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LSU Historical Dissertations and Theses
Graduate School
1980
Endosymbiosis in the Caecum of the Southern
Green Stink Bug, Nezara Viridula (L.).
Robert Paul West
Louisiana State University and Agricultural & Mechanical College
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Dissertations and Theses. 3575.
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8110429
WEST, ROBERT PAUL
ENDOSYMBIOSIS IK THE CAECUM OF THE SOUTHERN GREEN STINK
BUG, NEZARA VIRIDULA (L.)
The Louisiana State University and
Agricultural and Mechanical Col.
University
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Endosymbiosis in the Caecum o f the
Southern Green Stink Bug,
Nezara v irid u la ( L . )
A D issertation
Submitted to the Graduate Faculty o f the
Louisiana S tate U niversity and
A g ric u ltu ra l and Mechanical College
in p a rtia l fu lfillm e n t o f the
requirements fo r the degree of
Doctor o f Philosophy
in
The Department of Entomology
by
Robert Paul West
B .S ., Clemson U n iv e rsity, 1973
M .S ., Clemson U n iv e rsity, 1975
December 1980
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DEDICATION
To my Lord and S avior, JESUS CHRIST, through whom I
was able to complete th is work and to whom a ll the
honor fo r i t should be given.
n
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ACKNOWLEDGEMENTS
I would lik e to express my deepest appreciation to Dr. L. D. Newsom,
my major professor, and Dr. A. D. Larson (Department o f M icrobiology),
my minor professor, fo r c o -d ire c tin g th is work.
Dr. Larson was
responsible fo r in it ia t in g my in te re s t in th is study and fo r guidance
and encouragement a t those d i f f i c u l t times which occur during a ll
research.
Dr. Newsom allowed a great deal o f freedom in my studies and
was a constant source o f genuine in te re s t and encouragement.
I am also g ra te fu l to my committee members. Dr. A. M. Hammond,
Dr. B. H. Wilson, and Dr. S. D. Hensley fo r t h e ir advice and t h e ir
review of th is manuscript.
I would also lik e to express my g ratitu d e to Mrs. Karen Howard,
Dr. Rod Nelson, and Dr. M. D. Socolofsky ( a ll from the Department of
Microbiology) fo r t h e ir assistance with various facets of the electron
microscopy and in te rp re ta tio n of the re su ltin g micrographs.
I would
lik e to thank Dr. B ill Henk o f the School o f Veterinary Medicine fo r
the use o f t h e ir Zeiss 10 transmission electron microscope and fo r his
assistance and suggestions during th a t tim e.
I would lik e to express
special thanks to Kyle Hranitzky fo r his assistance in id e n tify in g the
caecal is o la te s .
I reserve my fin a l and inexpressible appreciation to my w ife , Judy,
who has p ersonified love during th is endeavor by bearing a ll thin g s,
believing a ll th in g s , hoping a ll thin g s, and enduring a ll things.
lo ve, she never f a i l s — to enrich and bless my l i f e c o n tin u ally.
iii
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Like
TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS ...................................................................................................
iii
TABLE OF CONTENTS...............................................................................................
iv
LIST OF TABLES......................................................................................................
v ii
LIST OF FIG U R E S ....................................................................................................... v i i i
ABSTRACT...................................................................................................................
ix
CHAPTER I - Electron Microscopy o f the Caecum o f the Southern
Green Stink Bug, Nezara v irid u la ( L . ) .............................
1
INTRODUCTION ...................................................................................................
2
MATERIALS AND METHODS
..............................................................................
5
Stink b u g s ...............................................................................................
5
Preparation fo r S E M ..........................................................................
5
Preparation fo r T E M ..........................................................................
5
RESULTS AND DISCUSSION ..............................................................................
7
SEM of
the midgut andh i n d g u t ........................................................
7
SEM o f
the c a e c u m ..............................................................................
12
SEM o f
the caecum fromdiapausing s tin k b u g s ...........................
18
TEM of
the c a e c u m ..............................................................................
19
REFERENCES CITED ...........................................................................................
23
CHAPTER I I - Is o la tio n and Characterization o f the Caecal Bacteria
of Nezara v irid u la
( L . ) .......................................................
25
INTRODUCTION ...................................................................................................
26
MATERIALS AND METHODS
..............................................................................
28
Is o la tio n o f the caecal bacteria ...................................................
28
Production o f antisera ......................................................................
29
iv
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Page
Agglutination t it e r s
..........................................................................
29
Characterization and id e n tific a tio n o f the iso lates . . . .
30
F e rritin -a n tib o d y la b e llin g studies ................................................
30
RESULTS AND DISCUSSION
..............................................................................
Is o la tio n o f the caecal bacteria
Agglutination t it e r s
33
.................................................
33
..........................................................................
33
Characterization and id e n tific a tio n o f the iso lates . . . .
34
F e rritin -a n tib o d y la b e llin g studies ................................................
37
REFERENCES CITED
..........................................................................................
42
CHAPTER I I I - E ffe c t of A n tib io tic s to Eliminate Caecal Bacteria
from a Stink B u g ......................................................................
44
INTRODUCTION
..................................................................................................
45
MATERIALS AND METHODS ..................................................................................
47
A n t ib io t ic s ..............................................................................................
47
Stink b u g s ..............................................................................................
47
Preparation fo r SEM..............................................................................
48
RESULTS AND DISCUSSION
..............................................................................
49
..........................................................................................
54
CHAPTER IV - Transmission of Caecal Symbionts to Offspring by
Nezara v irid u la ( L . ) ..................................................................
55
REFERENCES CITED
INTRODUCTION
..................................................................................................
56
MATERIALS AND METHODS ..................................................................................
58
Is o la tio n from e g g s ..............................................................................
58
A n t i s e r a ..................................................................................................
58
Slide agglutination tests ..................................................................
58
Preparation of eggs fo r SEM..............................................................
59
V
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Page
RESULTS...........................................................................................................
60
DISCUSSION.......................................................................................................
65
REFERENCES CITED ..........................................................................................
66
CHAPTER V - Apparent Absence of the Caecal Bacteria w ithin Some
Field-C ollected Specimens of Nezara v irid u la ( L .) . . .
67
INTRODUCTION ...................................................................................................
68
MATERIALS AND METHODS
..............................................................................
70
Stink b u g s ..............................................................................................
70
Is o la t io n s ..............................................................................................
70
S lide agglutination tests
..............................................................
70
RESULTS AND DISCUSSION ..............................................................................
71
REFERENCES CITED ..........................................................................................
76
V IT A ...........................................................................................................................
77
VI
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LIST OF TABLES
Page
CHAPTER IV
1.
Caecal b acteria on Nezara v ir id u la ( L .) eggs as
determined by s lid e agglutin ation te s ts .................................
61
CHAPTER V
F ield survey to confirm the presence of the two
caecal b acteria w ith in Nezara v ir id u la (L .) .........................
vn
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72
LIST OF FIGURES
Page
CHAPTER I
1.
Alimentary tr a c t o f Nezara v irid u la ...........................................
8
2.
The midgut and hindgut o f Nezara v ir id u la ..... ...........................
10
3.
The caecum and it s associated b a c te ria in
Nezara v i r i d u l a ...................................................................................
13
The caecal bacteria and t h e ir attachment
in Nezara v i r i d u l a ..........................................................................
16
Thin sections of the caecum and it s associated
bacteria from Nezara v ir id u la ......................................................
20
4.
5.
CHAPTER I I
1.
2.
Negative stains o f the caecal is o la te s
from Nezara v i r i d u l a .......................................................................
35
Thin sections o f the fe rr itin -a n tib o d y
la b e llin g of the caecum from Nezara v ir id u la
39
.....................
CHAPTER I I I
1.
A n tib io tic e ffe c ts on the caecum o f Nezarav irid u la . . .
51
CHAPTER IV
1.
Caecal bacteria inoculum on the surface o f
an egg of Nezara v i r i d u l a ..............................................................
V I 11
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62
ABSTRACT
Studies to in vestig ate possible endosymbiotic relationships w ithin
the southern green s tin k bug, Nezara v ir id u la ( L . ) , a major pest of
soybeans in Louisiana, were conducted.
Primary emphasis was placed on
the caecum, a region o f the d ig estive tr a c t previously associated with
m icrobial endosymbiosis in insects.
b a c te ria measuring 2 .5 by 0.5 /jni was
crypt w a lls .
By SEM, a monoculture of rod-shaped
observed adhering to the caecal
Examination o f additional specimens revealed various
morphological va riatio n s including cocci, elongated rods, and rods which
were apparently budding, often found w ith in the same specimen.
Dia­
pausing specimens appeared to be no d iffe r e n t from reproducing in d iv id ­
u a ls .
TEM o f th in sections provided fu rth e r evidence of the structure
o f the bacteria and t h e ir association with the caecal w a ll.
Subsequently, two d is tin c t bacteria of d iffe r e n t colonial types
were isolated from the caecum; both were rods.
One was coccoid in
appearance (designated ty p e -A ), while the other consisted o f longer rods
(designated type-B ).
Both were Gram negative and m o tile.
They were
id e n tifie d as Enterobacter aerogenes (type-A) and pro visio n ally as
Aeromonas sp. (ty p e -B ).
F e rritin -a n tib o d y la b e llin g of the caecum,
using antisera prepared against type-A and type-B b a c te ria , was used to
confirm th a t the is o la te s were id e n tic a l to those bacteria observed
w ith in the caecum.
Production of aposymbiotic specimens, to determine the value of the
symbiosis to the host, was attempted.
Stink bugs were fed a n tib io tic
trea te d soybeans, sampled a t 6 , 12, 24, 48, 72, and 96 h, and the
ix
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processed caeca were examined in the SEM.
The a n tib io tic s e ffe c ts could
be v isu alized as the bacteria became elongated, 5 to 8 pm long, and
d is tin c t detachment s ite s remained in the walls o f the caecum follow ing
b a c te ria l e lim in a tio n .
Their effectiveness peaked a t 24 to 48 h and
there was no evidence to in d icate th a t prolonged treatment beyond 96 h
would completely elim inate the f lo r a .
Therefore, the method was con­
sidered unsuitable fo r producing aposymbiotic specimens.
Confirmation th a t
v irid u la transm itted it s symbionts by smearing
it s eggs a t deposition was obtained by two approaches: (1) b a c te rial
is o la tio n s from eggs were tested by s lid e agglutination using the
previously produced an tis e ra; (2) eggs were processed and examined in
the SEM.
Both species o f caecal bacteria is o la te d (type-A and type-B)
were id e n tifie d from the surface of the eggs.
visualized on an egg in the SEM.
The bacteria were also
Both rods and coccoid forms were
present on the eggs and th e ir dimensions were comparable to those
previously observed w ithin the caecum.
A lim ite d f ie ld survey o f
v ir id u la was conducted to confirm the
consistent presence of both symbionts w ithin the caecum.
Is o la te s from
the insects' caeca were tested by s lid e agglutin ation as previously
done.
Both type-A and type-B bacteria were id e n tifie d from some f ie ld -
collected specimens, although only a single type was isolated in the
m ajo rity of caeca.
These re su lts were acceptable since i t is not un­
common fo r one b a c te rial species to outgrow and mask another in such
is o la tio n s .
However, the caecal bacteria were unexpectedly absent in
the remaining specimens, possibly in d ic a tin g th a t the symbiosis may be
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of m arginal, i f any, b e n e fit to the s tin k bug.
Nevertheless, the
cumulative data presented indicates an endosymbiotic re la tio n s h ip
between N. v ir id u la and it s caecal b a c te ria.
XI
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CHAPTER I
Electron Microscopy o f the Caecum of
the Southern Green Stink Bug,
Nezara v ir id u la (L. )
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INTRODUCTION
A discussion o f symbiosis should begin w ith a c le a r d e fin itio n of
the term.
By symbiosis, I r e fe r to the "appearances o f cohabitation
of unlike organisms", de Bary (1879).
Endosymbiosis re fe rs to "a
regulated cohabitation, occurring w ithout essential disturbances, bet­
ween two partners o f d iffe r e n t species, whereby the one is taken up in
the body o f the o th e r, usually more highly organized partn er, and the
mutual adaptation has attain ed such a degree of intim acy, th a t the
supposition, i t could be an arrangement b e n e fic ia l to the host organism,
is ju s tifie d " (Buchner 1953, p. 1 7 ).
Symbiosis research a c tu a lly began with observations o f Robert Hooke
in 1665 (in Koch 1967) on the symbiotic organ o f the human louse,
Pedicuius.
I t was much la te r th a t Blochmann (1 8 8 8 ), during his study
of b la t t id s , coined the term "bacteroids" to describe the symbionts
he observed.
Forbes (1892) studied the b acteria normal to the
hemipteroid d igestive organs and Glasgow (1914) s p e c ific a lly examined
the g a s tric caeca and t h e ir b a c te ria l inhabitants in the Heteroptera.
Kuskop (1 9 24 ), Rosenkranz (1939), and Schneider (1940) continued the
study of b a c te ria l symbiosis in the Heteroptera.
However, throughout
much of th is century, Buchner and his students have been in vestig ating
insect endosymbiosis and are responsible fo r the greatest part of our
present knowledge o f the subject.
In a more recent study, Goodchild
(1966) has developed some in te re s tin g hypotheses concerning b a c te rial
endosymbiosis in connection with the evolution o f the hemipteran
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alim entary canal.
A d e ta ile d histo ry o f th is f ie ld is treated by
Buchner (1965).
In general, the lit e r a t u r e agrees th a t the re la tio n s h ip between
host and symbiont often suggests mutual need.
Symbiotic associations
are in va ria b ly present when the insect host feeds upon specialized ,
or incomplete, d ie ts which lack some required n u tritio n a l component
(Boush and Coppel 1974; Brooks 1963a;
Buchner 1965; Koch 1956, 1967).
Schwemmler (1973) has c la s s ifie d animals (c e rta in hemipterans) and
plants into three d is tin c t types based on th e ir physicochemical com­
po sition .
A consumer, the in se c t, and a producer, it s food, can be
d ire c tly compared on the basis of th e ir type c la s s ific a tio n .
belong to the same type, no symbiosis e x is ts.
When they
I f the types d i f f e r ,
endosymbiosis exists to compensate fo r the d ifferen ce and the greater
the d iffe re n c e , the stronger the endosymbiosis.
Endosymbiosis may be c la s s ifie d as e x tra c e llu la r or in tr a c e llu la r .
In e x tra c e llu la r endosymbiosis, the symbionts may be lo calized in the
hosts' body in a number o f ways (Brooks 1963b, Buchner 1965, Koch 1967).
These include ferm entation chambers as in Tipula fla v o lin e a ta Meigen;
blind sacs o f the midgut as in the larvae of the o liv e - f ly , Dacus oleae
(Gmelin); and crypt guts as in Carpocoris fuscispinus Boheman which are
also called caeca ( s . , caecum).
Caeca are quite c h a ra c te ris tic o f the
Heteroptera, and in p a rtic u la r the Pentatomidae.
Steinhaus e t a l.
(1956) studied the b a c te ria l symbionts w ithin the caeca o f several
Heteroptera and successfully isolated the symbionts in several cases.
The southern green s tin k bug, Nezara v irid u la (L .) (Hemiptera:
Pentatomidae), is a major pest of soybeans in Louisiana (Jensen and
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Newsom 1972).
Adults overwinter in weeds and other cover to emerge in
the spring and develop on various w ild hosts, e.g. clovers.
During the
la te spring and summer they move to developing soybeans to feed which
re su lts in reduced bean y ie ld , o il content, and germination (Duncan and
Walker 1968, Todd e t a l. 1973, Thomas e t a l. 1974).
As a re s u lt o f studies by Foglesong e t a l. (1975) and Steinhaus
e t a l. (1956), and the established economic importance o f
v irid u la
in soybean production, I questioned what was known about the in te s tin a l
flo r a of th is pest and the possible symbiotic relationsh ips w ith in i t .
Since the lit e r a t u r e reported very l i t t l e
in it ia t e d .
in th is area, a study was
The primary emphasis was placed on the caecum, the s ite
associated with endosymbiosis in other Heteroptera.
This report is the f i r s t in a series which w ill deal with various
aspects o f endosymbiosis w ith in the caecum of
v ir id u la .
This work
consisted of a scanning electron microscopy (SEM) survey of the midgut,
hindgut, and caecum.
Transmission electron microscopy (TEM) was used
to elucidate the caecal u ltra s tru c tu re and it s associated symbionts.
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MATERIALS AND METHODS
Stink bugs
Specimens fo r electron microscopy were obtained from the f ie ld and
the labo ratory.
F ield specimens were co llected near E rw in v ille , Krotz
Springs, Port Barre, Angola, S t. G a b rie l, and Baton Rouge, LA, on such
hosts as clovers (T rifo liu m sp p .) , soybean, and corn.
Laboratory
specimens were obtained from a colony maintained by the Department of
Entomology, L.S.U . in Baton Rouge.
These s tin k bugs were fed corn
and green beans and the population was re g u la rly supplemented with
fie ld -c o lle c te d specimens a t the time o f th is study.
Both sexes were
processed and examined.
Preparation fo r SEM
The procedure used was modified from Foglesong e t a l. (1975) and
the id e n tific a tio n o f the regions o f the alim entary canal were
f a c ilit a t e d by the work of Malouf (1933).
Specimens were dissected in
0.1 M cacodylate b u ffe r (pH 7 .2 ) to expose the alim entary canal.
The
region o f in te re s t was lig a te d both a n te rio rly and p o s te rio rly and
perfused with 1% osmium te tro x id e - 2.5% glutaraldehyde f ix a tiv e in the
above b u ffe r.
A fte r 30 min, the lig a te d portion was excised and placed
in the f ix a tiv e fo r an addition al 20 min.
A fte r f ix a t io n , the tissue
was washed in b u ffe r fo r 1 h and dehydrated to 75% ethanol.
The caecal
bands were separated from the fourth stomach as short segments and cut
lo n g itu d in a lly .
The other tissues examined were cut in to doughnut­
shaped sections which were cut in h a lf to give two C-sections.
were dehydrated through 100% ethanol into acetone.
Tissues
A fte r c r it ic a l point
5
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drying, specimens were mounted on SEM stubs, coated w ith ca. 200 K
gold-palladium , and viewed in a HITACHI S-500 scanning electron
microscope a t 20 kV accelerating voltage and photographed with Polaroid
type 55 P/N film .
Preparation fo r TEM
Caeca were processed fo r TEM examination using both a standard
eucaryotic tissue schedule (Dawes 1971) and an abbreviated schedule
developed fo r th is work.
While the standard schedule u tiliz e d 3%
glutaraldehyde fo r fix a tio n and 1% osmium te tro x id e fo r p o s t-fix a tio n ,
both in Sorensen's b u ffe r, the abbreviated schedule combined these
steps by using the SEM f ix a t iv e described above.
The fix e d specimens
were dehydrated through 100% ethanol into propylene oxide and embedded
in Epon 812.
microtome.
Blocks were cut with a diamond knife on an LKB u lt r a ­
Sections were collected on 300-mesh copper grids with no
supporting film and stained with uranyl acetate fo r 5 min and Reynolds
lead c it r a t e (Reynolds 1963) fo r 15 min.
Sections were examined and
photographed with a RCA EMU-3G transmission electron microscope operated
a t 50 kV.
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RESULTS AND DISCUSSION
SEM o f the midqut and hindgut
Although the most lik e ly s ite o f endosymbiosis in N^. v ir id u la was
the caecum» the other regions o f the midgut and the hindgut were sur­
veyed fo r established microbial populations.
These included the
f i r s t , second, and fo u rth stomachs of the midgut and the ileum and
rectum o f the hindgut (F ig . 1 ).
The th ird stomach o f the midgut was
in d istin g u ish ab le from the second and they were examined together.
During dissections o f the s tin k bug, some measurements were noted.
The midgut averaged ca. 26 mm in length w hile the fourth stomach with
it s caecal bands was ca. 6 mm long.
Therefore, the fourth stomach, or
caecal region, averaged ca. 23% o f the length o f the midgut.
The in tern al structures of the f i r s t and second stomachs were
id e n tic a l
(F ig . 2A).
There were no established bacterial populations
present, although some digesting food m atter and possibly some m iscel­
laneous b a c te rial c e lls were observed.
The in tern a l stomach surface
consisted o f the protruding ends o f dig estive c e lls of the gut
epithelium (F ig . 2A).
The c e ll w alls o f several c e lls had ruptured to
expose and discharge th e ir granular contents.
Snodgrass (1935)
describes th is process as the simplest form of d is in te g ra tio n of the
dig estive c e lls .
Subsequently, the c e ll walls close and the c e lls
continue t h e ir d ig e s tiv e functions.
Protruding from the d ig estive c e lls
were spindle-shaped structures 5 to 10 jim long (F ig . 2B).
Such globules
were e ith e r s e lf-d is ru p tin g releasers o f secretion products (processes
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Fig. 1.
Alimentary t r a c t o f Nezara v ir id u la .
Crop (C) and
Malpighian tubules (Mt) are indicated by arrows.
F ir s t
stomach ( 1 ) , second and th ird stomachs ( 2 - 3 ) , fourth
stomach with caecal bands ( 4 ) , ileum ( I ) , and rectum (R)
are indicated by segments of the gut enclosed by pairs
o f arrows.
Bar = 2 mm.
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10
Fig. 2.
The midgut and hindgut o f Nezara v ir id u la .
(A)
D igestive c e lls o f the second stomach.
shaped globules a t high m agnification.
(D)
Ileum.
(E) Rectum.
Bars = 5 )jtn.
(B) Spindle-
(C) Fourth stomach.
Inset shows the re c ta l surface a t
higher m agnification.
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n
m
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12
o f holocrine secretion) or d isin te g ra tio n products follow ing extensive
normal secretion (Snodgrass 1935).
The globules were sparse in some
areas and quite dense in others, having the appearance o f "patches of
grass".
The lumen o f the fourth stomach was devoid o f any established
b a c te rial populations (F ig . 2C).
The surface was quite smooth and
the protruding ends o f the digestive c e lls observed previously were
absent.
Several miscellaneous b a c te ria , detached secretion globules,
and u n id e n tifie d digested matter were noted and apparently being
elim inated.
The hindgut lumen was also devoid o f any apparent bacterial popu­
la tio n s .
(F ig . 2D).
The ileum was s im ila r in appearance to the fourth stomach
Its surface was smooth except fo r some miscellaneous m atter
being elim inated.
The rectum, s p e c ific a lly the rectal sac, had an
irre g u la r surface (F ig . 2E).
At higher m agnification the expanding,
contracting nature o f th is structure was revealed (F ig . 2E, in s e t).
SEN of the caecum
The caecal region of the midgut includes the fourth stomach with
four caecal bands intertw ined around i t (F ig . 3A).
The bands are
attached to the stomach by th in connectives apparently with narrow
openings between them and the gut (Buchner 1965).
A longitudinal sec­
tio n o f a caecal band illu s tr a te s an in ternal structure of many cryp t­
lik e units which house the bacterial population (F ig . 3B).
The crypts
are separated by th in w alls ca. 0.5 to 2 |jm in thickness while the outer
caecal w alls may vary from ca. 1 to 6 jjm.
Fig. 30 represents part of
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13
Fig. 3.
The caecum and it s associated bacteria in Nezara v ir id u la .
(A) Caecal region cross section with a central fourth stomach
surrounded by caecal bands, indicated by arrows.
(B) Internal caecal crypts.
Bar = 50 ;jm.
wall pushed in to caecum proper.
tracheae.
Bar = 5 /im.
Bar = 100 ;jm.
(C) Outer caecal
Bar = 5 |im.
(D) Caecal
(E) B acterial rods w ithin the caecum.
Bar = 5 ^m.
Inset shows the rods a t higher m agnification.
Bar = 2 jjm.
(F) Coccoid forms w ithin the caecum.
Bar = 2 jum.
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14
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15
the outer wall which has been pushed into the caecum proper.
These
in tern a l crypts are responsible fo r the g a rla n d -lik e appearance of the
caecal bands.
The caecum has numerous tracheae on it s external surface
and in te r n a lly tracheae permeate the e n tire structure (F ig . 3D) perhaps
creating an aerobic environment.
The in tern a l supporting ridg es, or
ta e n id ia , w ith in each trachea are c le a rly evident.
I n i t i a l l y , an apparent monoculture o f rod-shaped bacteria were
observed adhering to the caecum (F ig . 3E).
They were ca. 2.5 by 0 .5 jjm,
well packed, and attached a p ic a lly to the wall o f the caecum.
Subse­
quently, coccoid forms 1 to 2 ^m in diameter were observed (F ig . 3F).
They were usually interspersed with the rods and thought to be
morphological varian ts o f them.
Continued examination o f the specimens
revealed more extreme morphological forms (F ig . 4A).
These included
la rg e r coccoid forms (3 to 4 ^m in diam eter), elongate rods (4 to 6 jjm
lo n g ), and rods which were apparently budding (F ig . 4B).
The extent of
budding was q u ite pronounced in some bacteria (F ig . 4 0 ).
The apparent polymorphism observed above was not unexpected.
The
lit e r a t u r e indicated th a t th is was present to varying degrees among
symbionts (Brooks 1963b, Buchner 1965, Richards and Brooks 1958).
Buchner (1965, p. 225) states th a t "Heteroptera symbionts often grow
in to formations which are f a r removed from the typ ical b a c te rial form".
The bacteria appear to be well attached to the caecum.
However, i f
a bacterium does detach fo r some reason, there remains a d is tin c t
depression a t the s ite of attachment (Fig. 4D).
In the same fig u re
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16
Fig. 4.
The caecal bacteria and t h e ir attachment in Nezara v ir id u la .
(A) Morphological forms o f the caecal b a c te ria.
Bar = 5 ^m.
(B) Elongate and budding rods w ith in the caecum.
(C) Budding caecal b a c te ria .
Bar = 2 ;jm.
remaining a fte r b a c terial detachment.
bacterium in the process o f detaching.
(D) Depressions
Arrow indicates a
Bar = 2 jtim.
shows the depressions a t higher m agnification.
(E) Diapausing stin k bug caecum.
Bar = 5 ^m.
Bar = 5 ^m.
Inset
Bar = 0.5 ;jm.
(F) Typical
rods and coccoid forms w ithin a diapausing caecum.
Inset shows
the fibrous glycocalyx m aterial prevalent in some specimens.
Bars = 5 ^m.
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17
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18
there is also a bacterium in the process o f detaching from the caecum.
At higher m agnification these depressions are quite smooth textured in
most cases (F ig . 4D, in s e t).
The mechanism of attachment is not known.
Yet in some specimens
(Figs. 3E, 4B, 4C) a fibrous m aterial was present which might represent
dehydrated polysaccharide fib e rs forming a glycocalyx used by bacteria
to adhere to substrates (Costerton e t a l. 1978).
J. W. Costerton
(personal communication) suggested th a t the strands (F ig . 48) were
ty p ic a l glycocalyx evident a fte r such specimen preparation in th a t the
strands are attached to the point o f o rig in .
He fu rth e r suggested th at
the detachment s ite s were not depressions in the caecum, but rath er in
a th ic k la y e r o f mucous, or g ly c o c a ly x -lik e , m aterial covering the
caecum and surrounding the c e lls .
Apparently these caecal bacteria are
p r o lif ic slime producers, probably comparable to his most p r o lif ic
slime producing Pseudomonas c u ltu res.
Further evidence o f th is slim e,
glycocalyx, production was observed in fe rr itin -a n tib o d y la b e llin g
studies.
Electron microscopy demonstrated an apparent th ic k mucous
layer between the c e lls and the f e r r i t i n la b e l.
The s itu a tio n as described would appear to be s im ila r to th a t in
cystic fib ro s is p atients where the causative bacteria conglomerate in
masses o f p ro te c tiv e slim e, or glycocalyx.
and adhere to the lung w a lls .
These masses are scattered
In the caecum, the masses of bacteria
l i t e r a l l y lin e the in tern a l c a v itie s of the s tru ctu re.
SEM of the caecum from diapausing s tin k bugs
Caeca from diapausing s tin k bugs appeared the same as non-diapausing
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19
specimens (F ig . 4E ).
The flo r a consisted o f comparable rods and coccoid
forms as well as the various polymorphic v a ria tio n s o f these (F ig . 4 F ).
The fibrous m a te ria l, suspected to be glycocalyx, was prevalent in
several specimens (F ig . 4F, in s e t).
TEM o f the caecum
Both the standard and the abbreviated tissue schedules resulted in
well preserved and d is tin c t th in sections.
Fig. 5A gives an o rie n ta tio n
to the general stru ctu re seen on the SEM and the micrographs to fo llo w .
The in ternal caecal stru ctu re is represented by the projection which
apparently is a section through a rid g e .
Mitochondria are present w ith ­
in th is tissue and the sectioned b acteria are in close association with
the caecum.
Although the attachment mechanism is probably a glycocalyx
composed o f polysaccharide, apparently i t is e ith e r not preserved or is
nondistinguishable by the embedding procedures used in th is study.
The
b acteria possess an outer c e ll w a ll, or outer membrane, and an inner
protoplasmic membrane in d ic a tin g they are probably Gram negative.
The
outer membrane, and in some places the inner membrane, was pulled away
from the c e lls in those sections using the standard tissue schedule.
This was believed to be due p rim a rily to the schedule used since the
membranes o f many c e lls from the abbreviated schedule sections were not
p u llin g away.
(F ig . 53).
Where i t did occur, only the outer membrane was involved
I t is in te re s tin g to note the differences in shape and
appearance of the b a c te ria , apparently due to sectioning a t d iffe r e n t
le v e ls .
The white regions represent the DNA and the black regions
represent the ribosomes.
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20
Fig. 5.
Thin sections o f the caecum and it s associated bacteria from
Nezara v ir id u la .
Bars = 1 ^m.
(A) Caecal ridge with several
attached b a c te ria , standard tissue schedule.
(B) Caecum and
attached bacterium, abbreviated tissue schedule.
and embedded bacterium, standard tissue schedule.
(C) Caecum
(D) Caecum
encircled by attached b a c te ria , standard tissue schedule.
(E) Budding bacterium without a septum, standard tissue
schedule.
Bacterium (b ), outer membrane (om), inner membrane
(im ), mitochondrion (m), and microtubules (m t).
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21
Æ
m
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22
In Fig. 5C the caecum was elevated above the surrounding area.
It
contains d is tin c t mitochondria and a bundle o f what appears to be
microtubules enclosed by a double membrane.
Within i t is an attached
bacterium which indicates th a t some b a c te ria l attachment does re s u lt
in an indentation of the surface o f the caecum.
In an enlargement of a s im ila r area (Fig. 5D), the caecum has two
d is tin c t bundles of microtubules surrounded by mitochondria.
Several
bacteria are in close association a t the periphery.
Fig. 5E illu s tr a te s a bacterium, apparently in the process of
budding.
However, no septum can be seen forming w ithin i t .
Micrographs
o f additional budding bacteria did not show developing septa.
At
present th is condition is unexplained.
In conclusion, the caecum of Nezara v ir id u la does possess closely
associated bacteria which form d is tin c t attachment sites and may have
an endosymbiotic re la tio n s h ip with the in sect.
Established b a c terial
populations are absent from the other regions of the midgut and the
hindgut.
Use o f the SEM has provided some in sig h t in to the in tern al
caecal structure and the associated b a c teria.
Thin sections have
fu rth e r elucidated the u ltra s tru c tu re of the caecum and it s adhering
b a c teria.
The data presented demonstrates a very in trig u in g aspect of
th is pest species' biology.
The resu lts add to our knowledge of th is
insect and may also be applicable to other species.
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REFERENCES CITED
Blochmann, F.,, 1888. Uber das regelmassige Vorkommen von
bakterienahnlichen Gebilden in den Geweben und Eiern verschiedener
Insekten. Z. B io l. 24: 1-15.
Boush, G. M ., and H. C. Coppel. 1974. Symbiology: Mutualism between
arthropods and microorganisms. In^ G. E. Cantwell ( e d .) . Insect
diseases, v o l. 2. Marcel Dekker, In c ., New York. 295 pp.
Brooks, M. A. 1963a. The microorganisms o f healthy insects.
L l E. A. Steinhaus ( e d .) . Insect pathology, v o l. 1. Academic Press
In c ., New York. 661 pp.
Brooks, M. A. 1963b. Symbiosis and aposymbiosis in arthropods.
Symp. Soc. Gen. M icrobiol. 13: 200-31.
Buchner, P.
1953. Endosymbiose der T iere m it pflanzlichen
Mikroorganismen. Verlag Birkhauser, Basel, Switzerland.
771 pp.
Buchner, P.
1965. Endosymbiosis of animals with plant microorganisms.
John Wiley and Sons, Inc. (In te rs c ie n c e ), New York. 909 pp.
Costerton, J. W., G. G. Geesey, and K. -J . Cheng.
s tic k . S c ie n tific American 238: 86-95.
1978.
How bacteria
Dawes, C. J. 1971. Biological techniques in electron microscopy.
Barnes and Noble, In c./H arp er and Row, Publishers, In c ., New
York. 193 pp.
de Bary, A.
France.
1879. Die Erscheinung der Symbiose.
Duncan, R. G ., and J. R. Walker.
green s tin k bug on soybeans.
Trübner, Strassburg,
1968. Some e ffe c ts o f the southern
Louisiana Agric. 12: 10-11.
Foglesong, M. A ., D. H. Walker, J r . , J. S. P u ffe r, and A. J. Markovetz.
1975. U ltra s tru c tu ra l morphology of some prokaryotic microorganisms
associated with the hindgut o f cockroaches. J. B a c te rio l. 123:
336-45.
Forbes, S. A. 1892. Bacteria normal to digestive organs o f Hemiptera.
I l lin o is State Lab. Nat. H is t. B u ll.
4: 1-7.
Glasgow, H. 1914. The g a s tric caeca and the caecalbacteria
Heteroptera. B io l. B u ll. 26: 101-70.
Goodchild, A. J. P. 1966.
Hemiptera. B io l. Rev.
Evolution of the alim entary
41: 97-140.
o f the
canal in the
23
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24
Jensen, R. L ., and L. D. Newsom. 1972. E ffect o f s tin k bug damaged
soybean seed on germ ination, emergence, and y ie ld . J. Econ.
Entomol. 65: 261-4.
Koch, A. 1955. The experimental elim ination o f symbionts and it s
consequences. E x p tl. P a ra s ito l. 5: 481-518.
Koch, A. 1967. Insects and th e ir endosymbionts. In_ S. M. Henry ( e d .) .
Symbiosis, v o l. 2. Academic Press In c ., New York. 443 pp.
Kuskop, M. 1924.
47: 350-85.
Bakteriensymbiosen bei Wanzen.
Arch. P rotistenk.
Malouf, N. S. R. 1933. Studies on the in ternal anatomy o f the s tin k
bug, Nezara v ir id u la ( L . ) .
B u ll. Soc. Roy. Entomol. Egypte
17: 96-119.
Reynolds, E. S. 1963.
The use o f lead c itr a te a t high pH as an
electron-opaque s ta in in electron microscopy. J. Cell B io l.
208-12.
17:
Richards, A. G ., and M. A. Brooks. 1958. In ternal symbiosis in
in sects. Ann. Rev. Entomol. 3: 37-56.
Rosenkranz, W.
1939. Die Symbiose der Pentatomiden (Hemiptera
H eteroptera). Z. Morphol. Okol. T iere 36: 279-309.
Schneider, G. 1940. Beitrage zur kenntnis der symbiontischen
Einrichtungen der Heteropteren. Z. Morphol. Okol. T ie re 36:
595-644.
Schwemmler, W.
1973. Ecological significance o f endosymbiosis:
o v e ra ll concept. Acta B iotheoretica 22: 113-19.
Snodgrass, R. E. 1935. P rinciples o f insect morphology.
H ill Book Co., In c ., New York. 667 pp.
An
McGraw-
Steinhaus, E. A ., M. M. Batey, and C. L. Boerke. 1956. B acterial
symbiotes from the caeca o f c e rta in Heteroptera. H ilg ardia
24: 495-518.
Thomas, G. D ., C. M. Ig n o ffo , C. E. Morgan, and W. A. Dickerson.
Southern green s tin k bug: Influence on y ie ld and q u a lity of
soybeans. J. Econ. Entomol. 67: 501-3.
1974.
Todd, J. W., M. D. Jellum , and D. B. Leuck. 1973. E ffects o f southern
green s tin k bug damage on f a t t y acid composition of soybean o i l .
Environ. Entomol. 2: 685-9.
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CHAPTER I I
Is o la tio n and C haracterization o f the
Caecal Bacteria o f Nezara v ir id u la (L .)
25
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INTRODUCTION
A fte r endosymbionts have been located w ithin an insect and studied
in s it u , the next logical step is to is o la te and grow the symbionts in
pure cultu re fo r more extensive in v itr o studies.
These would include
the characterization and id e n tific a tio n o f the microorganisms and
possibly the re in fe c tio n o f aposymbiotic specimens and the subsequent
re is o la tio n to f u l f i l l the c r it e r ia of Koch's postulates.
Attempts to is o la te endosymbionts have met with varying degrees of
success in past studies (Buchner 1965, Glasgow 1914, Koch 1967, Richards
and Brooks 1958, Steinhaus 1951, Steinhaus et a l. 1956).
In general,
there has been greater success in is o la tin g e x tra c e llu la r than in tr a ­
c e llu la r symbionts.
While basic cultu re media w ill often meet the
simpler requirements o f e x tra c e llu la r symbionts, in tr a c e llu la r symbionts
often require more specialized substrates to supply th e ir needs when
removed from w ithin the host's c e lls .
Brooks (1963) discusses some
generalizations to be considered in cultu ring in tra c e llu la r symbionts.
Is o la tio n o f the symbionts o f the plant-sucking Heteroptera is
rath er simple since they liv e e x tra c e llu la rly within the caecum o f the
host.
tr is tis
Glasgow (1914) succeeded in is o la tin g the symbionts o f Anasa
(De Geer), Alydus quinquespinosus Say, A. conspersus Montandon,
and Metapodius terminal is Dallas using squash ju ic e bo uillon.
Steinhaus
(1951) isolated the symbionts o f both Chelinidea tabulate (Burmeister)
and £ . v it t iq e r Uhler on ordinary n u trie n t agar and glucose n u trie n t
agar.
Subsequently, Steinhaus e t a l. (1956) isolated and characterized
26
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27
the symbionts o f Euryophthalmus cinctus c a lifo rn ic u s (Van Duzee),
Euschistus conspersus U hler, Anasa t r i s t i s
Uhler on the same is o la tio n media.
(De G eer), and £ . v it t ig e r
The bacteria o f C^. v it t iq e r and
JE. cinctus c a lifo rn ic u s were described as new species. Pseudomonas
excibis and £ . nactus, re sp ec tiv e ly .
In 1957, Huber-Schneider
(Koch 1967) is o la te d the symbionts o f Mesocerus marginatus L. using
liq u id bouillon plus 1% peptone, glucose liq u id b o u illo n , and n u trie n t
agar.
He described the various forms assumed by the bacteria in
d iffe r e n t media and conducted an extensive study o f th e ir physiological
c a p a b ilitie s .
Following the electron microscopy study of the caecal bacteria
w ith in the southern green s tin k bug, Nezara v irid u la ( L . ) , (West 1980),
the next step o f the research was to is o la te the symbionts fo r in v itr o
study.
Steinhaus et a l. (1956) provided an i n i t i a l approach to achieve
th is goal.
The work reported here consists o f three phases; the
is o la tio n o f the caecal b a c te ria , the ch aracterizatio n and te n ta tiv e
id e n tific a tio n of the is o la te s , and confirmation th a t the iso lates are
the symbionts observed w ith in the caecum by using a fe rritin -a n tib o d y
la b e llin g procedure.
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MATERIALS AND METHODS
Is o la tio n o f the caecal bacteria
Stink bugs were obtained from soybean fie ld s near Port Barre and
Angola, LA, and from a laboratory colony maintained on corn by the
Department o f Entomology, L.S.U.
The colony was supplemented re g u la rly
with fie ld -c o lle c te d specimens during th is study.
Is o la tio n s were
performed on both sexes on two separate occasions.
The f i r s t used
specimens from Port Barre and the colony; the second, specimens from
Angola and the colony.
Is o la tio n s were performed under aerobic conditions since the caecal
environment was probably aerobic as indicated previously (West 1980).
The procedure used was modified from Steinhaus e t a l.
(1956).
The legs,
antennae, and wings were removed, the specimen was mounted on an insect
pin, surface s te r iliz e d in 5.25% sodium hypochlorite, and rinsed in
s t e r ile water.
Dissecting instruments were held in 95% ethanol and
flamed before use.
mounted dry on wax.
The dorsal abdomen was removed and the insect was
The caecal region was removed and tran sferred
immediately to a p late o f n u trie n t agar or n u trie n t agar with 1% glucose.
The caecum (1 caecum/plate) was macerated w ith forceps and the contents
streaked with an inoculating loop.
followed.
Standard aseptic procedures were
The plates were incubated a t room T (ca. 70°F, 21°C) and
growth appeared w ithin 24 to 48 h.
The two d is tin c t colony types (designated type-A and type-B) on the
o rig in a l is o la tio n plates were tran sferred to fresh plates to assure
28
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29
pure cu ltu res.
The best representative o f each colony type from each
specimen location was tran s fe rred to a n u trie n t agar s la n t.
slants were subcultured every 14 to 21 days.
These
They were maintained a t
room T (ca. 70°F, 21°C).
Production o f antisera
B acterial subcultures from the caecal is o la tio n s were used fo r
production o f a n tis e ra .
B acteria were collected in 0.5% form alin and
adjusted to a K le tt reading (blue f i l t e r ) of 100 fo r immunization.
New Zealand white rabbits were in jected with antigen suspensions
according to the follow ing schedule:
on day 1, 0.2 ml intravenously
and 0 .5 ml subcutaneously; on days 4 , 8, and 12, with 1 .0 , 2 .0 , and
3 .0 ml re s p e c tiv e ly , in je c te d intravenously.
on day 18.
Test bleedings were made
On day 22, 3 .0 ml was injected intravenously followed by
exsanguination on day 29.
Pre-bleedings were by needle puncture of the
ear a rte ry and te s t bleedings were by the Bellco ear b o ttle system.
Antisera were enriched fo r immunoglobulins by 35% saturated ammonium
s u lfa te p re c ip ita tio n (Hebert 1976).
Anti sera were produced against a type-A and a type-B subculture
and designated A and B a n tis e ra .
The agglutination t it e r s o f the crude
antisera were 1024 and 2048, resp ec tiv e ly .
Antisera were also produced
against two a d d itio n a l, but d iffe r e n t, type-B subcultures and
designated Port Barre B and Colony B a n tis e ra.
These were not enriched
fo r immunoglobulins and t h e ir crude agglutination t it e r s were both 1024.
A gglutination t it e r s
A gglutination t it e r s were performed on a ll subcultures from the
caecal is o la tio n s .
A and B antisera were used and the antigen was the
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30
p a rtic u la r subculture t it e r e d , grown fo r 18 to 24 h.
The purpose was
confirm ation th at a ll type-A subcultures and a ll type-B subcultures
were each the same b a c te ria l species.
By checking each type culture
with the a lte rn a te a n ti serum,
the presence o f
cross reactions
determined.
reactions would
confirm th a t type-A and
Absence of cross
couldbe
type-B bacteria were d iffe r e n t.
C haracterization and id e n tific a tio n of the isolates
Type-A and type-B bacteria were studied by standard characterization
procedures.
Both were i n i t i a l l y stained with crystal v io le t and Gram
s ta in (18 to 24 h old c u ltu re s ).
M o tility was determined using m o tility
te s t medium (Anonymous 1971).
F la g e lla tio n of the caecal bacteria was determined by negative
stainin g o f 18 to 24 h old cultures with uranyl acetate (1% w t/v o l).
Stains were examined and photographed with a RCA EMU-3G transmission
electron microscope a t 50 kV.
Subsequently, a series of
biochemical tests (B la ir e t a l. 1970)
was performed on the type-A and type-B b a cteria.
The resu lting
physiological p ro file s were used to te n ta tiv e ly id e n tify the bacteria
(Buchanan and Gibbons 1974).
A ll tests were conducted a t room T (ca.
70°F, 21°C).
F e rritin -a n tib o d y la b e llin g studies
F e r r itin -la b e lle d antibody was used to v is u a liz e lo c a liz a tio n of
antibody on whole b a c te ria l c e lls .
This study used such la b e llin g to
v is u a liz e the in te ra c tio n between the caecal bacteria in s itu and the
antisera produced against the caecal is o la te s , thus confirming th at the
is o la te s were id e n tic a l to the bacteria observed w ithin the caecum.
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31
P rio r to la b e llin g o f the caecum, the caecal subcultures were
processed using two controls.
F ir s t , 5 mM phosphate-buffered salin e
(PBS) was substituted fo r the te s t antiserum to check fo r a nonspecific
f e r r i t i n reactio n.
Second, normal ra b b it serum (MRS) was substituted
undiluted to check fo r natural antibodies which might attach to the
b acteria and mimic species s p e c ific la b e llin g .
Since natural antibodies
were present w ithin NRS, a series o f d ilu tio n studies was conducted
using both NRS and A or B a n tisera on the type cultu res.
were 1:50, 1:100, and 1:200.
The d ilu tio n s
By comparing the concentration of f e r r i t i n
around the NRS treated c e lls with the anti serum treated c e lls , the
antiserum d ilu tio n re s u ltin g in species s p e c ific la b e llin g was d eter­
mined.
The fe rritin -a n tib o d y la b e llin g procedure was th a t of Yang e t a l.
(1977) with the follow ing m odifications.
to 24 h old when processed.
Caecal subcultures were 18
Antisera were d ilu te d as noted above.
Each
post-treatm ent wash was performed twice and the bacteria were suspended
to h a lf the o rig in a l volume follow ing antiserum treatment and mixed
with an equal volume o f a 1:4 d ilu tio n o f f e r r i t i n conjugated IgG
fra c tio n goat a n ti-r a b b it IgG, heavy and lig h t chains (Cappel Labora­
to r ie s , Downingtown, PA).
A ll specimens were examined and photographed
with a RCA EMU-3G transmission electron microscope a t 50 kV.
The fe rr itin -a n tib o d y la b e llin g o f the caecum consisted o f the
follow ing:
the f e r r i t i n la b e llin g and the f ix a tio n , dehydration, and
embedding.
The f e r r i t i n la b e llin g procedure was a m odification of the
one described above.
Stink bugs were dissected in 0.1 M cacodylate
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32
b u ffe r (pH 7 .2 ) , the caecal region removed, p re -fix e d in 0.5%
glutaraldehyde fo r 3 h a t 4°C, and washed 3 times in PBS.
were fo r 15 min each.
A ll washes
Caecal bands were removed, cross sectioned with
a razor blade, and washed in PBS.
Caeca were immersed in 0 .5 ml 1:50
d ilu tio n of antiserum, incubated a t 37°C fo r 1 h, and washed 3 times
in PBS.
Caeca were immersed in 0 .5 ml 1:4 d ilu tio n of f e r r i t i n
conjugate (see above), incubated a t 37°C fo r 1 h, and washed 4 times
in PBS.
Caeca were fix e d in 3.0% glutaraldehyde in 0.1 M cacodylate
b u ffe r (pH 7 .2 ) fo r 30 min a t room T and overnight a t 4°C.
The fo llo w ­
ing morning they were washed in b u ffer twice a t 15 min each and once
fo r 1 h.
Caeca were dehydrated in increasing concentrations o f acetone,
twice in 100%, (10 min each), propylene oxide twice (15 min each),
propylene oxide: Epon 812 (1 h ), Epon 812 (1 h ), fresh Epon (1 h ), a ll
a t room T, then overnight a t 4°C.
The follow ing day specimens were
allowed to reach room T, embedded, and cured 18 h a t 60°C.
Specimens
were sectioned on an LKB ultramicrotome and collected on 300-mesh copper
grids with no supporting film .
Sections were stained with uranyl
acetate (1% w t/v o l) fo r 5 min and f e r r i t i n enhancing stain (Ainsworth
and Karnovsky 1972) fo r 55 min.
Specimens were examined and photo­
graphed with a Zeiss 10 transmission electron microscope a t 60 kV.
Three caecal samples were processed concurrently using the above
described procedures.
One sample with A antiserum, one with B a n t i-
serum, and a PBS control to check fo r nonspecific f e r r i t i n la b e llin g
follow ing the p re -fix a tio n step.
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RESULTS AND DISCUSSION
Is o la tio n o f the caecal bacteria
Two d is tin c t b a cteria o f d iffe r e n t colonial types were isolated from
the caecum o f
v ir id u la .
One colonial type was c ir c u la r , cream-white,
opaque, smooth and was a r b it r a r ily designated type-A.
The other was
c ir c u la r , d u ll w h ite, tran slu cen t, smooth and a r b it r a r i ly designated
type-B.
These bacteria were consistently involved in the is o la tio n s
performed on both occasions.
The only other growth present were two
contaminants on a sing le p la te with the above is o la te s .
Caecal smears
were consistently marked by abundant growth.
Both bacteria were present on many p la te s , although one colonial
type was usually more abundant than the other.
contained only one o f the is o la te s .
However, some plates
This was probably due to unequal
inoculum w ithin the caecum re s u ltin g in one bacteria outgrowing and
masking the presence of the other.
Both n u trie n t agar and glucose
n u trie n t agar supported abundant growth.
There was apparently no
re la tio n s h ip between the presence o f one or both bacteria and the type
of media used.
A gglutination t it e r s
Agglutination t it e r s confirmed th a t a ll type-A subcultures were
the same b a c te ria , but only two o f fiv e type-B subcultures would
ag g lu tin ate .
Therefore, addition al antisera (P ort Barre B and Colony
B an ti sera) were produced against two o f the nonagglutinating sub­
c u ltu res.
This was based on the assumption th a t the three
nonagglutinating cultures were possibly antig en ic varian ts o f the other
33
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34
two.
A gglutination t it e r s using each of these antisera confirmed that
a ll type-6 subcultures were the same b a c te ria.
Subsequently, the type-
B subcultures were tested with A antiserum and the type-A subcultures
were tested with B, Port Barre B, and Colony B antisera to check fo r
cross reactions.
No cross reactions were observed indicating th a t
type-A and type-B b acteria were d iffe r e n t.
C haracterization and id e n tific a tio n of the isolates
Crystal v io le t staining o f the b acteria revealed th a t type-A were
coccoid to short rods while type-B were longer rods.
were Gram negative and m o tile.
Both bacteria
Negative staining demonstrated th at the
type-A bacteria were peritrichous (F ig . lA) and the type-B bacteria
were lophotrichous (F ig . IB ).
The biochemical tests performed involved te stin g fo r amino acid
metabolism, namely lysine decarboxylase, o rn ith in e decarboxylase,
arginine dihydrolase, and indole formation from tryptophan.
Sugars
(23) were tested to determine i f carbohydrate metabolism was oxid ative
or ferm entative.
Additional tests were Voges-Proskauer, K lig le r iron
agar, n itr a te reduction, and growth in the presence of KCN.
Careful consideration o f the resu lts led to the follow ing
id e n tific a tio n s .
Type-A bacteria were id e n tifie d as Enterobacter
aeroqenes Hormaeche and Edwards.
Type-B bacteria were pro visio n ally
id e n tifie d as Aeromonas sp. (Family Vibrionaceae) due p rim a rily to th e ir
lophotrichous fla g e lla tio n as well as th e ir ferm entative character.
When a ll the characters were analyzed with the data presented in
Buchanan and Gibbons (1974), th is genus was the one which f i t best.
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35
Fig. 1.
Negative stains of the caecal is o la te s from Nezara
v ir id u la .
Bars = 1 ^m.
the type-A b a c te ria .
(A) Peritrichous f la g e lla of
(B) Lophotrichous f la g e lla o f the
type-B b a cteria.
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36
K
:
I":,:
IE
%
^ y!-'%
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37
However, the type-B b acteria could possibly be an undescribed organism.
F e rritin -a n tib o d y la b e llin g studies
The preceding re su lts indicated th a t the iso lates from the caecum
were the caecal bacteria previously observed w ithin
1980).
v ir id u la (West
M orphologically, the caecal bacteria observed on the SEM were
very s im ila r to the is o la te s stained w ith crystal v io le t; both consisted
of coccoid forms and longer rods.
Iso latio n s consistently showed two
d is tin c t bacteria o f d iffe r e n t colonial types.
resulted in markedly abundant growth.
Caecal smears also
However, fin a l confirmation of
caecal b acteria is o la tio n s was derived from the fe rritin -a n tib o d y
la b e llin g of the caecum.
The PBS control revealed th a t there was no appreciable nonspecific
f e r r i t i n re ac tio n .
The NRS control demonstrated the presence of natural
antibodies which could mimic s p e c ific la b e llin g .
Subsequently in
d ilu tio n studies on the type-A b a c te ria , a 1:50 d ilu tio n of A antiserum
( t i t e r >512) resulted in f e r r i t i n la b e llin g which was d is tin c tiv e from
th a t using the same d ilu tio n of NRS.
1:50 d ilu tio n o f the B antiserum
As fo r the type-B b a c te ria , a
( t i t e r >512) resulted in la b e llin g
distingu ishable from th a t using NRS, though not as d is tin c tiv e as the
type-A b acteria but adequate fo r purposes of th is study.
Therefore,
1:50 d ilu tio n s o f the A and B antisera would produce s p e c ific la b e llin g
o f t h e ir respective b a c te ria.
During the la b e llin g of the caecum, the p re -fix a tio n step was to
firm the tissue so th a t the caecal bands could be separated from the
alim entary t r a c t .
The concentration was s u ffic ie n tly low so as not to
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38
adversely e ffe c t the antigen s ite s o f the b a c te ria.
Washes were
important during the procedure, especially follow ing the f e r r i t i n
treatm ent, since nonattached f e r r i t i n remaining in the specimens could
make i t d i f f i c u l t to confirm la b e llin g .
Sections were stained with
uranyl acetate to enhance the bacterial c e ll membranes.
stain was to enhance the f e r r i t i n la b e l.
The f e r r i t i n
This stain not only
in te n s ifie s the electron opacity of f e r r i t i n but i t also increases its
v is ib le size ca. 2 -fo ld (Ainsworth and Karnovsky 1972).
Although PBS
was used as a c o n tro l, i t was unnecessary to include a NRS control since
the natural antibody reaction had been adjusted fo r when the antisera
d ilu tio n rates were determined.
The re su lts o f the la b e llin g and embedding o f the caecum were con­
clu sive.
Sections o f the PBS control consisted of c e lls with e ith e r
no f e r r i t i n or a very lig h t accumulation d is trib u te d around the c e lls
(F ig . 2A).
The f e r r i t i n was present p rim a rily as single units and
represented a s lig h t nonspecific reactio n.
Sections of the caecum
treated with A anti serum contained both la b e lle d and nonlabelled c e lls
(F ig . 2B).
F e r r itin was present around la b e lle d c e lls as d is tin c tiv e
clumps lo c a lize d on the antigen sites (Shands 1965, 1966).
Nonlabelled
c e lls e ith e r had no f e r r i t i n or a lig h t accumulation of single units
as observed in the c o n tro l.
Sections of the caecum treated with B
a n ti serum had both la b e lle d and nonlabelled c e lls which were s im ila r
in appearance to the A treated c e lls (F ig . 2C).
The clumped f e r r i t i n
represented the species s p e c ific label while no f e r r i t i n and lig h t
fe rr itin
distinguished those c e lls representing the a lte rn a te bacterial
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39
Fig. 2.
Thin sections of the fe rritin -a n tib o d y la b e llin g o f the
caecum from Nezara v ir id u la .
(A) PBS control with lig h t
accumulation of f e r r i t i n around some b a cteria.
Bar = 1 /jm.
(B) A antiserum treated bacteria with f e r r i t i n clumps,
arrows, lo calized on the antigen sites o f lab elled c e lls .
Bar = 0 . 5 /im.
(C) B antiserum treated bacteria with f e r r i t i n
clumps around la b e lle d c e lls .
Bar = 0 . 5 /jm.
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40
B
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41
species present w ith in the caecum.
Higher concentrations o f f e r r i t i n
may have been present on la b e lle d c e lls , but i t was suspected th a t some
label may have been lo s t when capsular m aterial was stripped away
during processing.
Some capsular remnants with attached f e r r i t i n were
observed in the open areas between the c e lls .
These re su lts confirmed
th a t the caecal is o la te s were the same b acteria observed w ithin the
caecum using electron microscopy.
In conclusion, two d is tin c t bacteria o f d iffe r e n t colonial types
were is o la te d from the caecum o f Nezara v ir id u la .
is o la te s were rods.
M orphologically, both
One is o la te was coccoid in appearance (designated
ty p e -A ), w hile the other consisted o f longer rods (designated type-B ).
C haracterization studies id e n tifie d them as Enterobacter aerogenes
(type-A ) and the other p ro v is io n a lly as Aeromonas sp. (typ e-B ).
F e rritin -a n tib o d y la b e llin g o f the caecum, using antisera prepared
against type-A and type-B b a c te ria , confirmed th a t the is o lates were
id e n tic a l to b acteria previously observed w ithin the caecum.
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REFERENCES CITED
Ainsworth, S. K ., and M. J. Karnovsky. 1972. An u ltra s tru c tu ra l
staining method fo r enhancing the size and electron opacity o f
f e r r i t i n in th in sections. J. Histochem. Cytochem. 20: 225-9.
Anonymous. 1971. Difco manual o f dehydrated c u ltu re media and reagents
fo r m icrobiological and c lin ic a l laboratory procedures, 9th ed.
Difco Laboratories, In c ., D e tr o it, Michigan. 350 pp.
B la ir , J. E ., E. H. Lennette, and J. P. Truant (e d s .). 1970. Manual
o f c lin ic a l microbiology. Am. Soc. M ic ro b io l., Bethesda, Maryland.
727 pp.
Brooks, M. A. 1963. Symbiosis and aposymbiosis in arthropods.
Symp. Soc. Gen. M icrobiol. 13: 200-31.
Buchanan, R. E ., and N. E. Gibbons. 1974. Bergey's manual of
determ inative bacteriology, 8th ed. Williams and Wilkins Co.,
Baltim ore, Maryland. 1268 pp.
Buchner, P. 1965. Endosymbiosis o f animals with plan t microorganisms.
John Wiley and Sons, Inc. (In te rs c ie n c e ), New York. 909 pp.
Glasgow, H. 1914. The g a s tric caeca and the caecal bacteria o f the
Heteroptera. B io l. B u ll. 26: 101-70.
Hebert, G. A. 1976. Improved s a lt fra c tio n a tio n o f animal serums fo r
immunofluorescence studies. J. Dent. Res. (Special Issue A)
55: A33-7.
Koch, A. 1967. Insects and th e ir endosymbionts. %n S. M. Henry ( e d .).
Symbiosis, v o l. 2. Academic Press In c ., New York. 443 pp.
Richards, A. G ., and M. A. Brooks.
Ann. Rev. Entomol. 3: 37-56.
1958.
Internal symbiosis in insects.
Shands, J. W. 1965. Lo calization o f somatic antigen on Gram-negative
b acteria by electron microscopy. J. B ac te rio l. 90: 266-70.
Shands, J. W. 1966. Localization o f somatic antigen on Gram-negative
b acteria using f e r r i t i n antibody conjugates. Ann. N. Y. Acad.
Sciences 133: 292-8.
Steinhaus, E. A. 1951. Report on diagnoses of diseased insects 19441950. H ilg ard ia 20: 629-78.
Steinhaus, E. A ., M. M. Batey, and C. L. Boerke. 1956. B acterial
symbiotes from the caeca o f c e rta in Heteroptera. H ilg ardia 24:
495-518.
42
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43
West, R. P. 1980. Endosymbiosis in the caecum of the southern green
s tin k bug, Nezara v irid u la ( L . ) . Ph.D. D issertation . Louisiana
State U n iv ., Baton Rouge, Louisiana. 77 pp.
Yang, G. C. H ., G. D. Schrank, and B. A. Freeman. 1977. P u rific a tio n
o f fla g e lla r cores of V ibrio cholerae. J. B ac te rio l. 129: 1121-8.
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CHAPTER I I I
E ffe c t of A n tib io tic s to Eliminate Caecal
Bacteria from a Stink Bug
44
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INTRODUCTION
Elim ination o f the symbionts w ithin an in se c t, an aposymbiotic
specimen, is the primary means o f studying the e ffe c ts of symbionts
on the host (Richards and Brooks 1958).
The resu lts o f such studies
may in d icate the physiological sign ificance o f the endosymbionts.
Although many studies o f th is nature have been conducted, they have
met with varying degrees o f success (Buchner 1965; Koch 1956, 1960,
1967; Richards and Brooks 1958).
Numerous methods have been devised to elim inate endosymbionts from
insects.
The references cited above thoroughly document these methods
so a b r ie f lis t in g o f a few w ill s u ffic e here.
In those cases where
nature in terru p ts the symbiotic cycle, as in the smearing o f eggs with
inoculum, the re in fe c tio n may be prevented by a r t i f i c i a l means; such as
the d is in fe c tio n of the eggs.
Physical methods a v a ila b le are elevated
temperatures, depressed temperatures, centrifugal fo rc e , and temporary
s ta rv a tio n .
Chemical methods include numerous chemotherapeutics and
a n tib io tic s .
An approach may be used in d iv id u a lly or in some cases
combinations may prove to be more e ffe c tiv e .
Early in the study o f the caecal b acteria o f the southern green
s tin k bug, Nezara v irid u la ( L . ) , i t was re a lize d th a t aposymbiotic
specimens were desirable to determine the value of the bacteria to the
host.
Reports of studies using a n tib io tic s fo r th is purpose existed
in current lit e r a t u r e .
Consequently, with an incomplete knowledge a t
the t'.i.ie th is study was in it ia t e d , i t was concluded th a t th is was the
approach to use.
A procedure was devised to tr e a t s tin k bugs with a
45
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46
combination o f a n tib io tic s used by Breznak and Pankratz (1977).
Treated bugs were sampled a t timed in te rv a ls , processed, and examined
on a scanning electron microscope (SEM) to determine the effectiveness
of the drugs.
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MATERIALS AND METHODS
A n tib io tic s
For a n tib io tic treatm ent, stin k bugs were maintained on soybeans
pre-soaked fo r 1 h in an a n tib io tic solution consisting o f (in ^g/ml
deionized water) te tra c y c lin e , 800; p e n ic illin G, 900; chloramphenicol
and streptomycin s u lfa te , 1,000 each (Breznak and Pankratz 1977).
F ifte e n ml aliq uots o f the solution were maintained frozen u n til needed.
To confirm th a t the soybeans were absorbing the a n tib io tic s , they
were pre-soaked in a n tib io tic s o lu tio n , the outer bean was trimmed away,
and the remaining center was rinsed with d is t ille d water.
The dry
center was tran sferred a s e p tic a lly to a n u trie n t agar p la te , the e n tire
surface o f which was inoculated with an IE. c o li JHOOl culture 1 h
e a r lie r .
Three such plates were incubated a t 37°C and checked a t 12,
18, and 28 h.
Stink bugs
Eighteen s tin k bugs (9 (?, 9 .ÿ) were obtained from a laboratory
colony maintained by the Department o f Entomology, L.S.U.
They were
held in a 5 gal aquarium covered by cheesecloth without food or water
fo r 24 h p rio r to the study.
Since stin k bugs had been maintained under
comparable conditions previously with no apparent e ffe c t on the caecal
f lo r a , no controls were used.
Any differences from normal were
a ttrib u te d to the treatm ent.
The study was in itia te d by placing a p e tri dish of ca. 25 treated
soybeans into the aquarium as the only source of nourishment.
47
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Soybeans
48
were replaced with fresh treated beans a t 12 h in te rv a ls to insure
th a t active a n tib io tic s were con tin u ally present.
Those insects
observed feeding were marked with a small spot of n a il polish on the
scutellum to insure th a t the specimens sampled had fed.
Insects were
removed fo r SEM processing at 6, 12, 24, 48, 72, and 96 h a fte r
in it ia t in g feeding on treated beans.
Preparation fo r SEM
Caeca were excised from the stin k bugs and prepared fo r SEM
examination using the procedure outlined in West (1980).
Specimens were
viewed in a HITACHI S-500 scanning electron microscope a t 20 kV
accelerating voltage and photographed with Polaroid type 55 P/N film .
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RESULTS AND DISCUSSION
There was a d is tin c t in h ib itio n zone (> 0 .5 cm wide) around the
a n tib io tic trea te d soybean in each p late a t 12, 18, and 28 h post­
treatm ent.
Therefore, the a n tib io tic s were absorbed in to the center
of the beans and retained t h e ir a c t iv it y fo r a t le a s t 24 h.
Since the s tin k bugs were denied food and water fo r 24 h p rio r to
a n tib io tic treatm ent, they were more lik e ly to accept the treated soy­
beans.
Within the f i r s t 3 h, eight bugs were marked fo r feeding.
During the remainder o f the study, they fed in te r m itte n tly .
Six of the
eight were removed as samples a t the appropriate times.
SEM examination o f the specimens sampled revealed the progressive,
yet lim ite d , e ffe c t of the a n tib io tic s .
caeca previously examined (West 1980).
The 6 h specimen resembled the
Two b a c terial forms were
present; rods (ca. 2 .5 by 0 .5 jum) attached a p ic a lly and coccoid forms
ca. 1 to 2 pm in diameter.
Morphological v a riatio n s of these which had
been previously observed included rods which were apparently budding
and la rg e r coccal forms 2 to 3 ^m in diameter.
Some elongated rods 5
to 8 /im long were present which had not been observed previously.
These
may have resulted from the i n i t i a l e ffe c ts o f the a n tib io tic s on the
environment w ith in the caecum.
Yet, such e ffe c ts were not s u ffic ie n t
to cause large numbers of bacteria to detach from the caecum because few
detachment s ite s were seen.
The 12 h specimen contained bacteria comparable to the 6 h specimen.
The rods and cocci were dominant with the same morphological variations
present.
However, some areas were characterized by numerous detachment
49
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50
s ite s , p rim a rily oval-shaped, in d ic a tin g th a t the a n tib io tic s were
exerting an increased influence (F ig . lA ).
At 24 h, the b a c te ria l flo ra was e s s e n tia lly unchanged as compared
to the 12 h observations.
However, many areas possessed large numbers
o f detachment s ite s and few attached b acteria (F ig . IB ).
24 h specimens were s im ila r in appearance.
The 48 h and
The ty p ic a l b acteria were
present except f o r an increased proportion o f the elongated rods.
a t 24 h, there were many areas o f detachment s ite s (F ig . 1C).
As
In some,
the s ite s were more elongate ra th e r than oval-shaped as had been
observed previously (F ig . ID ).
Apparently the rods, elongated due to
the i n i t i a l a n tib io tic in flu e n c e , were detaching from the caecum and
leaving impressions which re fle c te d the elongate growth.
At 72 h (F ig . IE ) and 96 h (F ig . I F ) , the specimens appeared
unchanged as compared to 48 h.
No evidence was observed to in d ic a te an
increase in effectiveness o f the treatm ent beyond 48 h.
Throughout the examination o f specimens, there were areas where
b acteria had detached w hile there were corresponding areas w ith in the
same specimen where detachment was less pronounced or absent.
The
detachment s ite s remained d is tin c t in a ll specimens observed since they
are apparently embedded in a glycocalyx la y e r, secreted by the symbionts,
as well as in the surface o f the caecum i t s e l f (West 1980).
This
polysaccharide m aterial maintains it s form fo r some time subsequent to
b a c te ria l detachment.
In conclusion, although the a n tib io tic s did have an e ffe c t on the
caecal flo r a o f Nezara v ir id u la , i t was lim ite d .
There is no evidence
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51
Fig. 1.
A n tib io tic e ffe c ts on the caecum o f Nezara v ir id u la .
Arrows in d icate detachment s ite s .
12 h.
(B) 24 h.
(C) 48 h.
Bars = 5 ^m.
(A)
(D) Detachment s ite s re fle c tin g
the elongate growth of some bacteria follow ing 48 h of
a n tib io tic treatm ent.
(E) 72 h.
(F) 96 h.
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52
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53
to in d ic a te th a t prolonged treatment beyond 96 h would have resulted
in aposymbiotic specimens.
A fte r considering these re s u lts , two
possible explanations may be suggested.
E ith er the a n tib io tic s '
effectiveness peaked a t 24 to 48 h and were incapable o f complete
symbiont e lim in a tio n , or the s tin k bugs reduced t h e ir feeding and intake
s u ffic ie n tly a fte r 48 h to prevent acquisition o f the concentration
necessary fo r complete e lim in a tio n .
In e ith e r case, these a n tib io tic s
under these study conditions apparently o ffe r l i t t l e chance fo r success­
fu l production o f aposymbiotic s tin k bugs.
Another consideration deals with the m aterial in which the bacteria
are embedded.
I f th is s itu a tio n is comparable to th a t o f the b a c te rial
masses in the lungs o f the cystic fib ro s is p a tie n t, as discussed in
West (1 9 80 ), then the p a ra lle l may be c arried a step fu rth e r.
In cystic
fib ro s is treatm ent, the use of a n tib io tic s involves the careful
adm inistration o f several drugs in dosages ju s t short o f le th a l le v e ls
to be e ffe c tiv e against th is b acterial in fe c tio n (J. W. Costerton,
personal communication).
S im ila rly , i t might be necessary to approach
the to x ic le v e ls o f a n tib io tic s in the s tin k bugs fo r the treatment to
be e ffe c tiv e against the caecal b a c te ria.
Therefore, i t could be
extremely d i f f i c u l t to successfully elim inate the caecal symbionts
with a n tib io tic s without k illin g the insect in the process.
Subsequent to th is study, i t was re a liz e d th a t a simpler and more
e ffe c tiv e approach would involve d is in fe c tio n o f the eggs p rio r to
eclosion o f the f i r s t in s ta r nymphs.
I f an attempt to produce
aposymbiotic s tin k bugs is repeated, th is would be a possible approach.
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REFERENCES CITED
Breznak, J. A ., and H. S. Pankratz. 1977. In s itu morphology o f the
gut m icrobiota o f wood-eating term ites f R e tic u litermes fla v ip e s
(K o lla r) and Coptotermes formosanus S h ira k i]. Appl. Environ.
M icrobiol. 33: 406-26.
Buchner, P.
1965. Endosymbiosis o f animals with plant microorganisms.
John Wiley and Sons, Inc. (In te rs c ie n c e ), New York. 909 pp.
Koch, A. 1956. The experimental elim in atio n o f symbionts and it s
consequences. E x p tl. P a ra s ito l. 5: 481-518.
Koch, A. 1960. In tr a c e llu la r symbiosis in insects.
M icrobiol. 14: 121-40.
Ann. Rev.
Koch, A. 1967. Insects and th e ir endosymbionts. %n S. M. Henry (e d .).
Symbiosis, v o l. 2. Academic Press In c ., New York. 443 pp.
Richards, A. G ., and M. A. Brooks. 1958. In ternal symbiosis in
insects.
Ann. Rev. Entomol. 3: 37-56.
West, R. P.
1980. Endosymbiosis in the caecum o f the southern green
stin k bug, Nezara v ir id u la ( L .) .
Ph.D. D is s erta tio n . Louisiana
State U n iv ., Baton Rouge, Louisiana.
77 pp.
54
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CHAPTER IV
Transmission of Caecal Symbionts to
Offspring by Nezara v irid u la (L .)
55
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INTRODUCTION
Endosymbiosis w ith in insects includes a broad range o f relationsh ips
between the insect host and its symbionts, whether e x tra c e llu la r or
in tr a c e llu la r .
However, one re la tio n s h ip common to a ll cases is the
transmission o f the symbionts to the hosts' o ffs p rin g .
The mechanisms
th a t have evolved fo r th is purpose are as diverse as the examples of
endosymbiosis th a t may be studied (Brooks 1963a, 1963b; Buchner 1953,
1965; Carayon 1952; Koch 1960, 1967).
Koch (1967) discusses three possible methods fo r symbiont trans­
mission;
the oral uptake o f symbionts by the young brood; the smearing
o f eggs with symbionts a t the time of egg deposition; and the in fectio n
o f eggs, or embryos, before deposition.
The mechanism of transmission
is quite n a tu ra lly dependent upon the lo c a liz a tio n o f the guests w ithin
the host.
The f i r s t and second methods are found in insects which
house th e ir symbionts in the gut region, and may involve e x tra - or
in tr a c e llu la r symbiosis.
The th ird is found where there is no r e la tio n ­
ship between the symbiotic housing and the alim entary canal, usually
involving in tr a c e llu la r symbiosis.
The smearing o f eggs is p a rtic u la rly
prevalent among the m ajo rity o f bugs (H eteroptera), p rim a rily the
Pentatomidae.
F irs t in s ta r nymphs o f the southern green stink bug, Nezara v irid u la
( L . ) , remain aggregated on the egg mass follow ing eclosion.
This is
apparently a nonfeeding period and could be called a rest period.
Buchner (1965) discusses four d is tin c t mechanisms by which the
56
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57
Heteroptera transmit bacteria lo calized in the gut to progeny.
One
consists o f the release o f large masses o f the symbiont into the hindgut
a t the time of egg deposition, inoculating the eggs.
suck up the symbionts during the "rest period".
The young then
Rosenkranz (1939) had
previously come to th is same conclusion about the "rest period".
Consideration of the above facts leads to the p o s s ib ility th a t f^.
v irid u la transmits it s symbionts to o ffsprin g by smearing the eggs
during deposition.
approaches.
Confirmation o f th is hypothesis was secured by two
F ir s t , b a c te rial iso lates from egg masses were tested by
s lid e agglutination with antisera prepared against the previously
isolated caecal bacteria (West 1980) to v e rify the symbionts' presence
on the eggs.
Second, an egg mass was examined with a scanning electron
microscope (SEM) to attempt to v is u a liz e the inoculum on the surface
o f the eggs.
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MATERIALS AND METHODS
Is o la tio n from eggs
Egg masses were obtained from a laboratory colony maintained by
the Department o f Entomology, L.S.U.
Eggs were usually collected w ithin
24 h of deposition from recently cleaned containers and transferred
a s e p tic a lly to a s t e r ile p e tri p la te .
Individual eggs were separated
and tran sferred to p e tri plates o f n u trie n t agar to be pressed in to ,
or smeared over, the agar surface.
The remaining eggs were homogenized
in s t e r ile salin e (0.85% NaCl) and streaked on agar plates with a
tra n s fe r loop.
Standard aseptic procedures were followed.
I n i t i a l l y the plates were incubated a t room T (ca. 70°F, 21°C).
However, problems with fungal contamination necessitated incubation at
an elevated T o f 33°C.
Typical b a c te ria l colonies were transferred
to slants and tested by s lid e agglutination a fte r 18 to 24 h growth.
Antisera
Four antisera were used in the s lid e agglutination te s ts ; one to
id e n tify type-A caecal bacteria and three fo r type-B caecal bacteria.
Procedures fo r production and processing o f antisera are outlined in
West (1980).
Slide agglutin ation tests
Slide agglutinations were patterned a fte r Bailey and Scott (1970).
On a clean glass s lid e , a drop o f saline and a loop o f bacteria were
mixed, a drop o f antiserum added, the s lid e was t i l t e d back and fo rth
fo r 1 min, and the reaction noted.
A p o sitive reaction was a g g lu ti­
natio n, the prompt formation of fin e granules or large aggregates.
58
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59
A negative reactio n was continued homogeneous tu r b id ity .
Controls
consisting o f s a lin e and an ti serum were run each tim e.
Preparation o f eggs fo r SEM
Eggs were mounted on an SEM stub and pumped in an Edwards
Sputter U nit (Edwards High Vacuum In c ., Grand Is la n d , N. Y .) to
dessicate p rio r to coating with ca. 800 Â gold-palladium .
Eggs were
maintained in a dessicator under vacuum fo r several h p rio r to examina­
tio n in a HITACHI S-500 scanning electron microscope operated a t 25 kV
accelerating voltage.
They were photographed with Polaroid type 55
P/N film .
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RESULTS
I n i t i a l l y no problems were experienced with the is o la tio n s of
b a c te ria .
However, with the second, t h ir d , and fourth egg masses, a ll
but one p la te had gross fungal contamination and b acteria
be is o la te d .
could not
Two b a c te rial is o lates were obtained from the fungus-
fre e p la te , but by s lid e agglutin ation n e ith er proved to be the caecal
b a c te ria .
The source of contamination was determined to be the colony
rearing containers.
Therefore, the incubation T o f is o la tio n plates
was elevated from 21°C (70°F) to 33°C, providing the bacteria with a
growth advantage over the contaminating fungi.
Results o f s lid e agglutinations on iso lates from the remaining egg
masses processed are presented in Table 1.
The presence of type-B
caecal bacteria was confirmed fo r both egg mass 1 and 6.
Type-B was
demonstrated by each is o la tio n method used on egg mass 1, by the press
and the smear methods on egg mass 6, w hile the loop method yielded two
b a c te rial contaminants.
On egg mass 5, the presence of type-A caecal
bacteria was confirmed by the press and the smear methods.
The loop
method was not performed due to the inadvertent contamination o f the
egg mass p rio r to homogenization.
The e n tire observable surface o f the mounted egg mass was c a re fu lly
examined w ith the SEM.
located.
Two possible s ite s o f b a c terial adherence were
The f i r s t , located on the side of an egg, was questionable
because i t only consisted o f a few b a c te ria l c e lls in a small area.
However, the second s ite (F ig . lA ), located on the dorsal surface o f an
60
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61
Table 1.
Caecal bacteria on Nezara v irid u la (L .) eggs as determined
by s lid e agglutination te s ts .
Egg Mass No.
Press
Is o la tio n Method 1/
Smear
Loop
1
B 2/
B
B
5
A
A
- 3/
6
B
B
C
y
Press, egg pressed into agar surface; Smear, egg smeared over agar
surface in ta c t; Loop, homogenized egg mass streaked on agar
surface with tra n s fe r loop.
2/
A, type-A caecal b a c te ria; B, type-B caecal bacteria; C, misc.
b a c te ria l contaminants.
3/
Not performed.
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62
Fig. 1.
Caecal bacteria inoculum on the surface of an egg o f
Nezara v ir id u la .
(A) Egg a t low m agnification.
indicates inoculum.
Bar = 50 |im.
higher m agnification.
c e ll mass.
Bar = 5 ^m.
m agnification.
Bar = 10 ^m.
Arrow
(B) Inoculum a t a
(C) Caecal bacteria
(D) Individual c e lls at high
Bar = 5 ^m.
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63
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64
egg in the proxim ity o f the spines around the operculum, contained many
b acteria when observed a t higher m agnification (F ig . IB ).
I t consisted
o f a concentrated mass o f b a c te ria l c e lls confined to a r e la tiv e ly
small area.
Higher m agnifications (F ig . 1C, D) revealed th a t the
m a jo rity of the c e lls were coccoid forms measuring ca. 1 to 2 ^m in
diameter w hile the rod-shaped c e lls were ca. 2 to 3 by 0.5 to 1 ^m.
These dimensions were very s im ila r to those o f the c e lls observed w ithin
the caecum o f N. v ir id u la (West 1980).
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DISCUSSION
Both caecal bacteria previously isolated from the caecum of
v ir id u la (West 1980) were found on the surface o f egg masses.
Although
both species were not isolated from the same mass, th is was not an
unexpected re s u lt.
The d iffe r e n t bacteria on any p a rtic u la r mass of
eggs would not necessarily be evenly d is trib u te d , and i t would not be
uncommon fo r one species o f bacteria to outgrow and mask the other
during such is o la tio n s .
Therefore, only one species would be id e n ti­
fia b le by the s lid e agglu tin atio n te s t.
However, since both were
present on the eggs, the evidence supports the smearing of eggs as the
mechanism o f transmission to o ffs p rin g .
Additional evidence was pro­
vided by the v is u a liz a tio n by SEM o f an apparent mass of adhering
bacteria on the surface o f an egg.
w ithin the caecum of
Both morphological forms observed
v ir id u la were observed w ithin the suspected
inoculum and th e ir dimensions were very s im ila r.
In conclusion,
considering the above evidence in view o f previous reports on tra n s fe r
o f symbiotic bacteria from parent to o ffs p rin g , the caecal bacteria of
Nezara v ir id u la are transm itted to t h e ir offsprin g by smearing, or
in o c u la tin g , the external surface o f the eggs during deposition.
65
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REFERENCES CITED
B ailey, W. R ., and E. G. Scott. 1970. Diagnostic microbiology, 3rd
ed. C. V. Mosby Co., St. Louis, Missouri. 385 pp.
Brooks, M. A. 1963a. The microorganisms of healthy insects. Ij% E. A.
Steinhaus ( e d .) . Insect pathology, vol. 1. Academic Press In c .,
New York. 661 pp.
Brooks, M. A. 1963b. Symbiosis and aposymbiosis in arthropods.
Symp. Soc. Gen. M icrobiol. 13: 200-31.
Buchner, P. 1953. Endosymbiose der T iere mit pflanzlichen
Mikroorganismen. Verlag Birkhauser, Basel, Switzerland.
771 pp.
Buchner, P. 1965. Endosymbiosis of animals with plant microorganisms.
John Wiley and Sons, Inc. (In te rs c ie n c e ), New York. 909 pp.
Carayon, J. 1952. Les mécanismes de transmission h é ré d ita ire des
endosymbiontes chez les insectes. T ijd sch r. Entomol. 95: 111-42.
Koch, A. 1960. In tra c e llu la r symbiosis in insects.
M icrobiol. 14: 121-40.
Ann. Rev.
Koch, A. 1967. Insects and th e ir endosymbionts. In^ S. M. Henry (e d .).
Symbiosis, v o l. 2. Academic Press In c ., New York. 443 pp.
Rosenkranz, W. 1939. Die Symbiose der Pentatomiden (Hemiptera
Heteroptera). Z. Morphol. Okol. Tiere 36: 279-309.
West, R. P. 1980. Endosymbiosis in the caecum of the southern green
stink bug, Nezara v irid u la ( L .) . Ph.D. D issertatio n . Louisiana
State U n iv ., Baton Rouge, Louisiana. 77 pp.
66
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CHAPTER V
Apparent Absence of the Caeca! Bacteria
w ith in Some F ield-C ollected Specimens
of Nezara v ir id u la (L. )
67
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INTRODUCTION
The lit e r a t u r e on endosymbiosis suggests th a t the re la tio n s h ip
between insect host and symbiont is in many cases a v it a l one (Buchner
1953, 1965; Koch 1960, 1967; Richards and Brooks 1958).
Not only is
the symbiont apparently present in a ll in divid uals o f a p a rtic u la r
insect species, but a constant, c h a ra c te ris tic type of symbiont is
consistently involved in the re la tio n s h ip (Brooks 1963a, 1963b;
Steinhaus 1951).
Cases involving the loss o f symbionts during in divid ual l i f e have
been documented (Koch 1956, 1967) but as a ru le involve in tr a c e llu la r
symbiosis.
Some cases involve the loss of symbionts by one sex, usually
the male, a t a p a rtic u la r time during the l i f e cycle (th e Cerambycidae,
Bostrichidae, Pediculidae, Si tophi lus granarius ( L . ) ) . A few cases of
to ta l symbiont loss have been discovered such as the Egyptian v a rie ty
o f Sitophi lus granarius which has completely lo s t it s symbionts due to
c lim a tic factors of it s h a b ita t.
Other examples involve species which
have t o t a lly lo s t t h e ir symbionts but the presence of embryological
mycetome rudiments reveals th a t they once possessed them (Buchner 1965).
My studies have been lim ite d to e x tra c e llu la r endosymbiosis in the
caecum o f the southern green s tin k bug, Nezara v irid u la ( L . ) .
Electron
microscopy o f the caecum has revealed th a t apparently two b a c te ria l
symbionts are involved, and is o la tio n studies have confirmed th is
observation (West 1930).
I subsequently decided to conduct a lim ite d
68
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69
survey o f s tin k bugs w ithin Louisiana to confirm th a t these same two
species were consistently involved in each case o f endosymbiosis w ithin
th is in se c t.
My approach involved c o lle c tin g specimens from several
lo c a tio n s , is o la tin g the symbionts using the o rig in a l is o la tio n
procedures, and te s tin g the is o la te s by s lid e agglutin ation with
an tisera prepared against the o rig in a l is o la te s .
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MATERIALS AND METHODS
Stink bugs
Specimens were collected from fie ld s near Natchitoches, Krotz
Springs, Port Barre, Hamburg, and Bayou Goula, LA, on such hosts as
clovers, corn, green beans, and soybeans.
Specimens from a laboratory
colony maintained by the Department o f Entomology, L.S.U. were checked
as controls.
Iso latio n s
The same procedures were used as in the o rig in a l isolations from
the caecum (West 1980).
room T (ca. 70°F, 21°C).
The n u trie n t agar plates were incubated a t
Typical mature b a c te ria l colonies were
tran sferred to slants and tested by s lid e agglutin ation a fte r 18 to
24 h growth.
Isolations were made from a male and a female from each
location and on each c o lle c tio n date.
Five pairs were processed from
Hamburg, LA, collected June 25, 1980.
S lide agglutination tests
S lide agglutinations were patterned a f t e r B ailey and Scott (1970).
The procedure and antisera used were as described in West (1980).
70
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RESULTS AND DISCUSSION
The resu lts of the study were unexpected.
During electron
microscopy, a ll specimens apparently possessed two bacterial symbionts
and in i n i t i a l is o la tio n s
usually both, sometimes only one, of the
symbionts were present on the agar plates (West 1980).
was c h a ra c te ris tic o f the caecal smears.
Abundant growth
During th is study, is o la tio n
plates from some fie ld -c o lle c te d specimens remained s te r ile with no
growth (Table 1 ).
On the remaining plates (Table 1 ) , there was abundant
growth around the crushed caecum and along the accompanying is o la tio n
streak.
The growth appeared as a single species in each case and s lid e
agglutin ation tests confirmed th a t one of the caecal bacteria was
always involved.
As fo r the colony specimens, symbionts were isolated
from both sexes.
Furthermore, the plate with the female caecum was the
only instance where both symbionts were concurrently is o la te d .
The
i n i t i a l growth around the caecum and along the smear was type-B.
How­
ever, six microcolonies which appeared around the caecum a f t e r
additional
incubation
(24 h) were id e n tifie d as type-A by s lid e
a g g lu tin atio n .
The fa c t th at only one of the symbionts was isolated on the m ajority
o f plates was not expected since both were present on most of the
i n i t i a l is o la tio n plates.
However, such resu lts can be explained since
i t is not uncommon fo r one b a c te rial species to outgrow and mask
another when grown simultaneously on a r t i f i c i a l media.
My i n i t i a l response to the lack of growth on some plates was th a t
71
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72
Table 1.
Field survey to confirm the presence of the two caecal
bacteria w ith in Nezara v irid u la ( L .) .
Slide Agglutination Test 1/
C ollection S ite
Date Collected
$
?
Natchitoches, LA
6 /4 /8 0
-
-
Krotz Springs, LA
6 /6 /8 0
A
-
Hamburg, LA
6/12/80
B
-
Port Barre, LA
6/13/80
-
-
Bayou Goula, LA
6/15/80
-
-
Colony (C ontrol)
6/16/80
B
Hamburg, LA 3/
6/25/80
Port Barre, LA
B, A 2 /
1
-
-
2
-
-
3
-
B
4
-
B
5
-
-
-
B
6/26/80
1/
A, type-A caeca! b a c te ria ; B, type-B caecal b a c te ria ; - , no growth
on is o la tio n p la te .
2/
I n i t i a l growth, type-B; secondary growth, type-A.
V
Iso latio n s performed on 5 pairs.
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73
the is o la tio n procedure was a t f a u lt .
Closer examination revealed th at
the procedure was the same as during the o rig in a l is o la tio n s and was
not the cause of the negative re s u lts .
Therefore, some specimens of
f^. v ir id u la apparently do not possess the two symbionts w ithin t h e ir
caeca.
Steinhaus (1951) found larg e numbers o f e x tra c e llu la r symbionts
in the caeca of both sexes o f the cactus jo in t bug, Chelinidea tabulata
(B urm eister), but found no bacteria in one male specimen fo r some
unexplained reason.
e t a l.
In studying Euschistus conspersus U hler, Steinhaus
(1956) isolated the same bacterium, presumably the symbiont,
from 14 o f 59 caeca.
The remaining plates had no growth except fo r
three plates with contaminants present.
not present in some specimens.
Apparently the symbionts were
In each case the insect possessed a
caecum, the specialized stru ctu re evolved fo r housing symbiotic
b a c te ria .
When symbionts are needed in the e a rly stages o f development, i t
is generally assumed th a t they supply n u tritio n a l facto rs fo r growth.
I f needed in the a d u lt, i t is generally assumed they are needed fo r
reproduction (Richards and Brooks 1958).
A normally symbiotic insect
can be considered handicapped should i t be completely deprived o f th a t
re la tio n s h ip .
The handicap always seems to be a n u tritio n a l deficiency
and it s s e v e rity may vary w ith d iffe r e n t species (Brooks 1963b).
v ir id u la ,
In
i t is not known whether the symbionts are needed during
nymphal development or during the adult stage.
Since symbionts are
transm itted to o ffs p rin g follow ing eclosion (West 1980), t h e ir absence
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74
would occur a t th is tim e, in d ic a tin g th a t they were not v it a l to
development o f immatures.
The fie ld -c o lle c te d specimens appeared to
be normal, healthy adults.
There was no visual evidence to in dicate
th a t they were e ith e r su ffe rin g from a n u tr itio n a l deficiency or from
inadequate reproductive capacity.
Perhaps the symbionts are not needed
e ith e r fo r the development or fo r the reproduction of the s tin k bug.
Considering the above, i t might be concluded th a t th is is not a
case o f symbiosis w ithin N^. v ir id u la .
However, i t could be argued th at
the microorganisms might obtain more benefits from an association than
it s host (Richards and Brooks 1958).
In th is instance, the symbionts
apparently obtain several advantages.
The host is a protected environ­
ment which supplies a ll t h e ir metabolic needs as evidenced by th e ir
continued growth and reproduction.
They have a favorable abode in
which they are protected from dessication and are geographically
dispersed.
As fo r the s tin k bug, the b enefits of symbiosis, i f any,
might be m arginal.
As stated by Richards and Brooks (1958, p. 39) there
is "no reason why an association cannot be b e n eficial without being
necessary".
Any consequences o f loss might only be evident a f t e r more
extensive observations, the development o f subsequent generations, or
i f the f ie ld population was subjected to greater stress than was present
a t the time o f c o lle c tio n .
As suggested by Richards and Brooks (1958)
aposymbiotic and symbiotic in divid u als may liv e equally well under
optimal conditions but not under p a rtic u la r suboptimal conditions.
The
caecum apparently evolved as a stru ctu re to accommodate symbionts and
there must have been a s e le c tiv e advantage fo r the s tin k bug to maintain
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75
i t and the mechanism by which the symbionts were transm itted to the
o ffs p rin g .
In s p ite o f the absence o f caeca! bacteria w ithin some
fie ld -c o lle c te d specimens o f Nezara v ir id u la , the evidence would
in dicate th a t an endosymbiotic re la tio n s h ip between th is insect and
it s caecal bacteria is s t i l l an acceptable conclusion, but the b en efit
to the host is unknown.
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REFERENCES CITED
B ailey, W. R ., and E. G. S cott. 1970. Diagnostic microbiology, 3rd
ed. C. V. Mosby Co., S t. Louis, Missouri. 385 pp.
Brooks, M. A. 1963a. The microorganisms o f healthy insects.
In. E. A.
Steinhaus ( e d .) . Insect pathology, v o l. 1. Academic Press In c .,
New York. 661 pp.
Brooks, M. A. 1963b. Symbiosis and aposymbiosis in arthropods.
Soc. Gen. M icrobiol. 13: 200-31.
Buchner, P. 1953. Endosymbiose der Tiere m it pflanzlichen
Mikroorganismen. Verlag Birkhauser, Basel, Switzerland.
Symp.
771 pp.
Buchner, P. 1965. Endosymbiosis of animals with plant microorganisms.
John Wiley and Sons, Inc. (In te rs c ie n c e ), New York. 909 pp.
Koch,
A. 1956. The experimental elim ination of symbionts and it s
consequences. E x p tl. P a ra s ito l. 5: 481-518.
Koch,
A. 1960.
M icrobiol.
In tr a c e llu la r symbiosis in insects.
14: 121-40.
Ann. Rev.
Koch, A. 1967. Insects and th e ir endosymbionts.
S. M. Henry (e d .),
Symbiosis, v o l. 2. Academic Press In c ., New York. 443 pp.
Richards, A. G ., and M. A. Brooks. 1958.
Internal symbiosis in
insects. Ann. Rev. Entomol. 3:37-56.
Steinhaus, E. A. 1951. Report on diagnoses of diseased insects 19441950. H ilgardia 20: 629-78.
Steinhaus, E. A ., M. M. Batey, and C. L. Boerke. 1956. Bacterial
symbiotes from the caeca o f certain Heteroptera. H ilg ardia 24:
495-518.
West, R. P. 1980. Endosymbiosis in the caecum o f the southern green
stink bug, Nezara v ir id u la ( L .) . Ph.D. D is s erta tio n . Louisiana
State U n iv ., Baton Rouge, Louisiana. 77 pp.
76
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VITA
Robert Paul West was born on January 16, 1951, in Elizabeth C ity ,
North Carolina to Robert J. and Peggy Joyce West.
Since his fa th e r
was in the United States Coast Guard, he resided a t several locations
during his adolescence.
His fa th e r r e tire d from the service in 1966
and s e ttle d in Anderson, South C arolina.
Robert graduated from I . L.
Hanna High School in 1969.
That f a l l he entered Clemson U n iv e rsity and received a Bachelor
o f Science degree in Biology (Entomology) in May, 1973.
He immediately
entered graduate school and was awarded a Master o f Science degree in
Entomology in August, 1975.
In June, 1975, he entered Louisiana State
U niversity and is presently a candidate fo r the Doctor o f Philosophy
degree in Entomology.
77
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EXAMINATION AND THESIS REPORT
Candidate:
Robert Paul West
Major Field:
Entomology
Title of Thesis: Endosymbiosis in the Caecum of the Southern Green Stink Bug, Nezara
viridula (L.)
A p p ro v e d :
M a jo r P r o f e s s o r a n d C h a ir m a n
^
D e a n o f th e G r a d u a » S c h o o l
E X A M IN IN G
C O M M IT T E E :
X /'
D a te
o f E x a m in a tio n :
November 20, 1980
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