixture (1 ml paraformaldehyde, 1

ml glacial acetic acid and 3 ml saturated aqueous picric acid) for 4 to 5 hours.

The tissues were then washed with 70% ethanol until the yellowish color

disappeared and dehydrated in increasing concentrations of ethanol, (80%, 90%,

95%, 100% and 100%) for 15 min. each, then cleared in toluene once for 30 min.

and xylene twice for 30 min. Tissues were incubated at 60 °C in a mixture of 1:1

for 15 min. and then in 100% paraffin for 15 min. before being oriented and

allowed to cool in embedding paraffin.

The embedded tissues were sectioned at 7 jxm with a microtome and then

spread on a water bath at 45° C and affixed to slides that coated with one drop of

10 (xl 1% poly-L-lysine spread on a slide and held at room temperature for at least

24 hours before use. The coated slides were warmed on a slide warmer to 60° C

and 1 ml of H2O was applied to the slide creating a small water bath to which the

sections were transferred and incubated overnight on a slide warmer before

staining.

Peroxidase-antiperoxidase (PAP) section staining. The staining procedures

were according to Schooneveld and Veenstra (1989). The slides with tissue

sections were deparaffinized in xylene twice, for 3 min. each time and hydrated

through decreasing concentrations of ethanol by 3 min. in each (100%, 100%,

95%, 90%, 80% and 70%). The slides were then washed in water for 10 min.,

twice in PBS (0.15 M, pH 7.4) for 5 min. and finally in PBS for 15 min. Slides were

then transferred to petri dishes lined with wet paper to maintain humidity.

Sections were blocked by 10% normal goat serum for 10 min. Anti-leucokinin IV

antibody (diluted 1:4000 in PBS) was then applied to the slides and incubated

overnight at 4 °C. The slides were washed twice for 10 min. in PBS and

transferred back to the humidifer. After a second blocking step (10% NGS for 10

min.), the anti-rabbit immunoglobulins IgG (Sigma, Louis, MO, USA) (diluted

1:50 in PBS) were incubated with sections for 60 min. at room temperature.

Slides were washed in PBS for 10 min., and incubated with rabbit PAP complex

(Sigma, Louis, MO, USA) (1:200) for 60 min. at room temperature. Slides were

washed in PBS for 10 min. and Tris buffer (Tris-HCl 0.05 M, pH 7.6) for 5 min.

The substrate of 5 mg of 3,3'-di-amino-benzidine-tetra-hydrochloride (DAB) was

dissolved in 10 ml Tris buffer, and hydrogen peroxide was added to a final

concentration of 0.01% just before use, then applied to the slide for 5 min. After a

single rinse in water, stained tissues were dehydrated in increasing

concentrations of ethanol (70%, 80%, 90%, 95%, 100%, 100%) for 3 min. each,

cleared in xylene twice for 3 min. each, mounted with DPX mountant (Fluka

chemie AG, Switzerland) and covered with a cover slip. The slides were dried in

a 37 °C incubator for 20 hours.

32

Fluorescent labeled section staining. The nervous tissues were fixed in

Bouin's mixture as described above. Tissues were first incubated with antisera

against leucokinin-IV overnight, then they were washed in a PBS bath twice for

10 min. each and the anti-rabbit immunoglobulins IgG L- Rhodamine conjugate

(Boehringer Mannheim Biochemicals, Indianapolis, IN, USA) (1:200) were

applied and incubated for two hours. The slides were washed in a PBS bath

twice (10 min. each) and cleared in 55% glycerin diluted with carbonate buffer

(0.05 M carbonate-bicarbonate, pH 9.5) for 30 min., mounted with 80% glycerin,

covered with a cover slip and observed under a Nikon optiphot fluorescent

microscope. To determine co-localization, the glycerin was washed away after

the first observation and FITC-labeled anti-leucokinins or anti-C-terminal

diuretic hormone IgG (1:100) was applied to sections and incubated for 4 hours at

room temperature. Washing and further treatment was then repeated as above.

Whole mount staining

The staining procedures were those used by Davis (1989). The central

nervous system was dissected as described above and fixed in 4%

paraformaldehyde in po4 buffer [(paraformaldehyde (10 g) was first dissolved in

distilled H2O (110 ml), heated to 60° C with stirring for 10 min. Drops of NaOH

(IN) were added until the solution became clear and distilled H2O was added to

125 ml and mixed with PO4 buffer (125 ml, 0.2 M, pH 7.4).] Tissues were fixed

overnight at 4°C, then washed at room temperature with PBSAT (0.15M PBS, pH

7.4, 0.1% sodium azide and 0.5% Triton X-100) 6 times, 1 hour each with

agitation. Tissue remained in the final wash overnight at 4 °C. Tissue were

blocked with 2% (NGS) in PBSAT at room temperature for 5 hours. The tissues

33

were soaked in primary antisera (anti-leucokinin IV antibody) which was diluted

in PBSAT/2% NGS with agitation at room temperature overnight. The

preparation was washed 6 times with PBSAT for 1 hour each time and incubated

with fluorescent-labeled secondary antibody (anti-rabbit IgG 1:200) overnight at

4° C. Tissue was washed 6 times and then cleared in a glycerin series of 40%,

60% and 80%, covered with a cover slip and observed under a fluorescent

microscope. If co-localization was to be tested, the glycerin was washed out with

PBS and the tissues were incubated with the FITC labeled leucokinin antibodies

or C-terminal antibodies (1:100) over night at room temperature. After washing

with PBSAT 6 times for 1 hour each, tissue was cleared with a glycerin series of

40%, 60% and 80%, covered with cover slide and observed under a fluorescent

microscope.

Two types of controls were performed.

CI. Diluted leucokinin antisera (1:1000) was incubated with 10 nmol

leucokinin overnight before incubation with tissue; and

CII. During the staining process, the first antiserum was omitted. If the

staining was abolished, then the primary antibody was binding specifically.

Enzyme-linked immunosorbent assay (ELISA) of leucokinins

ELISA was used to test the specificity of the antiserum against leucokinins

and to determine the concentrations of leucokinins in the nervous tissues.

Leucokinin IV was conjugated to bovine serum albumin (BSA) using

iodoacetic acid anhydride as described by Wetzel et al. (1990). In this system,

peptides are first reacted with iodoacetic acid, which occurs preferentially at the

N-terminal of the peptide, and then the iodoacetyl-peptide is reacted with a thiol

group. Iodoacetic acid anhydride (250 mg) was dissolved in 2 ml of dry

tetrahydrofuran (THF) to a concentration of 350 mM and aliquots (200|il each)

were placed at -20 °C in Eppendorf tubes, which were stored in 50-ml plastic

screw-top conical tubes containing Drierite. Stored in this way, reagents have

been reported to be stable for at least 9 months. Leucokinin (0.2 mg) was

dissolved in 0.4 ml of 2-(n-morpholino) ethanesulfonic acid (MES) buffer (0.1 M

MES, pH 6.0). Iodacetic acid anhydride (4 |il) in tetrahydrofuran was added and

vortexed for 3 times for each for 3 min. BSA (5 mg) in 7 ml TRIS-HC1 buffer [(1

M Tris-HCl, 1 mM ethylenediaminetetraacetic acid (EDTA) and 1 mM DL-

dithiothreitol (DTT) (pH 7.5)] was added and incubated at room temperature for

1 hour. The mixture was dialyzed against 200 ml buffer (10 mM NaOAC, 0.25

mM EDTA, pH 5.4) for 24 hours at 4° C. A mixture of 4 ml was obtained after

dialysis was completed. 475 (il of a buffer (1M TRIS-HC1,100 mM EDTA, pH 8.0)

was added and incubated at 37 °C for 20 hours. The final volume of the

conjugate was about 5 ml and stored at -70 °C.

Testing the conjugate. The conjugate was diluted in the following series:

250,125,50,25,10,5, and 2 ng/50 |ol. The different concentration of conjugate

was loaded in a 96 well-ELISA plate at 50 (0,1/well and incubated for 3 hours with

agitation in a 37 °C incubator. After being washed with 0.05% Tween-20 in PBS

for 5 min, 200(4.1 of 1% NGS in PBS was added to the wells, the plates were

incubated for 1 hour at room temperature to block the non-specific binding.

Antisera against leucokinin were diluted 10,000 to 500,000 fold with 1% NGS.

Diluted antiserum (100 |nl) were added to each well to form different

35

combinations of concentrations between antiserum and conjugate. Plates were

incubated at 4 °C overnight with agitation. The plates were then washed with

0.05% Tween-20 in PBS three times for 5 min each with agitation at room

temperature. Anti-rabbit IgG (Kpl, MD, USA) diluted to 1:1000 (70 |xl) was

added in each well and incubated for one hour. The plates were washed with

0.05% Tween-20 in PBS four times for 5 min each with agitation at room

temperature. The developer (Kpl, MD, USA) (a mixture of 50%

tetramethylbenzidine (TMB) peroxidase solution and 50% peroxidase solution B)

(75 (ill) was added in each well and incubated for 30 min, then stopped with of 75

|xl of 1 M H3PO4. The optic densities were determined on the Biotek ELISA

reader at 450 nm.

Establishing a standard curve for leucokinins. The optimum concentrations

for the antiserum was 1:250,000 and the conjugate was 10 ng/well. The

competitive ELISA was then used to establish a standard curve. Conjugate in 50

[0.1 PBS volume (10 ng) was coated to each well of the ELISA plate as described

above. Standard concentrations of leucokinin IV or the test samples extracted

from nervous tissue were added in order to the well (50 jil/well). 50 |xl of

1:500,000 anti-leucokinin antiserum was then added to each well and incubated

at 4 0 C overnight. The subsequent procedures were followed as above. By

comparing the optical density of samples with the optical density of the standard

curve; the concentrations of leucokinins in the samples were calculated as fmol

per animal equivalent according to the standard curve.

36

HPLC fractionation of N. cinerea neuropeptides

Tissue preparation. The central nervous system of N. cinerea was dissected

in 0.9% saline and separated into brain, abdominal ganglia, optical lobes, and

combined corpora cardiaca and corpora allata complex and stored in Eppendorf

tubes separately on dry ice. Tissues were homogenized with a glass

homogenizer in 1 ml of Bennett's mixture (1% NaCl, 5% formic acid, 1%

trifluoroacetic acid (TFA) and 1 M HC1 in water) with the addition of 50 |xl of 25%

thiodiglycol (Veenstra 1989). The extract was centrifuged and the pellet re-

extracted in 0.5 ml of Bennett's mixture. The combined supernatants were loaded

on a previously activated and equilibrated C-18 Sep-Pak (Waters Associates, MA,

USA). The Sep-Pak was washed with 5 ml water and 5 ml of 13% acetonitrile,

and the peptides were eluted with 5 ml of 32% acetonitrile (all eluants contained

0.1% TFA). The eluant was reduced in volume to about 1 ml by vacuum

centrifugation, diluted with 2 ml 0.1% TFA and further separated by high-

performance liquid chromatography (HPLC).

HPLC. The HPLC apparatus consisted of a Beckman system Gold

programmable solvent module 126 and a Scanning Detector Module 167.

Separations were performed on an analytical Beckman 4.6 mm x 25 cm C18

column and precolumn. Solvents consisted of 0.1% TFA in water (A) and 0.1

TFA in 65% acetonitrile (B). The column was eluted with 20% B for 10 min at a

flow rate of 1 ml/min. Material from Sep-Pak was injected and eluted using 20%

B for 10 min, followed by a linear gradient to 70% B for 50 min and then a linear

gradient to 100% B for 10 min. The elution rate was at 1 ml/min. Fractions were

collected every minute and aliquots were analyzed by ELISA for leucokinin

37

immunoreactivity. The fraction of immunoreactive peaks were diluted in series

and test their affinity with the anti-leucokinin IV antisera by ELISA.

38

results

Specificity of antisera

The specificity of leucokinin antiserum was tested by

immunocytochemistry and the leucokinin IV ELISA. Pre-immunization sera did

not react with the sections of the brain. Weak immunoreactivity was observed

using antiserum from the first bleeding (i.e., the antiserum could only be diluted

to 1:250). At the second bleeding, the antibody titer increased such that the

antiserum could be diluted to 1:2000. Preabsorption of the antisera with

leucokinin always abolished immunoreactivity.

There was no apparent difference between the antisera of the two rabbits

in immunocytochemical staining. When antisera were tested with ELISA, there

were differences in titer between the two rabbits and between different

bleedings. The antiserum from the eighth bleeding of one rabbit could be diluted

to 1:250,000 and was used in the most sensitive ELISA, enabling detection of

leucokinin IV concentrations down to 10 fmol/well. Competitive ELISA showed

that antisera bound to leucokinin IV and a single, presumably homologous,

HPLC fraction from the corpora cardiaca-corpora allata complex of N. cinerea.

Neither corazonin or M. sexta allatotropin reacted in this ELISA (Fig. 1).

39

1.2-

u>

-O— Standard -B— immunofraction peak

•A— Allatotropin

-A— Corazonin

1.0-

0.8-

% o J—•

0.4-

0.2 mil .0001 .001 .01 10 1 1 100 1000 10000

Leucokinin (pm)/well

Fig. 1. Anti-leucokinin IV affinities to leucokinin IV, HPLC

immunoreactive peak fraction from corpora cardiaca of Nauphoeta cinerea,

allatotropin and corazonin. Shown are the ODs for leucokinin IV competitive

ELISA with various concentrations of the substances shown. See Materials and

Methods for details.

40

Leucokinin homogeneity in N. cinerea

Different parts of the nervous systems of N. cinerea were extracted and

separated by HPLC. Fractions collected at 1 min intervals (see Materials and

Methods above) were tested for leucokinin-IV immunoreactivity by competitive

ELISA. Brain, corpora cardiaca and abdominal ganglia all showed a single

immunoreactive peak at an elution time of 35 min, indicating that the same

neuropeptide was detected by the antiserum (Fig. 2). The immunoreactive peaks

at 35 min from the brain, corpora cardiaca and abdominal ganglia were diluted

serially (concentration is calculated to tissue equivalent per well) to test their

affinity with anti-leucokinin antisera and the results showed that they all

specifically bound the antisera. Three fractions have the same shapes (Fig. 3)

further indicating that the same peptide was recognized by antiserum.

41

0.4

Brains 0.5

0.67

2.0

-*—f

'<n C 0.5 4> •o « °-67

To

Corpora cardiaca

Q. O

2.0;

0.4 Abdominal ganglia

0.5

0.67

2.0 30 40 50 60 10 20 70

Retention time (min)

Fig. 2. Leucokinin IV immunoreactivity of HPLC fractions from brains,

corpora cardiaca and abdominal ganglia of adult Nauphoeta cinerea. See text for

details. ODs have been transformed using the inverse function for greater

clarity. The major peak in all tissues was from fraction 35.

42

Leucokinin IV Brain frac.35 CC frac.35 Abd frac.35

0.9

0.8

0.7

3 £ 0.6 «3

I 0.5 o

0.4

0.3

0.2 .0001 ,01 10 .001 .1

L-IV pm/ well or tissue equivalent/ well

Fig. 3. Anti-leucokinin IV affinities with immunoreactive peaks in 35 min

from brains, abdominal ganglia and corpora cardiaca. Shown are the ODs for

leucokinin IV competitive ELISA with various concentrations of the leucokinin

IV or tissue equivalent of brain, corpora cardiaca and abdominal ganglia. See

detail for text.

43

Distribution of leucokinin and vasopressin immunoreactive patterns in

N. cinerea

The central nervous system of N. cinerea consists of the brain and ten

segmental ganglia; including the subesophageal ganglion, three thoracic ganglia

and six abdominal ganglia (Fig. 4). The retrocerebral complex includes the

paired corpora cardiaca and corpora allata. The cardiac nervous system contains

paired lateral cardiac nerve and segmental nerves. Anti-Lys-vasopressin and

anti-leucokinin-IV bind to identical neurosecretory cells in the brain and

abdominal ganglia.

Brain and retrocerebral complex. There were several groups of leucokinin-

positive cells and immunoreactive processes in the brain:

(1) a group of 100-120 median neurosecretory cells in the pars

intercerebralis (MNSC Figs. 4,5A) and their projection axons to the retrocerebral

complex through the nervi corporis cardiaca I (NCCI Fig. 4);

(2) a group of four lateral neurosecretory cells (LNSC) in each par laterum

and projection axons in the nervi corporis cardiaca II (NCCII) (Figs. 4,5B);

(3) a group of 10-15 inter-neurons at the base of each optic lobe (Fig. 5A);

(4) three pairs of neurons in each protocerebral hemisphere, two of which

were close to the optic lobes (Figs. 4,5A) and one pair in the posterior

protocerebrum which had the same location as cells in L. maderae as described by

Nassel et al. (1992), who called them dorsal neurons. Their axons appeared to

descend through all the neuromeres to the terminal ganglion;

(5) numerous processes in the central body complex, posterior

deutocerebrum, tritocerebrum and optical lobes (Fig. 4);

44

(6) the immunoreactive processes in the frontal ganglion;

(7) the axon terminals and some intrinsic cells in the corpora cardiaca

(Fig. 4).

Subesophageal ganglion. There were no leucokinin-immunoreactive cell

bodies in the subesophageal ganglion but processes within the ganglion were

stained.

Thoracic ganglia. In the prothoracic ganglion, three pairs of intensely

stained neurons were present in the anterior part of the ganglion, and a group of

lightly stained cells were present in the posterior (Fig. 5B). In the mesothoracic

ganglion, only four neurons were leucokinin-positive. The metathoracic

ganglion is fused with the first abdominal ganglion. In addition to the four

neurons which stained at the same position of the neurons as in the mesothoracic

ganglion, there were two pairs of leucokinin-positive neurosecretory cells in the

posterior ganglion (one pair in each side), which corresponds ontogenetically to

the first abdominal ganglion (Figs. 4,5D). The branches of the descending axons

also stained in all thoracic ganglia (Fig. 4).

Abdominal ganglia and perivisceral organs. In each of the abdominal ganglia,

one posterior pair of leucokinin-positive neurosecretory cells were found in each

side of the ganglion (Figs. 4,5E). Like the neurosecretory cells in metathoracic

ganglion, the axons of these cells projected from the ganglion through ventral

nerves to the transverse nerve, where they bifurcated. One branch entered the

transverse nerve and then faded away, while the other extended to the lateral

cardiac nerve, which also stained strongly (Fig. 6). The terminal abdominal

ganglion is fused with the penultimate abdominal ganglion, and had three

45

neurosecretory cells in each side of the ganglion (Figs. 4,5F).

46

Fig. 4. Distribution of leucokinin positive cells and processes in Nauphoeta

cinerea. AG 1-9: abdominal ganglia. MNSC: median neurosecretory cells. MSG:

mesothoracic ganglion. MTG: metathoracic ganglion. LNSC: lateral

neurosecretory cells. OLIN: neurons in optic lobes. PDN: posterior dorsal

neurons. TAG: terminal abdominal ganglion.

47

PTG

AG2-6

MSG

TAG

MTG

Hsif:

48

Fig 5. Leucokinin positive neurosecretory cells and processes in Nauphoeta

cinerea. (A) Brain tissue showing median neurosecretory cells (MNSC), lateral

neurosecretory cells (LNSC) and various processes. (B) First thoracic ganglion

showing six strongly immunoreactive neurons (arrows) and a group of weakly

reactive neurons in the posterior of the ganglion. (C) Second thoracic ganglion

showing a similar pattern. (D) Fused third thoracic and first abdominal ganglia

showing patterns similar to other thoracic ganglia and posteriorly, similar to

other abdominal ganglia. (E) Abdominal ganglion showing two pairs of

neurosecretory cells in each side of the ganglion and their axons projecting along

the ventral nerve (arrows). (F) Last abdominal ganglion showing three

neurosecretory cells at each side of the ganglion and immunoreactive processes.

Scale bars: all of them are 100 |j,m.

H|^HHHjB |nH^nnn

^H^^BHH|H NPIHHHMH HHBM n|Hn H^HB

HHHj nMHHB^nnn|^H|HiHS

•H^HB

HHHI HH HH hhh bbhhh H^HHI KHHHHHH| •

49

50

Fig. 6. Distribution of leucokinin positive cells and processes of

abdominal ganglia and their neurohemal release sites of Nauphoeta cinerea. AG:

abdominal ganglion; LCN: lateral cardiac nerve.

51

AG5

LCN

AG6 f 1 :< r -*/

52

Distribution of leucokinin and vasopressin immunoreactive patterns in

A. domesticus

The central nervous system of A. domesticus is very similar to N. cinerea

except that A. domesticus has seven ganglia in the abdomen.

Brain and retrocerebral complex. In the brain, the distribution of leucokinin-

positive neurons was similar to that for N. cinerea. There was a group of 40-60

median neurosecretory cells stained in the pars intercerebralis (Figs. 7, 8A) and

four lateral neurosecretory cells in each pars lateralis (Fig. 8A). The median

neurosecretory cells did not stain as intensely as lateral cells. There were two

pairs of neurons in each side of the posterior protocerebrum. Two of them

projected their axons to the subesophageal ganglion and descended through the

neuromeres to the terminal abdominal ganglion located as observed in N.

cinerea. A pair of interneurons and their axon branches close to the base of each

optic lobe were positive. Immunoreactive processes were also observed in the

central bodies and tritocerebrum of the brain. However, there were no

leucokinin-immunoreactive neurons observed in the optic lobes. Processes in the

frontal ganglion and corpora cardiaca also stained (Fig. 8F) but none were seen in

the corpora allata (Fig. 8G).

Segmental ganglia and their perivisceral organs. In the subesophageal

ganglion, several groups of neurons and their axons stained (Fig. 7). Six neurons

stained lightly in the prothoracic ganglion. The neuropiles and descending axons

stained heavily in all thoracic ganglia (Fig. 7). In the metathoracic ganglion,

which is fused with the first two abdominal ganglia, four pairs of

immunoreactive neurosecretory cells were observed in the posterior part of the

53

ganglion (Fig. 7), which corresponds ontogenetically to the first two abdominal

ganglia. In the abdominal ganglia, one pair of leucokinin-positive

neurosecretory cells were observed in each posterior part of the ganglion (Figs. 7,

8E). These cells projected axons out of the ganglion through ventral nerves to the

link nerve, where they bifurcated, one branch entering the transverse nerve, and

the other extended toward the dorsal nerve in the direction of the lateral cardiac

nerve (Figs. 8E, 9). In the first five abdominal ganglia, the cardiac nerves were

only lightly stained. In the last five abdominal ganglia, the axon branch to the

dorsal nerve extended to the cardiac nerve producing varicose branches that

form a neurohemal meshwork on each side of the heart (Figs. 8B, 9).

Antiserum against Lys-vasopressin recognized the same neurosecretory

cells as those stained by the antiserum against leucokinin (Figs. 8C, D).

Fig 7. Distribution of leucokinin positive neurosecretory cells and

processes in Acheta domesticus. SEG: subesophageal ganglion. Other

abbreviations as in Fig 4.

AG4-8

56

Fig. 8. Leucokinin and vasopressin positive cells and processes in Acheta

domesticus. (A) Anti-leucokinin reactive median and lateral neurosecretory cells

of the brain. (B) Processes in cardiac nerves. (C) Anti-leucokinin and (D) anti-

vasopressin reactive cells in the same tissue preparation from the third

abdominal ganglion. (E) Anti-leucokinin neurosecretory cells and processes of

abdominal ganglion. (F) Processes in the frontal ganglion. (G) Processes in the

corpora cardiaca. Scale bars: all of them are 100|xm.

HK|

WB&

HH WaM HHHB

D

Fig. 9. Distribution of leucokinin positive neurosecretory cells in

abdomiiial ganglia and their neurohemal release sites oiAcheta domesticus.

Abbreviations as in Fig 6.

AG6

LCN

AG7

60

Distribution of leucokinin immunoreactive pattern in S. americana

Central nervous system. The central nervous system of S. americana is very

similar morphologically to that of N. cinerea. But, the distribution of leucokinin-

positive neurons in the brain was quite different. Only one pair of

immunoreactive neurons on each side of the pars intercerebralis was observed,

and their axons could not be followed (Figs. 10,11A). No immunoreactive

neurons were found in the pars intercerebralis. There was one pair of positive

neurons in each lateral part of procerebrum. A group of interneurons close to the

base of the optic lobes, and processes of these neurons also stained (Fig. 10).

Another group of three interneurons and their axons stained in the tritocerebrum

(Fig. 10). Immunoreactive processes were also observed in the central body. In

the subesophageal ganglion, three groups of positive neurons were observed

(Figs. 10,11C). Two descending axons were found in the subesophageal

ganglion, however, the origin of these axons could not be determined. In the first

two thoracic ganglia, a pair of positive bilateral neurosecretory cells in the

anterior part of the ganglion and a group of positive neurons were present in the

middle of the ganglion (Fig. 10). The metathoracic ganglion is fused with the

first three abdominal ganglia. This fused ganglion has three bilateral triplets of

positive neurosecretory cells in the posterior region (Figs. 10,11B). In each

abdominal ganglion, a pair of leucokinin-positive neurosecretory cells was

stained in posterior region of each side of the ganglion (Figs. 10,11D). In the

terminal abdominal ganglion, there were no leucokinin immunoreactive neurons.

Neuropils and descending axons stained in all segmental ganglia.

Peripheral nervous system. The first bilateral triplet of cells in the

metathoracic ganglion projected their axons through the first ventral nerve to the

61

link nerve and returned via the transverse nerve, terminating in varicose

branches within the transverse nerve between the metathoracic ganglion and the

first abdominal ganglion. The whole structure has a shape like an inverted "Y"

(Fig. 12). The second bilateral triplet of cells project their axons through the

second ventral nerves to the link nerve and again return and form a ipsilateral,

dendritic-like arborization near the first abdominal ganglia (Fig. 12). The third

group of bilateral triplet neurons projected their axons through the third ventral

nerve and terminated in varicose branches within the third transverse nerve

which was located between the first and second abdominal ganglion (Figs. HE,

12). In each abdominal ganglion, leucokinin-positive neurons projected their

axons via the ventral nerves to the link nerve and terminated in the transverse

nerve, which is located in the anterior part of the next posterior abdominal

ganglion (Fig. 12).

62

Fig. 10. Distribution of leucokinin positive cells and processes in

Schistocerca americana. Abbreviations as in Fig. 4.

63

MTG

AG4-8

PTG TAG

MSG

64

Fig. 11. Leucokinin positive cells and processes of Schistocerca americana.

(A) Brain showing two pairs of strongly immunoreactive neurons which are not

the median neurosecretory cells, and two pairs of neurons in the lateral part of

the brain. (B) The last fused thoracic ganglion showing three groups of

immunoreactive neurosecretory cells and processes. (C) Subesophageal ganglion

showing immunoreactive processes. (D) Third abdominal ganglion showing two

pairs of immunoreactive neurosecretory cells and processes. (E) Neurohemal

release sites on the fifth transverse nerve. Scale bars: (A), (B), (C) and (D), 50)i.m;

(E), 100|xm.

66

Fig. 12. Distribution of leucokinin positive neurosecretory cells in

abdominal ganglia and their neurohemal release sites of Schistocerca americana.

NHO: neurohemal organ. Other abbreviations as in Fig 4.

MTG

AG4

AG5

68

Distribution of leucokinin immunoreactive pattern in A. aegypti

The distribution of leucokinin-immunoreactive neurons was very similar

between the larvae and adult in A. aegypti. In the brain, about ten

neurosecretory cells stained in the pars intercerebralis (Fig. 14A) and three

neurosecretory cells stained in each side of the pars lateralis (Fig. 13A).

Immunoreactive processes were observed in the central bodies and posterior part

of the procerebrum, but no immunoreactive interneurons were observed. The

median neurosecretory cells did not stain as strongly as the lateral

neurosecretory cells. This pattern was very similar to A. domesticus. There were

no leucokinin-immunoreactive neurons in the subesophageal ganglion. In the

posterior part of the fused thoracic ganglia of the larvae, two staining axons

ascended from the first abdominal ganglion but no cell bodies were stained (Fig.

13B) Some immunoreactive processes were observed in the thoracic ganglia of

the larvae (Fig. 13B). In the thoracic ganglia of the adult, there were two groups

of neurosecretory cells stained in the posterior part of the ganglion, which

corresponded to the first abdominal ganglion. The immunoreactive processes

were also observed in the thoracic ganglion of the adult. Three neurosecretory

cells stained in each side of the first abdominal ganglion of larvae; but in the

adult, two cells disappeared so that only four staining neurosecretory cells were

present in the first abdominal ganglion which fused to the thoracic ganglion

mass (Fig. 13A). For both larvae and adults, each abdominal ganglion had two

pairs of stained neurosecretory cells in the posterior part (Figs. 13,14B). In the

terminal abdominal ganglion of the adult, the location of the four positive

neurons differed slightly, one bilateral neuron was present in the anterior part

the ganglion and another median pair of neurons in the posterior part (Fig. 14C).

69

Axons of all immunoreactive neurons in each abdominal ganglion projected out

via ventral nerves to form a diffuse pattern.

Distribution of leucokinin immunoreactive pattern A. mellifera

No leucokinin immunoreactive neurons were found in the brain,

subesophageal ganglion and thoracic ganglion in adult A. mellifera. In each

abdominal ganglion, four positive neurosecretory cells were observed in each

side of the ganglion (Figs. 14E, 15). The axons of these neurons projected out via

the ventral nerve and terminated in a bulbous-like neurohemal site which was

very close to the ganglion. Leucokinin-positive material accumulated there (Figs.

14D, 15).

70

FIG. 13. Distribution of leucokinin positive cells and processes in Aedes

aegypti. (A) Adult. (B) Larva.

TG TG

AG1 AG2-7

72

FIG 14. Leucokinin positive cells and processes in Aedes. aegypti. and Apis

mellifera. (A) Brain of adult A. aegypti showing a group of immunoreactive

median neurosecretory cells and later neurosecretory cells. (B) 1st abdominal

ganglion of A. aegypti larva showing 6 immunoreactive neurosecretory cells. (C)

last abdominal ganglion of adult mosquito showing 2 pairs of immunoreactive

neurosecretory cells and neurohemal release sites. (D) Neurohemal release site of

leucokinin in A. mellifera. (E) 2nd abdominal ganglion of A. mellifera showing 8

leucokinin immunoreactive neurosecretory cells and location of neurohemal site.

Scale bars: (A), 100|j.m; (B), 50|im; (C), 100|im; (D), 25 |im; (E), 50|xm.

A B

, :• L N-S C

74

FIG 15. Distribution of leucokinin positive cells and processes in the

abdominal ganglia of Apis mellifera. Abbreviations as in Fig 12.

75

NHO

AG5

AG6

76

Distribution of leucokinin and diuretic hormone immunoreactive

neurons in M. sexta

In the larvae, a group of four cells stained lightly in the pars intercerebralis

(Fig. 16A). The location of DH-positive cells was very similar to the cells found

previously by Veenstra and Hagedorn (1991). However, the anti-leucokinin

staining was inconsistent, making it difficult to decide whether both peptides

were localized in the same four cells or not. The intrinsic cells of the corpora

cardiaca showed leucokinin immunoreactivity (Fig. 16G). No leucokinin-positive

neurons were found in the subesophageal ganglion, but immunoreactive

processes were observed. In each of the thoracic ganglia and first abdominal

ganglion, one of the leucokinin positive neurons in each side of the ganglion was

observed. From the second to the sixth abdominal ganglia, two pairs of

leucokinin positive neurosecretory cells were observed in the posterior part of

each ganglion (Figs. 16D, 17). The seventh abdominal ganglion is fused to the

terminal abdominal ganglion and each part had two pairs of bilateral leucokinin-

positive neurosecretory cells. The location of leucokinin-positive neurons in the

terminal abdominal ganglion was very similar to that of A. aegypti: one bilateral

pair was present in the anterior region of the ganglion and another pair in the

posterior region (Figs. 16B, 17). The peripheral nervous system also stained with

leucokinin antiserum. The positive neurons in the abdominal ganglia projected

their axons via the ventral nerves to the link nerve and then bifurcated with one

branch going to the dorsal region before fading away and another branch

projecting to the transverse nerve that stained heavily (Fig 18).

77

In the adult, a group of median neurosecretory cells in the pars

intercerebralis also stained lightly and inconsistently as in the larvae. The

intrinsic cells and some axons stained heavily in the corpora cardiaca. No

immunoreactive processes were observed in the subesophageal and thoracic

ganglia. In each abdominal ganglion, two pairs of leucokinin-positive neurons

were observed in the posterior ganglion. In the terminal abdominal ganglion,

four pairs of leucokinin positive neurons were observed. The peripheral nervous

system was not investigated in the adult.

The distribution of the diuretic hormone-positive neurons in M. sexta was

compared with leucokinin-positive neurons in the nervous system using double-

labeling methods. Because staining of leucokinin antiserum in the brain of both

larvae and adult was inconsistent, co-localization of these two peptides could not

be determined in the brain. Both antisera stained the corpora cardiaca but

occupied different sites: Antiserum against leucokinin stained intrinsic cells of

corpora cardiaca and their axons, antiserum against diuretic hormone stained the

terminal of NCCI (Figs. 16G, H). In the abdominal ganglia, the antiserum against

diuretic hormone stained the same cells as the antiserum against leucokinin

except in the terminal abdominal ganglion (Figs. 161, J). However, one pair of

cells that was only lightly stained with anti-diuretic hormone was not easily

observed in larvae (Fig. 16J) or adults. This staining difference could be observed

in the staining of the transverse nerve of the larvae (Fig. 16K, L). In terminal

abdominal ganglion, only two cells in anterior part of the ganglion stained both

leucokinin and diuretic hormones. Two peptides localized different cells in

posterior part of the ganglion (Figs. 16C, D).

78

Fig. 16. Leucokinin and Mrtwduca-diuretic-hormone immunoreactive

neurons of Manduca- sexta. (A) Anti-leucokinin reactive neurons in the brain of a

third instar larva showing three of four median neurosecretory cells. (B) Fourth

abdominal ganglion of larva with two pairs of neurosecretory cells and

transverse nerves. (C) Terminal abdominal ganglion of larva with leucokinin

positive neurons and (D) Terminal abdominal ganglion of larva showing diuretic

hormone immunoreactive neurons at different locations. (E) Rhodamine anti-

leucokinin staining and (F) fluorescein anti-diuretic hormone staining of the

brain of the pharate adult. (G) Rhodamine anti-leucokinin staining and (H)

Fluorescein anti-diuretic hormone staining of the corpora cardiaca of the pharate

adult. (I) Rhodamine anti-leucokinin staining and (J) fluorescein anti-diuretic

hormone staining of the fourth abdominal ganglion of the pharate adult. (K)

Rhodamine anti-leucokinin staining and (L) Fluorescein anti-diuretic hormone

staining of the third transverse nerve of larva. Scale bars: (A), 50|U.m; (B), (C) and

(D), 100 (xm; (E), (F), (G), (H), (I), (J), (K) and (L), 100 |im.

F

ogm*

Fig. 17. Distribution of leucokinin positive neurosecretory cells in

abdominal ganglia and their neurohemal release sites oiManduca sexta larva.

Abbreviations as in Fig. 11.

AG3

NHO

AG4

AG5

AG6

TAG

82

discussion

Conservation of the leucokinin system

The significant differences and similarities among the species examine are

summarized in Figure 18. All six species had leucokinin-positive neurosecretory

cells. The distributions of two other peptides, Lys-vasopressin and the Manduca

diuretic hormone, were also compared to the distribution of leucokinin positive

neurons using double staining. The two peptides co-localized in neurons of

some species.

The leucokinins are a group of peptides originally isolated from the

cockroach L. maderae(Holman et al. 1986a, 1986b, 1987a, 1987b). Some these

structurally related peptides also have been isolated from the cricket A.

domesticus (Holman et al. 1991), the locust L. migratoria (Schoofs et al. 1992) and

the mosquito, A. aegypti (Hayes et al. unpublished data). Antiserum against

leucokinin I showed the same immunoreactive patterns in L. maderae (Nassel et

al. 1992) as in N. cinerea (Results).

The anti-leucokinin IV antiserum used in these experiments was found to

be highly specific in a competitive ELISA, to bind the same HPLC fractions from

several tissues of one species (N. cinerea), and to stain similar cells in abdominal

ganglia of all species tested. From these results, it seems reasonable that the

peptides that have been detected by anti-leucokinin IV all belong in the same

functional class, to which I will refer simply as "leucokinin," rather than

"leucokinin-hTce."

83

Fig. 18. Major similarities in the locations of leucokinin positive cells

in the six species examined. Also shown are the different patterns of

leucokinin release sites from the abdominal ganglia. Leucokinin and

vasopressin positive cells are shown as •. Vasopressin reactivity was

examined only in N. cinerea, S. americana and A. domesticus. For M. sexta

only: cells positive for leucokinin and M. sexta diuretic hormone are shown

by •; for diuretic hormone only by +, and for leucokinin only by 0.

Inconsistent staining for leucokinin is shown by A. Cells positive for

leucokinin only (with vasopressin and DH reactivity untested) are shown by

*. Fig. 18. Major similarities in the locations of leucokinin positive cells in

the six species examined. Also shown are the different patterns of leucokinin

release sites from the abdominal ganglia. Leucokinin and vasopressin

positive cells are shown as •. Vasopressin reactivity was examined only in

N. cinerea, S. americana and A. domesticus. For M. sexta only: cells positive

for leucokinin and M. sexta diuretic hormone are shown by •; for diuretic

hormone only by +, and for leucokinin only by 0. Inconsistent staining for

leucokinin is shown by A. Cells positive for leucokinin only (with

vasopressin and DH reactivity untested) are shown by *.

N.cinerae A.domesticus S.americana A.mellifera M.sexta A.aegypti

• 0 •• AA

85

Interspecific comparisons of the anatomy of leucokinin secretion,

transport and release

In the six insects examined, there were similarities and differences in the

distribution patterns of neurosecretory cells. The whole distribution pattern of

leucokinin in nervous system of N. cinerea is very similar to the L. maderae (Nassel

et al. 1992). In the brain of N. cinerea, A. domesticus, A. aegypti, there are median

neurosecretory cells and lateral neurosecretory cells showed immunoreactivity.

The distribution patterns among these immunoreactive neurosecretory cells were

very similar, except that in A. domesticus and A. aegypti, the median

neurosecretory cells stained not as strongly as lateral neurosecretory cells. In the

brain, the immunoreactive process in central bodies, posterior deutocerebrum

and tritocerebrum were observed and indicated that leucokinin peptides could

function as both neurotransmitter and neuromodulator in these species. The

neurosecretory cells in the brain are one of the major synthesis sites of these three

species. In S. americana, there were groups of cells staining in the brain but given

their location, it was difficult to decide if they were neurosecretory cells. In M.

sexta, a group of median neurosecretory cells showed immunoreactivity.

However, the staining was inconsistent. In A. mellifera, no immunoreactivity was

observed in the brain. So, it is difficult to decide if the brain is the major source

of leucokinin in these three species.

Although immunoreactivity was observed in corpora cardiaca in N.

cinerea, A. domesticus and M. sexta, immunoreactive intrinsic cells were only

observed in N. cinerea and M. sexta. So, corpora cardiaca function as the site of

synthesis as well as neurohemal release sites of the leucokinins in N. cinerea and

86

M. sexta. The corpora cardiaca of A. domesticus only function as a neurohemal

release site for leucokinins.

No immunoreactive neurons were observed in the subesophageal ganglia

and thoracic ganglia of A. aegypti, M. sexta and A. mellifera. Some

immunoreactive neurons and processes were observed in N. cinerea, A. domesticus

and S. americana. However, the distribution patterns were different. The

descending axons were observed in all segmental ganglia in these three species.

In N. cinerea and A. domesticus, the descending axons come from the dorsal

neurons, but in S. americana, the origin of these neurons could not be traced.

In the abdominal ganglia, the distribution patterns of neurosecretory cells

were very similar among the six species. It is likely that leucokinins are

synthesized in homologous neurosecretory cells in the abdominal ganglia.

The neurohemal release sites of leucokinin peptides in abdominal ganglia

were very different from one another. In N. cinerea, lateral cardiac nerves appear

to be the major release site of leucokinins because the lateral cardiac nerves

stained very heavily whereas the neurohemal organ in the transverse nerves did

not. In A. domesticus, the neurohemal release site also seems to be the lateral

cardiac nerve in the last five abdominal segments. In S. americana, the release

site seems to be the neurohemal organ in the transverse nerves in the abdominal

ganglia. In M. sexta, the neurohemal release site is also in the transverse nerve in

the abdominal ganglia. In A. aegypti, the neurohemal release site had a diffuse

pattern in the peripheral nervous system of each abdominal ganglion. In A.

mellifera, the neurohemal release sites formed a bulbous shape region near each

abdominal ganglion. Although the neurohemal release sites were different in

87

different species, the peptides were all transferred via ventral nerves to these

different release sites in abdominal areas. Given this distribution, leucokinin IV

seems mainly to function as a neurohormone with the difference of releasing

sites possibly reflecting their evolutionary history. Leucokinins were suggested

to be related to diuresis (Hayes et al. 1989, Coast et al. 1991). Early studies also

demonstrated that the diuretic factors are released from nerves in the abdominal

ganglia of Calliphora erythrocephala (Schwartz & Reynolds 1979), Rhodnius prolixus

and Glossina morsitans (Maddrell 1966; Maddrell & Gee 1974). Diuretic factors

present in abdominal ganglia have been investigated in P. americana (Mills 1967).

It is possible that groups of ubiquitous neurosecretory cells in the abdominal

ganglia are involved in diuresis. Since leucokinin immunoreactive

neurosecretory cells in the abdominal ganglia seem to be homologous cells, it is

possible that these cells are original source of diuretic peptides. The observation

that both vasopressin peptide and diuretic hormone of M. sexta localized the

same abdominal ganglion cells as leucokinin further strengthen this argument.

Significance of leucokinin and vasopressin co-localization

The leucokinins were suggested to be related to the diuresis of insects

based on bioassays (Hayes et al. 1989,1992, Coast et al. 1991). Vasopressin-like

peptides were also demonstrated to be involved in diuresis of A. domesticus and

L. migratoria (Strambi et al. 1978, Morgan 1987). Both leucokinin and vasopressin

was present in the different species tested. The results of my experiments

indicate that the distribution patterns of leucokinins were very similar to those of

Lys-vasopressin in the American cockroach, P. americana (Davis et al. 1992) and

the locust, S. americana (Davis person comun.). The co-localization experiment

88

presented here indicated that both peptides localized in the same neurosecretory

cells in abdominal ganglia of N. cinerea and A. domesticus.

In the locust, the leucokinin-like peptides are mainly synthesized in the

neurosecretory cells of the abdominal ganglia. The immunoreactive

neurosecretory cells in abdominal ganglia in S. americana also immunoreact to

Lys-vasopressin (Davis, personal comm.). Again these results indicate that

leucokinins and vasopressins are produced by the same cells in the abdominal

ganglia. Neurosecretory cells in abdominal ganglia related to diuresis were also

found in S. gregararia by Delphirt (1965) and L. migratoria by Proux (1978). The

position of leucokinin-immunoreactive cells in abdominal ganglia of N. cinerea is

very similar to the cells described in S. gregaria.

In the cricket, A. domesticus, the neurosecretory cells in the brain and

abdominal ganglia are the major sources of the leucokinin-like peptides. The

achetakinins are structurally related to leucokinins and were also isolated from

the cricket brain and corpora cardiaca which have been shown to be involved in

stimulating hindgut contraction of cockroach (Holman et al. 1990) and fluid

secretion by Malpighian tubules of crickets (Coast et al. 1991); thus the antiserum

might have recognized this group of peptides in these experiments. A

vasopressin-like factor has been demonstrated to cause diuresis in A. domesticus

(Strambi et al. 1978). The result of the co-localizing experiment presented here

suggests that both vasopressin-like peptide and leucokinins are present in the

same neurosecretory cells in the brain and abdominal ganglia. These neurons

might also be involved in secreting other diuretic factors.

89

The distribution patterns of leucokinins and vasopressins have been

investigated in grasshoppers, cockroaches, blowflies, and crickets. The results

indicate that both peptides are produced by the same group of neurosecretory

cells in each insect species. The neurosecretory cells involved in diuretic

hormone secretion suggested by Baudry (1968), and Berlind and Maddrell (1979)

were also immunoreactive to leucokinin antiserum (Veenstra, unpublished data).

According to the location of these neurosecretory cells in the abdominal ganglia,

these cells appear to be homologous. Since both leucokinins and vasopressins

affect diuresis (Hays et al. 1989, Proux & Rougon-Rapuzzi,1980), it is possible

that this group of neurosecretory cells secretes leucokinins and vasopressin-like

peptides as diuretic hormones for different specific functions related to diuresis.

Leucokinin and diuretic hormone neurosecretion in M. sexta

Leucokinins are present in both larvae and adults of M. sexta. The

distribution pattern of neurosecretory cells and axons was very similar between

larvae and adults. The major source of leucokinin in M sexta appears to be the

intrinsic cells of the corpora cardiaca and neurosecretory cells in the abdominal

ganglia.

The diuretic hormones of M. sexta have been isolated (Kataoka et al. 1989,

Blackburn et al. 1991), and their sources have been localized by antibodies raised

to these peptides (Veenstra & Hagedorn 1991). The results of the co-localization

experiments indicate that the diuretic hormone of M. sexta colocalized with

leucokinin in the abdominal ganglia, but one of the cells contained less diuretic

hormone than leucokinin. The results of co-localization further suggested that

these groups of neurosecretory cells in abdominal ganglia are involved in

90

diuretic hormone secretion. The poor staining of the brains by leucokinin

antiserum made it difficult to determine whether or not leucokinin co-localizes

with diuretic hormone in the brain. Both peptides were present in the corpora

cardiaca. However, they localized to different places. The intrinsic cells stained

for leucokinin only. The functional significance of the differences in distribution

has yet to be determined.

Comparison of the distribution pattern of leucokinin-like peptides with

FMRFamide-like peptides in insects

Leucokinin-like peptides have now been investigated in two cockroaches

(L. maderae, and N. cinerea, locust (S. americana), cricket (A domesticus), blowfly

(Phormia terraenovae, Nassel & Lundquist 1991) turnip moth (Agrotis segetum,

Cantera et al. 1992), mosquitoes (A aegypti) and honey bees (A mellifera). They

may be commonly present in insects as is the case with other groups of peptides

such as FMRFamide-like peptides, proctolin and cardioactive peptides.

Comparing the distribution patterns of FMRFamide-like peptide in the different

insects including the American cockroach P. americana (Endo et al. 1982), the

blowfly, Calliphora erythrocephala and C. vomitoria (Duve & Thorpe 1980,1982), the

locusts, S. gregaria (Myers & Evans 1985) and the locust L. migratoria (Ferber &

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Leptinotarsa decemlineata (Veenstra 1984), it is very interesting that the distribution

of immunoreactive neurons of FMRFamide was not unique as was found for the

leucokinins. This may be because the structure of the leucokinins is more

conserved, so immunoreactive neurons more closely reflect the physiological

differences.

92

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