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Symbiotic interrelationships between microbes and ambrosia beetles
JOURNAL
OF INVERTEBRATE
PlTOHLOOY
Symbiotic VII.
(1972)
Interrelationships Ambrosia
Bacterial
Spmbionts BENAMI
Deparfment
20, r,9-65
of Entomology,
between Beetles
Associated ~'ELEG
AND
C:niversity 1Zrceived
with DALE
Xyleborus :\I.
of Wisconsin,
December
Microbes
and
ferrugineus
S~RRIS
Madison,
Wisconsin
53706’
13, 1971
Transovarially transmitted bacterial symbionts, classified in the genus StaphyloCOCCIS, were isolated and pure cultured from the gut lumen of all stages of the ambrosia beetle, Syleborus ferrugineus. These bacterial symbionts were shown to invade the oocytes, and this invasion was associated with the initiation of nuclear division while the oocytes were surrounded by the follicular wall in the ovariole and mfertilized by sperm.
interrelations with the host’, isolation, classiIn recent years, there has been consider- fication, and possible roles of transovarially able discussion and research on the eco- transmitted bacterial symbionts of the logical and nutritional import’ances of the scolytid, X. ferrugineus. fungi associated with ambrosia beetles in ~IATERIALS AED METHODS their woody substrates. This type of mutualism has been especially studied in Xyleborus The insects utilized in thcsc investigations ferrugineus. Ra!;cr and Korris (1968) clemon- were reared with symbionts in sawdust diet strated that this ambrosia bcetlc posscssc~s(diet “A”, Saunders and Knoke, 1967). paired oral mgcangia (i.c., anatomical strucHistological studies. Male and female tures) in both scxcs in lvhich it,s mut,ualistk adults, eggs, various instars of larvae, and fungi are perpetuated. They found that, the pupae wcrc fixed in formalin-glacial acetic mycangia contain a complex of fungi, yeast, acid-50 % ethanol (1: 1: 18) for 24 hr under and bacteria. In another report, Norris and vacuum. Dehydration n-as through the Baker (1967) stated that most fungus-free TBA series (Jensen, 1962), and the deadults that emerged from surface-sterilized hydrated specimenswere embedded in “Tispupae st,ill bore bacteria, apparently from suemat” (Fisher Co., melting point, 56.5’C). the midgut of the surface-sterilized pupa. Serial 7-p sections of each cmbeddcd speciEarly research 011 this symbiotic complex men werr rut with a rotary microtomr. emphasized thr cctosymbiot,ic fungi of X. Sections were mounted and hydrated jerrugineus and especially focused on the through st,andard histological procedures dependence of the insect pupation on sterol and st’ained by either I,illie’s quick method produced by these mutualistic fungi (Norris for Gram st’aining or the Gram-Weigert and Baker, 1967; Baker and Norris, 196s; technique (Lillic, 1965). Norris et al., 1969; Iirolr et al., 1970; Chu Isolatiojl of symbiotic bacteria. To isolate et al., 1970). the bacterial symbionta from thr adult, The bacteria associatedwith this ambrosia female pupae were collected from brood beetle have remained essentially unstudied tunnels of stock cultures and surfare steriuntil very rwently. This paper reports some lized following the t’cchnique developed by of our findings regarding the occurrence, Norris and Baker (1967). The transformed INTRODUCTION
Copyright AU rights
1972 by Academic Press,lnc. DP reproduction in any form reserved.
59
60
PELEG
AND
adult from such a treated pupa was awpt)ically transfrrrcd to the surfaw of nutrient1 agar medium (Difco Laboratories) in plates for 4S hr at 28°C’. After 4s hr, the beetles were aseptically rc~mowd and cacsh plate was further incubated at 37°C: for .i days. Gram-staiwd smear slides \\-erc prcparcd from each bactrrial colony. Isolatrs wwe cultured 011 nutrient’ agar platw and slants. In attempting isolation of t,hc bactJerium from eggs, they ww surfaccl skrilizcd (Korris and Ba!wr, 1967), aseptically placed singly on nutrient agar slants, and incubated for 4s hr at 37°C. Thaw eggs that proved surfaw sterile \verc then crushed on the medium using a stcrilizc~d nwdlc. Each slant was held at, 37°C for an nddit~ional 5 days and \vas obscrvcd for bac*tcrial growth. In ot,her investigations of the owurrenw of endosymbiot’ic bacteria in the eggs, they were surfaw stcrilizcd (Norris and Baker, 1967) and their internal c~mt cats were squeczcd aseptically onto slides, smeared, heat fixed, and Gram stained. The criteria of Baird-Packer (1966) wcrc used in bact’erial classification. Associatio~ls
betweetr
bacteria
awl
fmgi.
Associations between bacteria and fungi were studied in sample: of ambrosial growth taken from the insect tunnels. Earh sample was removed asept,ically with a bacteriological needle, placed in a drop of sterile water, smeared, heat fixed, and Gram stained. RESULTS Smear slides of ambrosial growth taken aseptically from the brood tunnels of stock cultures revealed that mutualistic fungi may be associated with several types of bact’eria. However, tht prominent bacterium was a gram-positive coccus (Fig. 1) which often appeared in pairs. Following t’he methods given by Baird-Packer (1966), it was placed in the genus Staphylococcus. Histological sections of male and female adults, larvae, and pupae revealed the
NORRIS
presenceof these gram-positive wcci, usually in pairs, in the gut, lumen of the insects (Figs. 2, 3). Serial swtions of both virgin and mated females showed that, gram-positive coccuslike bodies, penetrate the dweloping owytes in the ovariole and bwomc, int,imatclJ associatedwith the ocwytc nwlci (E’ig. 4). In the prcscncc of thew cwwi, maturation of the ooqkcs and nuclear division in the embryo consistently occurred while oocytes were still surroundrd by the follicular layer in t’lie ovariole (Fig. 5). Smearing and C;ram staining the internal contents of surfacr-sterilized (Jggs establishcd the prcscnw of gram-positive cocci in the eggs (I’ig. 6). Thus, thew symbionts are t’ransovarinlly transmittrd to th(ht’ggs. Hi&ologic~al scct~ions of first-instar laryae, collcctcd and fixed upon hatching, rrvealed that the symbionts are transmitted from the egg to the hatching larva and app(‘ar initially confined to the gut hmwn (Figs. 7, s). The Staphylococcus symbionts \\-cre isolatcd and pure cultured from ad&s which transformed under aseptic (Aonditions from surface-sterilized pupae. WC fnilcd in om atbempts to cukurc thr symbionts from surface-sterilized rggs.
An unniated female X. jer,,nyi/leus beetle normally lays eggs which yield haploid malt progeny. Following mating, most eggs arc sperm-fertilized, and these produce diploid females (Norris and Chu, 1970). We have demonstrated that following the grampositive cocci penekation of the oocyte, the nucleus of the oocyte initiates division while st,ill surrounded by the follicwlar layer in the ovariole. We postulate that the bacterial symbionts prevent the haking, or near halting, of protein and/or other related synthesis and metabolism that typically occurs at some stage in t’he maturation of unfertilized oocytes (Monroy and Tyler, 1967), and that in this complex mutualistic
BACTERIAL
FIG. 1. Associatiolrs 8tnph&xwcc~/t.Y sp. (b),
SPMBIONTS
between the fungus Pusarium in the diet of their mutualistic
FIG. 2. View of longitudinal gram-positive hnctt+nl symbionts, gut epithelirlm J.
section some
through in pairs
OF
sol& host,
hindgut (arrow),
61
~~lJkh*US
(h, hypha; s, spores) X!/leborus ferrugineus.
of a third-&tar present in the gut
larva lumen
and
the
bacteri
showing namer (gl, gut lumen;
urn
ous ge,
62
F ‘IG. 3. Section ous gram-positive
PELEG
through the midgut cocci, many in pairs
I PIG. 4. Section through a young Noi ;e the gram-positive cocci, some Y, 9rolk) .
AND
NORRIS
of a female beetle during oogenesis. Note (arro\v), in the gut lumen (gl, gut lumen;
oocyte still surrounded by the follicular in pairs (arrows), in the oocyte nucleus
the presence of num lerge, gut epithelium)
layer (f) (n, nucleus;
in the ovaril 3le. nu, nucleol lus;
FIG. 3. Section through initiated before fertilization layer, f). Note that many met,aphase; nt,, telophase).
a mature by sperm nuclei (n)
oocyte in the ovariole showing that nuclear division has bee11 has occurred (i.e., the oocyte is still surrounded by the folliclllar alresdy have been formed, and some show division figures (nm,
6
FIG. 6. Internal rmbryonic nurlc~~s
contents of a surface-sterilized (11) and pairs of gran-positive
oviposited symbiotic
egg showing cocci (b).
the associat,ions
between
an
F kc;. 7. Longitudinal section of a newly hatched (gl) which were transmitt,ed ( arr .ow) in the gut lumen
FIG. 8. Higher of gram-positive
magnification of a portion symbiotic cocci.
larva. Note to the larva
of the bacterial
rrlat~ionship t,llt> bacterium apparmt ly supc~ ccdcs the sperm in such an oocy+c~ wtivatiorl funkm in t)hr rcproduc%ion prowss of its host ?r’. ferrugirrrus, and thus onablw thcl beetlr to rtrproduw part~hc~lo~c~nc~tic~~ll~~.
t.he cluster of bacterial from t,he egg.
symhionts
shown
in Fig.
7. Note
symhic
#nt,s
the pairs
I\C!KNO\VLEI)GMENT~
This study was supported by the College of i\gricultrtral and I,ife Sciences. Universitjy of Wisconsin, Rladison; and in part by a research grant ?;(I. AI O(i195.06 from the National Institutes
of Health P’olmdntion,
and by flmds from the Rlilwartkee, Wisconsin.
Sichornlebrr
REFEIZENC’ES
B.IKF;K, J. hl., .\xn Soaars, of fungi mutualistically tion of t,he ambrosia gineus.
J.
Inzwrfehr.
I). hr. l!lG3. A complex involved in the nutribeetle .\uIebol.~.y ferricPalhol..
11,
M-250.
B.uRD-~‘.\cIcEI~, .4. C’. MO. Methods for classifying Staphylococci and ;\Iicrorocci. In “Identificat,ion Methods for Microbiologists. I’art A,” (B. RI. (Gibbs, and F. A. Skinner, eds.). pp. 5Wi4. Academic Press, Sew York, 10Gli. CHU, H. M., No~ars, 1). RI., \KD KOK, I,. T. 1970. Pupation requirement of the hertlr, S!//ebortrs ferrngineus: sterols other than cholesterol. J. JICNSF:N,
I7LS(',~/
I'hysiol.,
W. A. lBK2. pp. Frreman,
16,
1379.138s.
“Botanical TIisto~hernistr~,” San Francisco, California,
MX 1%X2. KOIC, L. T., NORRIS, I). >I., .\xn C~iu, H. h1. 1!)70. Hterol metabolism as a basis for a mutllalistic symbiosis. .\‘crf~re ilonrlon), 225, 661.GU.
LIMPID:, I:. 1). lM5. “Histopathology Technic and Practical Histochemistry,” 715 pp. McC;rawHill, New York, 1965. hIONKoY, A., .\ND TYLFX, A. 1967. The activation of the egg. In “Fertilization” (C. H. Met,z and A. hlonroq-, c>ds.), Vol. 1, pp. 3X-412. .4cademic I’ress, New York, 19ti7. NOMUS, I). >I., .\ND B.KKH, J. &I. 1967. S,vmhiosis: effects of a mrltllalistic fungus upon the growth and reprod~~ction of Syleborfts jcr,,,cgincrrs. Scic~e, 156, 1120-1122. NOIUUS, I). hI., .\ND CHT, H. hl. 1970. Nrltriticln of Sylehorus Srrrlrginctts. II. A holidic diet for t tie aposymbiotic insect. .I 1111. En/onlo(. 80~~. .l,rler., 63, ll+L-1145. NOKRIS, I). hl., B.\I
Sot.
.Ivwr.,
62,
413-414.
s \LJx~~;lts, J. I,., .AND ~NOKI’:, J. Ii. 1967. 1)iets fat rearing the ambrosia heetle, Syleborus ferruginrits (Fahr.), in vi/m. Science, 157,460-i%.
OF INVERTEBRATE
PlTOHLOOY
Symbiotic VII.
(1972)
Interrelationships Ambrosia
Bacterial
Spmbionts BENAMI
Deparfment
20, r,9-65
of Entomology,
between Beetles
Associated ~'ELEG
AND
C:niversity 1Zrceived
with DALE
Xyleborus :\I.
of Wisconsin,
December
Microbes
and
ferrugineus
S~RRIS
Madison,
Wisconsin
53706’
13, 1971
Transovarially transmitted bacterial symbionts, classified in the genus StaphyloCOCCIS, were isolated and pure cultured from the gut lumen of all stages of the ambrosia beetle, Syleborus ferrugineus. These bacterial symbionts were shown to invade the oocytes, and this invasion was associated with the initiation of nuclear division while the oocytes were surrounded by the follicular wall in the ovariole and mfertilized by sperm.
interrelations with the host’, isolation, classiIn recent years, there has been consider- fication, and possible roles of transovarially able discussion and research on the eco- transmitted bacterial symbionts of the logical and nutritional import’ances of the scolytid, X. ferrugineus. fungi associated with ambrosia beetles in ~IATERIALS AED METHODS their woody substrates. This type of mutualism has been especially studied in Xyleborus The insects utilized in thcsc investigations ferrugineus. Ra!;cr and Korris (1968) clemon- were reared with symbionts in sawdust diet strated that this ambrosia bcetlc posscssc~s(diet “A”, Saunders and Knoke, 1967). paired oral mgcangia (i.c., anatomical strucHistological studies. Male and female tures) in both scxcs in lvhich it,s mut,ualistk adults, eggs, various instars of larvae, and fungi are perpetuated. They found that, the pupae wcrc fixed in formalin-glacial acetic mycangia contain a complex of fungi, yeast, acid-50 % ethanol (1: 1: 18) for 24 hr under and bacteria. In another report, Norris and vacuum. Dehydration n-as through the Baker (1967) stated that most fungus-free TBA series (Jensen, 1962), and the deadults that emerged from surface-sterilized hydrated specimenswere embedded in “Tispupae st,ill bore bacteria, apparently from suemat” (Fisher Co., melting point, 56.5’C). the midgut of the surface-sterilized pupa. Serial 7-p sections of each cmbeddcd speciEarly research 011 this symbiotic complex men werr rut with a rotary microtomr. emphasized thr cctosymbiot,ic fungi of X. Sections were mounted and hydrated jerrugineus and especially focused on the through st,andard histological procedures dependence of the insect pupation on sterol and st’ained by either I,illie’s quick method produced by these mutualistic fungi (Norris for Gram st’aining or the Gram-Weigert and Baker, 1967; Baker and Norris, 196s; technique (Lillic, 1965). Norris et al., 1969; Iirolr et al., 1970; Chu Isolatiojl of symbiotic bacteria. To isolate et al., 1970). the bacterial symbionta from thr adult, The bacteria associatedwith this ambrosia female pupae were collected from brood beetle have remained essentially unstudied tunnels of stock cultures and surfare steriuntil very rwently. This paper reports some lized following the t’cchnique developed by of our findings regarding the occurrence, Norris and Baker (1967). The transformed INTRODUCTION
Copyright AU rights
1972 by Academic Press,lnc. DP reproduction in any form reserved.
59
60
PELEG
AND
adult from such a treated pupa was awpt)ically transfrrrcd to the surfaw of nutrient1 agar medium (Difco Laboratories) in plates for 4S hr at 28°C’. After 4s hr, the beetles were aseptically rc~mowd and cacsh plate was further incubated at 37°C: for .i days. Gram-staiwd smear slides \\-erc prcparcd from each bactrrial colony. Isolatrs wwe cultured 011 nutrient’ agar platw and slants. In attempting isolation of t,hc bactJerium from eggs, they ww surfaccl skrilizcd (Korris and Ba!wr, 1967), aseptically placed singly on nutrient agar slants, and incubated for 4s hr at 37°C. Thaw eggs that proved surfaw sterile \verc then crushed on the medium using a stcrilizc~d nwdlc. Each slant was held at, 37°C for an nddit~ional 5 days and \vas obscrvcd for bac*tcrial growth. In ot,her investigations of the owurrenw of endosymbiot’ic bacteria in the eggs, they were surfaw stcrilizcd (Norris and Baker, 1967) and their internal c~mt cats were squeczcd aseptically onto slides, smeared, heat fixed, and Gram stained. The criteria of Baird-Packer (1966) wcrc used in bact’erial classification. Associatio~ls
betweetr
bacteria
awl
fmgi.
Associations between bacteria and fungi were studied in sample: of ambrosial growth taken from the insect tunnels. Earh sample was removed asept,ically with a bacteriological needle, placed in a drop of sterile water, smeared, heat fixed, and Gram stained. RESULTS Smear slides of ambrosial growth taken aseptically from the brood tunnels of stock cultures revealed that mutualistic fungi may be associated with several types of bact’eria. However, tht prominent bacterium was a gram-positive coccus (Fig. 1) which often appeared in pairs. Following t’he methods given by Baird-Packer (1966), it was placed in the genus Staphylococcus. Histological sections of male and female adults, larvae, and pupae revealed the
NORRIS
presenceof these gram-positive wcci, usually in pairs, in the gut, lumen of the insects (Figs. 2, 3). Serial swtions of both virgin and mated females showed that, gram-positive coccuslike bodies, penetrate the dweloping owytes in the ovariole and bwomc, int,imatclJ associatedwith the ocwytc nwlci (E’ig. 4). In the prcscncc of thew cwwi, maturation of the ooqkcs and nuclear division in the embryo consistently occurred while oocytes were still surroundrd by the follicular layer in t’lie ovariole (Fig. 5). Smearing and C;ram staining the internal contents of surfacr-sterilized (Jggs establishcd the prcscnw of gram-positive cocci in the eggs (I’ig. 6). Thus, thew symbionts are t’ransovarinlly transmittrd to th(ht’ggs. Hi&ologic~al scct~ions of first-instar laryae, collcctcd and fixed upon hatching, rrvealed that the symbionts are transmitted from the egg to the hatching larva and app(‘ar initially confined to the gut hmwn (Figs. 7, s). The Staphylococcus symbionts \\-cre isolatcd and pure cultured from ad&s which transformed under aseptic (Aonditions from surface-sterilized pupae. WC fnilcd in om atbempts to cukurc thr symbionts from surface-sterilized rggs.
An unniated female X. jer,,nyi/leus beetle normally lays eggs which yield haploid malt progeny. Following mating, most eggs arc sperm-fertilized, and these produce diploid females (Norris and Chu, 1970). We have demonstrated that following the grampositive cocci penekation of the oocyte, the nucleus of the oocyte initiates division while st,ill surrounded by the follicwlar layer in the ovariole. We postulate that the bacterial symbionts prevent the haking, or near halting, of protein and/or other related synthesis and metabolism that typically occurs at some stage in t’he maturation of unfertilized oocytes (Monroy and Tyler, 1967), and that in this complex mutualistic
BACTERIAL
FIG. 1. Associatiolrs 8tnph&xwcc~/t.Y sp. (b),
SPMBIONTS
between the fungus Pusarium in the diet of their mutualistic
FIG. 2. View of longitudinal gram-positive hnctt+nl symbionts, gut epithelirlm J.
section some
through in pairs
OF
sol& host,
hindgut (arrow),
61
~~lJkh*US
(h, hypha; s, spores) X!/leborus ferrugineus.
of a third-&tar present in the gut
larva lumen
and
the
bacteri
showing namer (gl, gut lumen;
urn
ous ge,
62
F ‘IG. 3. Section ous gram-positive
PELEG
through the midgut cocci, many in pairs
I PIG. 4. Section through a young Noi ;e the gram-positive cocci, some Y, 9rolk) .
AND
NORRIS
of a female beetle during oogenesis. Note (arro\v), in the gut lumen (gl, gut lumen;
oocyte still surrounded by the follicular in pairs (arrows), in the oocyte nucleus
the presence of num lerge, gut epithelium)
layer (f) (n, nucleus;
in the ovaril 3le. nu, nucleol lus;
FIG. 3. Section through initiated before fertilization layer, f). Note that many met,aphase; nt,, telophase).
a mature by sperm nuclei (n)
oocyte in the ovariole showing that nuclear division has bee11 has occurred (i.e., the oocyte is still surrounded by the folliclllar alresdy have been formed, and some show division figures (nm,
6
FIG. 6. Internal rmbryonic nurlc~~s
contents of a surface-sterilized (11) and pairs of gran-positive
oviposited symbiotic
egg showing cocci (b).
the associat,ions
between
an
F kc;. 7. Longitudinal section of a newly hatched (gl) which were transmitt,ed ( arr .ow) in the gut lumen
FIG. 8. Higher of gram-positive
magnification of a portion symbiotic cocci.
larva. Note to the larva
of the bacterial
rrlat~ionship t,llt> bacterium apparmt ly supc~ ccdcs the sperm in such an oocy+c~ wtivatiorl funkm in t)hr rcproduc%ion prowss of its host ?r’. ferrugirrrus, and thus onablw thcl beetlr to rtrproduw part~hc~lo~c~nc~tic~~ll~~.
t.he cluster of bacterial from t,he egg.
symhionts
shown
in Fig.
7. Note
symhic
#nt,s
the pairs
I\C!KNO\VLEI)GMENT~
This study was supported by the College of i\gricultrtral and I,ife Sciences. Universitjy of Wisconsin, Rladison; and in part by a research grant ?;(I. AI O(i195.06 from the National Institutes
of Health P’olmdntion,
and by flmds from the Rlilwartkee, Wisconsin.
Sichornlebrr
REFEIZENC’ES
B.IKF;K, J. hl., .\xn Soaars, of fungi mutualistically tion of t,he ambrosia gineus.
J.
Inzwrfehr.
I). hr. l!lG3. A complex involved in the nutribeetle .\uIebol.~.y ferricPalhol..
11,
M-250.
B.uRD-~‘.\cIcEI~, .4. C’. MO. Methods for classifying Staphylococci and ;\Iicrorocci. In “Identificat,ion Methods for Microbiologists. I’art A,” (B. RI. (Gibbs, and F. A. Skinner, eds.). pp. 5Wi4. Academic Press, Sew York, 10Gli. CHU, H. M., No~ars, 1). RI., \KD KOK, I,. T. 1970. Pupation requirement of the hertlr, S!//ebortrs ferrngineus: sterols other than cholesterol. J. JICNSF:N,
I7LS(',~/
I'hysiol.,
W. A. lBK2. pp. Frreman,
16,
1379.138s.
“Botanical TIisto~hernistr~,” San Francisco, California,
MX 1%X2. KOIC, L. T., NORRIS, I). >I., .\xn C~iu, H. h1. 1!)70. Hterol metabolism as a basis for a mutllalistic symbiosis. .\‘crf~re ilonrlon), 225, 661.GU.
LIMPID:, I:. 1). lM5. “Histopathology Technic and Practical Histochemistry,” 715 pp. McC;rawHill, New York, 1965. hIONKoY, A., .\ND TYLFX, A. 1967. The activation of the egg. In “Fertilization” (C. H. Met,z and A. hlonroq-, c>ds.), Vol. 1, pp. 3X-412. .4cademic I’ress, New York, 19ti7. NOMUS, I). >I., .\ND B.KKH, J. &I. 1967. S,vmhiosis: effects of a mrltllalistic fungus upon the growth and reprod~~ction of Syleborfts jcr,,,cgincrrs. Scic~e, 156, 1120-1122. NOIUUS, I). hI., .\ND CHT, H. hl. 1970. Nrltriticln of Sylehorus Srrrlrginctts. II. A holidic diet for t tie aposymbiotic insect. .I 1111. En/onlo(. 80~~. .l,rler., 63, ll+L-1145. NOKRIS, I). hl., B.\I
Sot.
.Ivwr.,
62,
413-414.
s \LJx~~;lts, J. I,., .AND ~NOKI’:, J. Ii. 1967. 1)iets fat rearing the ambrosia heetle, Syleborus ferruginrits (Fahr.), in vi/m. Science, 157,460-i%.