Title: Relations of the blue stain fungus, Ceratocystis ips (Rumbold) C. Moreau, to Ips bark beetles (Coleoptera: Scolytidae) occurring in Florida
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Title: Relations of the blue stain fungus, Ceratocystis ips (Rumbold) C. Moreau, to Ips bark beetles (Coleoptera: Scolytidae) occurring in Florida
Physical Description: v, 81 leaves : ; 28 cm.
Language: English
Creator: Yearian, William C., 1937-
Copyright Date: 1966
 Subjects
Subject: Beetles   ( lcsh )
Beetles   ( lcsh )
Ceratocystis   ( lcsh )
Entomology and Nematology thesis Ph. D
Dissertations, Academic -- Entomology and Nematology -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
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Statement of Responsibility: by William C. Yearian.
Thesis: Thesis (Ph. D.)--University of Florida, 1966.
Bibliography: Includes bibliographical references (leaves 80-81).
General Note: Typescript.
General Note: Vita.
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Bibliographic ID: UF00098218
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: alephbibnum - 000430507
oclc - 37498950
notis - ACH9844

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RELATIONS OF THE BLUE STAIN FUNGUS,
CERAT[OCYSTIS IPS (RUMBOLD)
C. MOREAU, TO IPS BARK BEETLES
(COLEOPTERA: SCOLYTIDAE)
OCCURRING IN FLORIDA












By
WILLIAM C. YEARIAN, JR.


A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL !OF
THE UNIVERSITY OF FLORIDA ( ( /, \
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOP. THE
DEGREE OF DOCTOR OF PHILOSOPHY








UNIVERSITY OF FLORIDA


June, 1966












ACKNOWLEDGMENT


The writer wishes to acknowledge his indebtedness to the

Co-Chairmen of his Supervisory Committee, Dr. R. C. Wilkinson and

Dr. L. A. Hetrick, for their advice and assistance throughout the

course of this study and the preparation of the manuscript.

Special thanks are also due Dr. J. T. Crelghton, Dr. C. M. Kaufman,

and Dr. K. R. Swinford for their criticism on the preparation of

the manuscript. The writer also wishes to express his appreciation

to Dr. L. C. Kuitert for his interest and encouragement. Sincere

gratitude is extended to Mr. W. J. Coleman for assistance in both

the field and laboratory phases of the work.

Thanks are accorded Dr. J. W. Kimbrough, Department of Plant

Pathology, University of Florida and Dr. C. L. Wilson, Department of

Plant Pathology, University of Arkansas for confirmation of Cerato-

cystis Ips (Rumboid) C. Moreau.

The generous assistance of Mr. Charles C. Russell In the

preparation of photographs that appear in the manuscript Is gratefully

acknowledged. Special thanks are also due Miss Linda Weir for typing

of the manuscript.

This study was conducted under Florida Agricultural Experiment

Station State Project 1188. Additional funds were made available by

the Southern Forest Disease and Insect Research Council, Southern

Pulpwood Conservation Association.









A special kind of thanks is due my wife, LaVerne, for her constant

encouragement during the course of this work. To the above and all

others who have given help during this study, the writer wishes to

express deepest appreciation.














TABLE OF CONTENTS

PAGE
ACKNOWLEDGMENT ......................... ...... ...... ....... II

LIST OF TABLES.............................................. vl
LIST OF FIGURES ......................... .............. .... Villa

INTRODUCTION........ ............. ...........................

LITERATURE REVIEW................. .... .............. 3

Transmission of Ceratocystis Species................... 3

Specificity of Ceratocystis spp. Bark Beetle
Association .......................................... 6

Characteristics of Bark Beetle Associated
Ceratocystis Species.................................. 8

Nature of the Ceratocystis spp. Bark Beetle
Association....................................... .... 10

Pathogenicity of Ceratocystis Associates of
Bark Beetles........... .............. .............. ... 12

MATERIALS AND METHODS ... ................. ............... .... 15

Fungus Isolations ...... ......... ..... ...... .... 15

Frequency of C. Ips Transmission....................... 16

Rearing Ips Species in Laboratory Colonies............. 19

Rearing Ips Species Free of C. Is .................. 21

Mass Attraction........................................ 24

Ips Development in C. Ips Stained Bolts .... 25

Brood Development and Fecundity........................ 27








RESULTS AND DISCUSSION...................................... 32

Fungus Isolations........................ .......... 32

Frequency of C. Ips Transmission...................... 37

Larval and Pupal Development on Phloem-Based
Rearing Medium ...... .... ...... .. ............... .... .. 39

Mass Attraction....................................... 42

Ips Development In C. Ips Stained Bolts ............... 44

Effect of C. Ips on Brood Development and
Fecundlty ................. ........................ 45

Egg gallery construction......................... 45

Ceratocystis Ips In brood development
test bolts....................................... 48

Brood size and mortality.......................... 49

Brood composition.................................. 53

Pupal weight ............................ ......... 62

Sex ratio ......................................... 62

Fecundity ........................... ........... 62

Discussion of the Effect of C. ips and Other
Organisms on Brood Development and Fecundity.......... 64

CONCLUSIONS ................ ........ ... ..... ....... ....... 73

LITERATURE CITED............................................ 75

BIOGRAPHICAL SKETCH ......................................... 80














LIST OF TABLES
PAGE
I. Phloem-based, semi-artificial rearing medium for
Ips bark beetles. Amounts sufficient to make
100 grams ........................................... 23

2. Relative frequency of Ceratocystis ps
isolation from Ips avulsus, fps callgraphus,
and Ips grand lcoll Is .. ............................... 36

3. Relative frequency of Ceratocystis Ips
transmission by Ips avulsus, Ips calllgraphus,
and Ips grandicolls ...... ......... ................ 38

4. Mean larval and pupal development of Ips avulsus,
Ips calllgraphus, and Ips grandlcollls on phloem-
based semi-artificial rearing meadlum at 300 C........ 40

5. Attractiveness of W and BSF Ips calllgraphus
male infested slash pine bolts........................ 43

6. Mean egg gallery length and spacing between
eggs of W, BSF, and BS Ips avulsus, Ips
calligraphus and Ips grandlcollis females
In slash pine bolts at 300 C.......................... 47

7. Mean size and mortality of W, BSF, and BS Ips avulsus,
Ips calllgraphus, and Ips grandlcolls broods In
slash pine bolts at 30v C..... ..... ... ............ 52

8. Mean composition of W, BSF, and BS Ips avulsus, Ips
calligraphus, and Ips grandlcollls broods In slash
pine bolts at 5, 10, 15, and 20 days at 300 C......... 57

9. Relative composition (percentage) of W, BSF, and BS
Ips avulsus, Ips calllgraphus, and Ips grandlcollis
broods In slash pine bolts at 5, 10, 15, and 20
days at 30 C................... ..................... 59

10. Chi-square comparison of W, BSF, and BS Ips avulsus,
Ips calllgraphus, and Ips grandlcollls brood
composition at 5, 10, 15, and 20 days at 300 C........ 61








II. Mean weight (mg.) of 100 W, BSF, and BS Ips avulsus,
Ips calllgraphus, and Ips grandlcollls pupae reared
Tn slash pine bolts at 30 C.......................... 63

12. Sex ration of W, BSF, and BS ps avulsus, Ips
calllgraphus, Ips grandcoll ls adults reared In
slash pine bolts at 30" C............................ 64

13. Mean length of egg gallery, total number of eggs
laid, and number of eggs deposited per centimeter
of gallery by W, BSF, and BS Ips avulsus,
Ips calllgraphus, and Ips grandicollls females
at 30 days In slash pine bolts at 30 C.............. 65















LIST OF FIGURES


PAGE
I. Screen cage, 6' X 6' X 6', containing male
infested loblolly pine bolts used to attract
wild beetles ...... ................................... 18

2. Gelatin capsule used to hold bark beetles prior
to entrance Into pine bolt. Capsule held In
place with "Duxsea l".................... ........... 20

3. Rotating turntable used to expose male Infested
pine bolts In attraction studies...................... 26

4. Pine bolt rearing unit used In brood
development studies................................... 30

5. Perltheclum of Ceratocystis Ips. Approx. 75X.......... 33

6. Ascospores of Ceratocystis Ips. Approx. 2000X......... 34

7. Section of 15-day old BSF Ips grandlcollls gallery
5 centimeters from nuptial chamber. Note
perlthecia of Ceratocystls Ips lining gallery and
masses of ascospores at tips of perlthecla.
Approx. 40X..................... .................... 50

8. Section of 15-day old BSF Ips grandlcollls gallery
5 centimeters from nuptial chamber. Note absence
of Ceratocystis Ips. Approx. IOX.................... 51

9. Mean composition of W, BSF, and BS Ips avulsus
broods In slash pine bolts at 5, 10, 15, and
20 days at 300 C...................................... 54

10. Mean composition of W, BSF, and BS Ips calll-
graphus broods In slash pine bolts at 5, 10,
15, and 20 days at 300 C.............................. 55

II. Mean composition of W, BSF, and BS Ips grandlcollis
broods In slash pine bolts at 5, 10, 15, and 20
days at 30 C................................... ...... 56


vill








12. Ips avulsus pupal chamber covered with masses
of conitda, probably Tubercularlella Ips.
Approx. 5X.............................................. 71













INTRODUCTION


The association between blue stain fungi, particularly of the

genus Ceratocystis Ellis and Halstead, and scolytld bark beetles In

the genera Dendroctonus Erichson and Ips DeGeer has long been known.

Because of the constant presence of blue stain fungi In the phloem

and sapwood of trees attacked by bark beetles, early workers

generally assumed the fungi were commonly introduced under the

bark by Insects. Leach, Orr, and Chrlstensen (1934) used a series

of caged and uncaged logs of red pine, PInus reslnosa Alt., to

prove that Ips grandlcollls (Eichh.) and Ips pini (Say) transmitted

the blue stain fungus, Ceratocystis Ips (Rumbold) C. Moreau. They

concluded that certain blue stain fungi, such as C. Ips, were rarely,

if ever, transmitted In any other way.

Most previous research on blue stain fungi and bark beetles was

directed at determining the species of fungi associated with various

bark beetles and the effect of the fungi on the host trees and their

wood products. No comprehensive study was made of the relationship

between the organisms involved, and the nature of the association

was open to considerable question. Cralghead (1928), Leach (1940),

Leach et al. (1934) and Nelson (1934) considered the association to

be one of mutualism, with the fungi benefiting from transmission by

the beetles to a suitable host and the Insects benefiting from

creation of a more favorable environment for brood development by








action of the fungi on the wood and inner bark. Hoist (1937) in the

United States and Grosmann (1930) In Germany reared certain bark

beetles from egg to adult free of their blue stain associates and

concluded the fungi were not essential for development within a

single generation of the insects. Hetrick (1949) observed an

Infestation of the southern pine beetle, Dendroctonus frontal s ZImm.,

In which the trees showed no signs of blue staining. Since beetle

brood development appeared to be normal, he concluded that blue

stain organisms were not essential for successful attack by the

beetles.

In light of the uncertainty of the exact nature of the blue

stain fungus-bark beetle association, a project was initiated to

study the Ips bark beetles occurring In Florida; Ips avulsus (Eichh.),

Ips calligraphus (Germ.), and Ips grandicollis (Eichh.), and their

blue stain fungus associate, Ceratocystis Ips (Rumbold) C. Moreau.

The principal objective of the study was to compare development of

all stages of the beetles in one or more generations in the presence

or absence of the fungus. The results of this investigation

together with observations on the biology and behavior of the beetles

are reported herein.













LITERATURE REVIEW


Thatcher (1960) reviewed current knowledge of the bark beetles

attacking southern pines which included the Ips species occurring

in Florida. These beetles have not been studied intensively and no

detailed data were available on their biology and habits. A

discussion of blue stain in timber was recently presented by Findlay

(1959a, b) In which he covered the fungi Involved and their effect

on strength and quality of wood, dissemination, prevention, and

control.

Bark beetle-associated blue stain fungi have been described

in several genera, but most have belonged to the genus Ceratocystis.

Species of Graphlum and Leptographium, many of which were shown to

be Imperfect forms of Ceratocystis, and Tubercularlella Ips Leach,

Orr, and Christensen also have been found to cause blue stain

(Methre, 1964a). The present study was restricted to the Ceratocystis

associates of Ips species bark beetles In Florida. Hunt (1956)

revised the genus Ceratocystis, and all previous citations from the

literature have been given in terms of his monograph.


Transmission of Ceratocystis Species


The bluing of timber was first described by Hartig in 1878

(Hartig, 1894). He recognized the fungal nature of the stain,

Ceratocystis pillfera (Fries) C. Moreau, and noted that the fungus








commonly occurred In trees attacked by insects. Von Schrenk (1903)

studied blue stain fungi In ponderosa pine, Pinus ponderosa Laws,

attacked by Dendroctonus ponderosae Hopk. He isolated Ceratocystis

pillfera from the galleries of the beetle which suggested the beetle

transmitted the fungus. Von Schrenk was unable to isolate the fungus

from the intestinal tract and feces of the beetle, however, and

discounted the probability of insect transmission. He concluded

that the spores of the fungus were distributed by the wind and

entered the region between the bark and wood through holes made

by the beetles.

Little attention was given the blue stain fungus-bark beetle

association until Craighead (1928) suggested that the fungi were

transmitted by bark beetles. Supported by isolations of Ceratocystis

species from wood adjacent to beetle galleries and directly from the

beetles, subsequently workers (Bakshi, 1950; Bramble and Hoist, 1935,

1940; Davidson, 1935, 1955, 1958; Ellis, 1939; Grosmann, 1930;

Leach et al., 1934; Nelson, 1934; Nelson and Beal, 1929; Robinson,

1962; Roblnson-Jeffery and Grinchenko, 1964; Rumbold, 1929, 1931,

1936, 1941; Verrall, 1941; and others) generally assumed that bark

beetles played an Important role In dissemination of blue stain

fungi. Rumbold (1931) showed that C. pilifera was a secondary

Invader. She was of the opinion that wood used by Von Schrenk (1903)

to start his cultures was too old, and the primary fungus, probably

Ceratocystis minor (Hedgc.) Hunt, had lost Its vitality by the time

the wood reached the laboratory. Grosmann (1930) stated that

Von Schrenk's failure to isolate the fungus from the beetle could









be explained In that he used liquid agar which, owing to its high

temperature of solidlflcation, was too hot at the time of

innoculation. She also added that Von Schrenk had obviously

neglected to prevent bacterial growth by the addition of acid to

the culture medium and to punctually Isolate the germinating

fungus spores. Grosmann noted that even though no exact proof

had been presented, repeated observations that scolytids appeared

mostly In association with blue stain fungi led to the recognition

that bark beetles played a significant role as transmitters of

blue stain fungi.

Leach et al. (1934) studied the interrelationships of bark

beetles and blue stain fungi In felled red pine, Plnus resinosa Alt.,

and demonstrated transmission of Ceratocystls Ips (Rumbold) C. Moreau

by Ips grandlcollls (Elchh.) and Ips pini (Say). In a series of

caged and uncaged logs with sealed and unsealed ends, C. Ips

occurred only in logs attacked by the bark beetles. The fungus

was abundant In all uncaged logs and absent from the caged logs

except when a few beetles accidentally gained entrance. In logs

lightly attacked by the beetles, the fungus was limited to the

Immediate vicinity of the beetle tunnels. Histological preparations

of adult beetles revealed ascospores of the fungus adhering In

clumps to various parts of the body wall and distributed throughout

the intestinal tract. Fragments of perithecla were also found In

the gut, and examination of fecal pellets showed viable ascospores

of the fungus to be present. In view of their findings, Leach at al.

(1934) concluded that these bark beetles universally Introduced

blue stain fungi Into logs and trees.








Not all Ceratocystis species have been found to be disseminated

by bark beetles. Verrall (1941) studied dissemination of various

blue stain fungi In lumber yards In Louisiana and Mississippi and

found air currents, insects, milling machinery, and rainwater to be

disseminating agents. C. Ips was occasionally Isolated from the

air, but Verrall stated Ips species bark beetles were the most

important transporters of the fungus since he Isolated C. Ips

from 62 per cent of the bark beetles he captured in the yards.

Leach et al. (1934) also presented evidence that mites associated

with bark beetles often carried C. Ips spores on their bodies and

distributed the spores through the beetle gallery system.


Specificity of Ceratocystis spp. Bark Beetle Association


Depending upon their association with bark beetles, Mathlesen

(1950) divided the Swedish Ceratocystis species Into the following

types: (I) those species spread primarily by wind and only rarely

by bark beetles: e.g., C. coerulescens (MUnch) Bakshl, C. floccosa

(Mathlesen) Hunt, C. pillfera, and C. plurlannulata (Hedgc.) C.

Moreau; (2) those species associated with several bark beetles

usually In older material, but also spread by wind: e.g., C. minor,

C. minute (Slem.) Hunt, C. plceae (Munch) Bakshl, and C. polonlcum

(Slem.) C. Moreau; and (3) those always associated with certain

species of bark beetles: e.g., C. brunneo-cillata (Mathlesen-K.)

Hunt with Ips sexdentatus Boern., C. cana (MUnch) C. Moreau with

Blastophagus minor Hart, and C. clavata (Mathlesen) Hunt with Ips

acumlnatus Gyll. In North America, Dendroctonus and Ips species








have been considered the most Important bark beetles of coniferous

trees. Several Ceratocystis species have been found associated with

Dendroctonus bark beetles (Bramble and Hoist, 1940; Davidson, 1955,

1958; Mathre, 1964a; Robinson, 1962; Robinson-Jeffrey and Grinchenko,

1964; and Rumbold, 1931, 1936, 1941), but C. minor was the most common

found species. Ceratocystis ips has been the only species of the

genus consistently associated with Ips bark beetles.

Ceratocystis Ips was described by Rumbold (1931) as an associate

of Ips calligraphus and Ips grandicollls. The fungus was also found

In association with Ips avulsus (Rumbold, 1931), Ips confusus (Lec.)

(Mathre, 1964a), Ips emarginatus (Lec.) (Mathre, 1964a; Rumbold, 1936),

Ips Integer (Elchh.) (Rumbold, 1936), Ips lecontel (Lec.) (Ellis,

1939), Ips oregoni (Eichh.) (Mathre, 1964a; Rumbold, 1936), Ips pini

(Leach et al., 1934), Ips plastographus (Lec.) (Rumbold, 1936) and

Ips ponderosa Sw. (Mathre, 1964a). C. Ips was also found associated

with Orthotomlcus caelatus (Elchh.) (Verrall, 1941), Dendroctonus

valens Lec. and Dendroctonus ponderosa (Mathre, 1964a) in this

country. In addition to North America, C. Ips was reported In

association with Ips sexdentatus in Germany (Francke-Grosmann, 1963)

and Poland (Slemaszko, 1939), Ips species In Japan (Nislkado and

Yamautl, 1933), and Orthotomicus proximus (Elchh.) In Sweden

(Mathlesen-KOrik, 1960). Certain ips species in Europe were found

to be associated with several Ceratocystis species other than C. Ips,

however.








Characteristics of Bark Beetle Associated Ceratocystis Species


The blue stain fungi associated with bark beetles character-

Istically have conldia and ascospores covered with a sticky mucilage:

a characteristic associated with adherence of these reproductive

bodies to the body wall of the Insects. This trait has not been

observed in wind-borne Ceratocystis species. Leach et al. (1934)

showed the sticky covering also protected the spores as they passed

through the beetles' digestive tract. Mathlesen-Kiarik (1960)

demonstrated that the sticky layer rendered the spores resistant to

desiccation. She found the spores remained viable for several

months on beetles in hibernation.

The bark beetle associated Ceratocystis have been shown to be

ecologically adapted to their vectors. MethIeson-KAirlk (1960)

and Grosmann (1930) studied the development of certain European

bark beetles and found developmental time for the fungi corresponded

very closely with that of the beetles. Leach et al. (1934) made

similar observations in the United States on C. Ips In association

with Ips grandicollis and Ips pini. The fungus began to grow

immediately after Introduction by the beetles and was isolated from

wood near the galleries within a week. Isolation was possible before

visible signs of staining were present. The fungus rapidly spread

radially and longitudinally from the egg gallery. Tangential

growth was slow and took place primarily through the larval galleries.

Isolations showed that the larvae moved ahead of C. ips and very

little, If any, of the fungus was consumed. Shortly after the

larvae pupated the fungus began to sporulate. Perlthecla and









coremla were abundant In the old egg galleries, larval tunnels, and

pupal chambers. Perlthecia also formed In large numbers In the inner

bark not consumed by the larvae. Francke-Grosmann (1963) noted that

C. Ips was especially adapted to endozoic dissemination since the

spores of embedded perithecia could be spread only when young

beetles ate the inner bark and carried the spores In their gut to

new breeding places. Newly formed Ips grandicollls and Ips pinl

adults began feeding before leaving the logs in which they had

developed, and according to Leach et al. (1934) the young beetles

wandered through old egg galleries and tunneled through Inner bark

containing embedded perlthecla. Many spores were ingested or

adhered to the outside of the body wall. They stated that the

beetles emerged from the logs thoroughly Infested with spores of

the fungus.

Through studies on the physiology of certain Ceratocystis

species, Mathlesen-Kaarik (1960) attempted to differentiate

between the Insect and non-Insect associated species. She found

Insect associated forms to be deficient of certain vitamins while

wind-blown forms were vitamln-autotrophic. The Insect associated

species were able to utilize a greater number of carbohydrates and

showed a wider enzymatic capacity than the wind-borne species. She

was not able to demonstrate consistent physiological differences

between the two groups of fungi, but she stated each fungus had its

own special moisture and nutritional requirements and developmental

time. Mathlesen-Kaarlk (1960) concluded that all these factors

together might produce optimal conditions for some particular fungus








or fungi in the galleries of a specific bark beetle; other fungi

might occasionally be Introduced Into the galleries of this Insect,

but were not compatible for long periods of time and would disappear

In succeeding Insect generations. She noted that this would fully

explain the existence of non-specialized associations between certain

widespread bluing fungi and a number of different but ecologically

similar bark beetles.


Nature of the Ceratocystis spp. Bark Beetle Association


The blue stain fungus-bark beetle association has been referred

to as mutualism by some workers and commensalism by others. It has

been clearly demonstrated in some Instances that the fungi benefit

from the association. Leach et al. (1934) and Mathlesen (1950)

proved that certain Ceratocystis species were almost exclusively

disseminated by bark beetles. Leach et al. stated that the fungi

obviously benefited from dissemination by the beetles and from

introduction into the Inner bark of logs and susceptible trees.

St. George and Beal* indicated that the action of Dendroctonus

frontalls on the Inner bark created conditions favorable for

C. minor growth since the fungus failed to become established when

Insect broods failed to develop. Pathogenicity trials (Mathre,

1964b, and Nelson, 1934) with C. Ips and C. minor added further





*St. George, R. A. and J. A. Beal. 1927. Progress report on
the southern pine beetle (Dendroctonus frontalls ZImm.), U. S. Bur.
Entomol. Plant Quar. Div. Forest Insect Invest. Ashville, N. C.,
(typewritten 40 pp.) (Cited from Dixon and Osgood, 1961, p. 18).








support to this assumption. The fungi were unable to Infect trees

when the Innoculum was Introduced through needle holes Intended to

simulate Initial bark beetle attack. Grosmann (1930) noted that

blue stain fungi rapidly spread longitudinally and radially in the

wood and phloem from the egg galleries of the beetles, but tangential

spread was slow and took place primarily through tangentially

extended larval galleries In the phloem.

Benefits derived by the beetles from the association have not

been clearly defined. Cralghead (1928) suggested that blue stain

fungi created conditions favorable for brood development and that

the fungi possibly furnished essential nourishment for the beetles.

Nelson (1934) found C. minor Infection resulted In a reduction of

the water content of the wood, and concluded the fungus was probably

necessary for optimum brood development of Dendroctonus frontalls.

Leach et al. (1934) stated:

... The blue-staining fungi, by Inhibiting the flow of
sap, in all probability, make living trees more favorable
for beetle development and by aiding In decomposition of
the Inner bark cause It to separate from the wood, creating
a more favorable environment for development of Insect
broods.

They pointed out that until a brood of beetles was reared In a fungus-

free log, it could not be safely concluded that blue stain fungi

were not necessary for normal development of the beetles.

Other workers have expressed the opinion that both blue stain

fungi and bark beetles can exist and develop fully without their

associates. Grosmann (1930) found both Ips typographus L. and

Blastophagus plnlperda L. to be Independent of their blue stain

associates. She noted that larvae of the beetles moved ahead of








the spread of the fungi through the Inner bark and very little, If

any, fungal material was consumed by the larvae. She observed young

adults of both beetles feeding on blue stain fungi, but she was

unable to detect any difference In the maturation period or vigor

of beetles that had or had not fed on the fungi. Hoist (1937)

aseptically reared Dendroctonus frontalls, Ips calligraphus and

Ips grandicollis In small numbers on strips of phloem sealed between

two glass plates. Since the reared Insects were within size limits

given by Blackman (1922), Hoist concluded that blue stain fungi

were not essential for development from egg to adult within a single

generation. Hetrick (1949) observed a Dendroctonus frontalls Infes-

tation In Florida In which the trees showed no sign of blue staining.

He stated that the trees died rapidly and normal broods developed.

Based upon this and other field observations, Hetrick concluded

that blue stain fungi were not essential for successful attack by

the bark beetle.


Pathogenicity of Ceratocystis Associates of Bark Beetles


Cralghead (1928) pointed out that the girdling action of bark

beetles alone did not fully explain the rapid death of attacked

trees since mechanically girdled trees usually remained green for a

year or more. The constant presence of blue stain fungi In beetle-

attacked trees prompted Cralghead to suggest that the fungi played

a role In causing death of the tree. Subsequent pathogenicity trials

with certain Ceratocystis species proved Cralghead's suggestion to

be correct. Nelson (1934) and Nelson and Beal (1929) were able to








kill small loblolly (Pinus taeda L.), pitch (P. rigida Mill.), short-

leaf (P. echlnata Mill.), and Virginia (P. virginlana, Mill.) pines

with C. minor. Bramble and Hoist (1940) and Calrd (1935) were also

able to kill shortleaf pines with the fungus. Nelson (1934) Innocu-

lated trees with C. Ips, but the results were Inconclusive since the

trees were attacked by bark beetles. Mathre (1964b) recently demon-

strated that C. Ips, as well as C. minor, C. monlta (Rumbold) Hunt,

and C. schrenklana (Hedgc.) C. Moreau killed small ponderosa pines,

P. ponderosa. In order to kill the trees, Mathre (1964b) and Nelson

(1934) found It necessary to apply the Innoculum over a large area

which completely encircled the tree. Trees Innoculated in small

patches or needle holes, so as to simulate initial bark beetle

attacks, were resistant to infection. Additional tests with

secondary fungi associated with bark beetles; C. pilifera (Mathre,

1964a), Trichoderma spp. (Mathre, 1964a and Bramble and Hoist, 1940),

and Dacromyces spp. (Bramble and Hoist, 1940) showed them to be non-

pathogenic.

Mathre (1964b) found oleoresin exudation pressure (o.e.p.) to

be a good indicator of the susceptibility of large ponderosa pines

to infection by C. Ips and C. minor. Trees with an o.e.p. greater

than 35 p.s.I. were resistant to infection. Small trees were

susceptible to infection regardless of the o.e.p., probably because

of the small resin volume to the exposed surface area ratio In such

trees.

Mathre (1964b) Indicated the role played by C. minor In killing

trees attacked by the western pine beetle, Dendroctonus brevicomis

Lec., was not clear. It was shown that the beetle preferred to







attack large trees with an o.e.p. below 60 p.s.I., but was able to

mass attack and kill large, high o.e.p. trees (Miller and Keen, 1960

and Vlte' and Wood, 1961). Mathre speculated that in such cases, the

reduction of o.e.p. was not due to C. minor since the fungus probably

would not Infect the trees until the o.e.p. was reduced to near zero.

He added further that the trees were usually stained in scattered

patches and wedges only and that water conduction was probably not

stopped until the bole was completely Invaded by the fungus. Ips

bark beetles, on the other hand, seldom attacked trees unless the

o.e.p. was near zero; a condition suitable for rapid infection by

C. Ips. Trees attacked by Ips species were completely stained by
the fungus and water conduction was stopped. Mathre (1964b) stated

"these observations suggest C. minor may not be necessary to kill

trees attacked by the western pine beetle, whereas C. ips may play

a role In killing trees attacked by Ips beetles."













MATERIALS AND METHODS


Fungus isolations


Blue stain fungus Isolations were made from the wood and inner

bark of loblolly, Pinus taeda L.; longleaf, P. palustris Mill.; pond,

P. serotina Michx.; sand, P. clause (Chapm.) Vasey; spruce, P. glabra

Walt.; and typical slash, P. elllottlil var. elliotti Engelm. pines

infested with one or a combination of the three species of ips bark

beetles occurring in Florida. Additional violations were made from

surface sterilized eggs, larvae, pupae, and adults; non-sterilized

adults; and galleries, including frass, of the three beetle species.

All isolations were made on a malt extract, yeast extract agar

medium consisting of 10 grams malt extract, 2 grams yeast extract,

and 20 grams agar per liter of water. Isolations from wood were made

by cutting small blocks of stained wood from beetle infested host

material, dipping them in 70 per cent ethyl alcohol, flaming to

reduce surface contamination, cutting small chips from the blocks,

and placing the chips In 2 per cent water agar. Sufficient nutrients

were present in the wood chips to support fungus growth, yet yeast

and bacterial contaminates were confined to the chips and immediate

vicinity. After a 10-day Incubation period at 240 C., a small piece

of water agar bearing fungus mycella was removed and transferred to

the culture medium. Inner bark isolations were carried out as above,

with the exception that surfaces were not decontaminated by dipping








In alcohol and flaming. Cultures from beetle galleries were obtained

by touching a small piece of sterile agar to the spore bearing tips

of perlthecia extending Into the galleries and placing the seeded

agar on the medium. Frass plugs were teased from the egg niches and

placed on the culture medium. Isolations from surface sterilized

eggs, larvae, pupae, and adults and nonsterilized adults were made

by crushing the specimens with sterile forceps and streaking the

fragments on the surface of the medium. Surface sterilization was

accomplished by washing the specimens for 5 minutes In the following

modification of the sterilizing solution given by Vanderzant and

Davlch (1958); 0.25 grams mercuric chloride, 6.5 grams sodium chloride,

1.25 milliliters hydrochloric acid, 250 milliliters 95 per cent ethyl

alcohol and 750 mllllllters distilled water. The wash was followed

with a 3-mlnute rinse In sterile water.


Frequency of C. Ips Transmission


Beetles used In studies on the frequency of Ceratocystis Ips

transmission were collected by two methods. Ips calligraphus and

Ips grandicoills adults were collected utilizing the male attraction

phenomenon (Anderson, 1948, and Wilkinson, 1964). Longleaf or lob-

lolly pine bolts, 5 feet long and 4 to 8 Inches inside bark diameter,

were cut from felled trees. The bolts were transferred to an Insect-

tight room held at 40 to 50 per cent relative humidity and cured

(dehydrated) until the phloem moisture content was approximately

100 per cent (o.d.w.). After curing, which usually required 7 days,

5 bolts were removed and placed In a 6' X 6' X 6' screen (16 mesh)









cage (Figure I) located in a pine woods on the University of Florida

Agronomy Farm. A minimum of 100 wild Ips calligraphus or Ips grandl-

collis males were released in the cage and allowed to Infest the

bolts. After a 48-hour period, the cage was visited hourly during

the light period of the day, and the attracted beetles were Individ-

ually collected from the outer surface of the cage and placed In 000

gelatin capsules. Ips avulsus adults were collected In 00 gelatin

capsules as they emerged from Infested loblolly pine slash collected

In Marion County, Florida.

Slash pine bolts, 6 Inches long and 3 to 5 Inches in diameter, were

cut from the clear portion of the bole of felled trees. Loose bark

scales were removed from the bolts with a pruning knife, and the ends of

the bolts were surface-decontaminated by dipping them for 30 seconds In

70 per cent alcohol containing the fungistatic agents, methyl parahydroxy-

benzoate and sorbic acid at rates of 1.0 per cent and 1.5 per cent,

respectively. The bolts were placed In an open rack and cured for 3 days

at 300 C. and 40 to 50 per cent relative humidity. Following curing,

the ends of the bolts ware again dipped for 30 seconds. After evapora-

tion of the alcohol, ends of the bolts were painted with a commercially

available, fungicldal and bactericidal, asphalt based pruning paint*

containing 0.84 per cent metallic-copper derived from brown copper oxide.

To determine the frequency of C. Ips transmission, the bolts

ware artificially Infested with the collected beetles. "Starter




*"Seealskln Pruning Coat." Florida Agricultural Supply Company,
Jacksonville, Florida.

















P - -i


Figure I. Screen cage, 6' X 6' X 6', containing
male Infested loblolly pine bolts used to attract
wild beetles.








holes" large enough to admit a given Ips species, 1/16, 5/64, and

7/64 Inches for ips avulsus, Ips grandicollls, and Ips calligraphus,

respectively, were drilled obliquely Into the outer bark only and

care was taken not to score the phloem. Two holes were drilled per

bolt, on opposing sides, and midway the length of the bolt. The

drill bit was dipped In 70 per cent ethyl alcohol before boring each

hole. The beetles were sexed according to the presence (female) or

absence (male) of a strldulatory organ on the top of the head

(Wilkinson, 1962). A single adult was placed In the long half of an

00 gelatin capsule lined with sterile filter paper. The capsule was

placed over the starter holes and held In place with a ring of "Dux-

seal,"* an asbestos-based plastic sealing compound (Figure 2). One

hundred individuals of each sex of the three Ips species were Intro-

duced In such manner. The Infested bolts were placed In sterile

12-pound Kraft paper bags and held for 10 days at 300 C. Bolts

treated in the same manner, with the exception of introduction of

beetles, served as checks. Following the 10-day holding period, the

bolts were dissected and examined for the presence of C. Ips.

Galleries from which the fungus was Isolated were considered positive.


Rearing Ips Species in Laboratory Colonies


For the most part, It was not necessary to maintain laboratory

colonies of the three Ips species. Natural infestations in all

stages of development were usually accessible throughout the study




*Johns-Mansville Corporation, Chicago, Illinois.





















































Figure 2. Gelatin capsule used to hold bark beetles
prior to entrance into pine bolt. Capsule held in
place with "Duxseal."








period. When beetles were reared In the laboratory, a modification

of the techniques described by Clark and Osgood (1964a, b) and

Hopping (1961) was used. Slash or loblolly pines were used as the

host material. Felled trees were cut into bolts 12 inches long,

transferred to the laboratory, and cured for 5 days. The ends of

the bolts were dipped In molten paraffin, after curing, and placed

In 25- or 50-pound lard cans with two glass emergence Jars mounted

on ventilation screens on opposing sides of the cans. From 2 to 4

bolts, depending upon their diameter, were placed In each can.

From 10 to 20 males of a given species were introduced into the

can, and 20 to 40 females were added I to 2 days later. The cans

were held at 300 C. Reared beetles were either collected In the

emergence Jars or dissected from the bolts 20 to 25 days after Intro-

duction of the females. The beetles were held until needed at 70 C.

In petri dishes lined with moist filter paper and containing fresh

phloem.


Rearing Ips Species Free of C. Ips


Several methods were tested as a means of obtaining adult Ips

bark beetles free of C. Ips. Surface sterilization of field

collected pupae was unsatisfactory. A 5-minute bath in the

previously mentioned sterilizing solution proved lethal to most of

the pupae, and isolations showed that some of the pupae were internally

infected with the fungus. Surface sterilization of adults followed

by a 10-day feeding period on macerated phloem containing sorbic

acid, methyl parahydroxybenzoate, and streptomycin sulfate








failed to rid the beetles of the fungus. Some beetles, free of

C. Ips, were reared from surface sterilized eggs utilizing the

"phloem sandwich" technique described by Hoist (1937). Larval

survival, however, was less than 5 per cent and all of the plates

became contaminated with mold and bacteria. Use of larval rearing

media provided a means of obtaining beetles free of C. Ips and

other contaminates. Among 18 experimental media tested, the phloem-

based medium listed In Table I proved to be the most satisfactory

for larval development and was used to obtain adults for brood

development and fecundity studies.

Slash pine phloem was used as the phloem source In the diet.

The phloem was thoroughly macerated In a blender containing the

prescribed amount of water to be used In preparing the diet. The

phloem was separated from the liquid by filtration through a

double thickness of cheesecloth, and the water-phloem filtrate

was used In preparation of the medium. Procedures for preparing

the medium were as follows: (I) the agar was placed In the water-

phloem filtrate and heated until dlsolved; (2) the agar solution

was poured into a blender and the combined dry Ingredients

(Including macerated phloem) were slowly added and the whole

homogenized; (3) the hot medium was poured from the blender Into

20 X 90 millimeter petri dishes at approximately 40 milliliters

per dish; (4) a disc of blotting paper the same diameter as the

dish was pressed to the surface of the medium and the medium

allowed to cool. The acidity of the medium was approximately

pH5.











Table I. Phloem-based, semi-artificial rearing medium for Ips bark
beetles. Amounts sufficient to make 100 grams. From
Yearlan and Wilkinson (1965).


Constituent


Amount


Pine phloem (fresh weight)
Agar
Sucrose
Fructose
Vitamin Diet Fortification Mixture
(In dextrose)*
Brewer's Yeast
Soybean protein
Wesson's salts
Sorbic acid
Methyl parahydroxybenzoate
Streptomycin sulfate


Water


(Grams)
46.00
2.00
1.00
1.00

0.50
1.00
1.00
0.10
0.20
0.15
0.01

(Milliliters)
47.00


*Nutritional Biochemlcals Corporation, Cleveland 28, Ohio.


Eggs of the three Ips species were obtained from infested host

material. The eggs were teased from the oviposition niches with a

flattened dissecting needle and transferred to a petrl dish with a

sable brush. The eggs were surface-sterilized, as previously

described, and placed In a petri dish lined with sterile, moist

filter paper to incubate at 300 C. Ten newly-hatched larvae were

aseptically transferred to a rearing dish. Each larva was inserted

Into a slit cut In the paper covering. The slit was then closed

and sealed with excess agar coated on the paper surface. The

dishes were held at 300 C. and 40 to 50 per cent relative humidity.








When the adults were approximately 7 days old, they were removed from

the medium in a sterile isolation chamber and transferred to dishes

containing fresh medium, according to sex and species. The beetles

were held at 70 C. until needed.

Isolations from beetles reared as described above showed them

to be free of fungal, bacterial and yeast contaminates.

Observations were made on the duration of the larval and pupal

stages of the Ips species reared on the phloem-based medium. Five

larvae were Implanted per dish. The dishes were examined daily and

the stage of development recorded. Head capsule width was used as

the criterion for differentiating the larval Instars (Wilkinson, 1963).

When a larva was not visible through the bottom of the dish, it was

dissected from the medium, examined, and transferred to a new dish.

Pupal weight and survival were also recorded.


Mass Attraction


The attractiveness of pine bolts artificially Infested with wild

Ips calllgraphus and Ips grandicollis males to both sexes of the same

species was demonstrated by Wilkinson (1964). A test was conducted

to compare the attractiveness of bolts Infested with males freed of

C. ips with bolts Infested with wild males. Slash pine bolts, 12

inches long and 6 to 8 inches in diameter were cut and cured as .

previously described. Following curing, the ends of the bolts were

dipped in molten paraffin. Six bolts each were Infested with two

wild Ips calligraphus males, Introduced 4 and 6 Inches, respectively,

from the top of the bolts. Six bolts were similarly infested with two

Ips calligraphus males reared on the phloem-based rearing medium.









After a 24-hour holding period, the bolts were exposed for 24

hours on a constantly rotating turntable (Figure 3) located in a

pine woods on the University of Florida Agronomy Farm. The 46-inch

diameter turntable was mounted on an "H" frame support and suspended

6 feet above ground level. A 120-volt generator served as the power

source for a 1/4 h.p. electric motor coupled to the turntable. A

series of gears reduced the turntable speed to 24 revolutions per

hour. The two types of bolts were alternately suspended around the

edge of the turntable, as shown in Figure 3.

At the end of the exposure period, the bolts were dissected and

the number of males, females, and new attacks present wore recorded.


Ips Development in C. Ips Stained Bolts


A study was conducted to ascertain (1) whether the three Ips

species would attack, construct egg galleries, and oviposit In bolts

completely stained with C. Ips; and if oviposition occurred, (2)

whether broods developed normally. Bolts 6 inches long were cut from

felled slash pine trees and cured for 3 days. Following curing, holes

0.25 inches In diameter and 3 inches deep were drilled longitudinally

In the xylem of both ends of the bolts. The holes were spaced 0.5

inches apart in a circular pattern and 0.5 Inches from the outer edge

of the phloem. The bolts were innoculated with a mixture prepared by

blending ten petrl dish cultures of C. Ips with 500 milliliters of

water. The holes in the bolts were filled with the liquid mixture

(using a water dropper) and plugged with 00 corks. The ends of the
















1~4r 4b1k


Figure 3. Rotating turntable used to expose male
infested pine bolts In attraction studies.









bolts were painted with an asphalt pruning compound* to reduce

desslcation. Bolts treated in the same manner, but Innoculated

with sterile liquid, served as a check. Sample C. Ips innoculated

bolts were dissected at 2-day intervals to detect movement of the

fungus into the phloem and after 8 days an estimated 95 per cent of

the phloem was stained.

Two beetle "starter holes" were drilled in the bark 0.5 Inches

from one end and on opposing sides of each bolt. Each hole in five

stained and unstained bolts was infested with one male and two

females of the three ips species. After 15 days, the bolts were

dissected and observations made.


Brood Development and Fecundity


Slash pine bolts were used exclusively for brood development

and fecundity studies. The bolts, 12 Inches long and 3 to 8 Inches

in diameter were selectively cut so as to be free of limbs and scars.

The bolts were surface decontaminated in the following manner: (I)

Immediately after cutting, loose bark scales were removed from the

bolts, and the ends of the bolts were beveled and dipped for 30

seconds in 70 per cent alcohol containing 200 p.p.m. mercuric chloride;

(2) the bolts were placed In an open rack and cured for 5 days at

300 C. and 30 to 40 per cent relative humidity; (3) the ends of the

bolts were painted with pruning paint; (4) the bolts were stored In

an Insect-tight room held at 210 C. until needed; and (5) immediately




*"Tree Kote." Walter E. Clark and Son. Orange, Connecticut.








prior to use, the bolts were sprayed, to the point of run-off, with

70 per cent ethyl alcohol containing the fungistatic agents, methyl

parahydroxybenzoate (1.0 per cent) and sorbic acid (1.5 per cent).

Comparison of brood development and fecundity was made between

three experimental types of beetles within each Ips species occurring

In Florida. The types were as follows:


(1) Blue-stain-free beetles (BSF): Beetles derived from surface

sterilized eggs and reared through the larval, pupal, and callow

adult stages on the phloem-based medium (Table I), thus free of

C. Ips.

(2) Blue stain beetles (BS): Beetles similarly reared on the phloem-

based medium, but Innoculated with C. Ips by placing them In

petri dishes containing sporulating cultures of the fungus for

24 hours.

(3) Wild beetles (W): Beetles collected in the field from naturally

Infested trees, logs, or slash.


In order to obtain beetles reared on similar host material and

approximately the same age, beetles used in the studies were the FI

progeny of BSF, BS, and W beetles reared in surface decontaminated

slash pine bolts. In general, bolts 3 to 4 Inches in diameter were

Infested with Ips avulsus, bolts 4 to 6 inches In diameter were

Infested with Ips grandicollis and bolts 6 to 8 Inches In diameter

were Infested with Ips calligraphus. "Starter holes" were drilled

in the bark as previously described. Four holes were located mid-

way along the length of the bolts and equidistant from each other









on the circumference. A male was Introduced into an 00 gelatin capsule

lined with sterile filter paper, and a capsule was placed over each of

the holes and attached to the bark with "Duxseal." After the male was

established for 24 hours, two females were Introduced Into each capsule.

To reduce the possibility of contamination of the bolts through the

"starter holes," the above procedures were carried out In an Isolation

chamber. Each Infested bolt was placed In a sterile 25-pound Kraft

paper bag. The top of the bag was folded several times and stapled.

The bolts were held at 300 C. and 40 to 50 per cent relative humidity.

The bolts were dissected either 20 days (Ips avulsus) or 25 days

(Ips calligraphus and Ips grandicollis) following Introduction of the

females. The general adults were removed, sexed, and placed in petrl

dishes lined with moist, sterile filter paper. Isolations were made

from BSF and BS beetles and wood as a check against accidental Intro-

duction of contaminates.

In brood development studies, two males, followed by two females

per male, were introduced Into surface sterilized bolts as described

above. The "starter holes" were drilled midway along the length on

opposing sides of the bolts (Figure 4). Lots of 20 bolts each were

Infested with BSF, BS, or W beetles of the three Ips species. Two

additional lots were infested either with BSF or W Ips grandicollis

adults. At 5-day intervals (up to 20 days) after Introduction of the

females, five bolts were selected at random from each of the treat-

ments and dissected. The length of egg galleries and number of egg

niches, eggs, larvae, pupae and adults were recorded. Pupal weight

was also recorded. Observations were made on C. ips development in

BS and W bolts, and Isolations were made from BSF, BS, and W bolts.




















































Figure 4. Pine bolt rearing unit used in brood develop-
ment studies.









Bolts used In fecundity studies were prepared as those for brood

development. Only one female, however, was introduced with each male.

Lots of ten bolts each were infested with each of the three experi-

mental types of each Ips species. The bolts were dissected 10 days

after Introduction of the females. The females were removed and

re-introduced into fresh bolts with newly established males. Obser-

vations were carried out over a 30-day period. At each dissection,

the length of egg gallery, number of egg niches, and number of eggs

were recorded for each female.















RESULTS AND DISCUSSION


Fungus Isolations


Ceratocystls Ips (Rumbold) C. Moreau was the only member of

the genus found associated with Ips avulsus, Ips calligraphus and

Ips grandicollls In Florida. A peritheclum and spores of C. Ips

are shown in Figures 5 and 6, respectively. Another fungus,

probably Tubercularlella Ips Leach, Orr, and Christensen, was

commonly found in association with Ips avulsus. In addition,

several undetermined fungi, yeasts, and bacteria were frequently

Isolated from the wood and galleries of trees attacked by the three

beetle species.

Ceratocystis Ips was easily isolated from the wood and phloem

of beetle Infested trees. Perithecla of the fungus were abundant In

old egg galleries of all three Ips species. The fruiting structures

were commonly found In unlngested phloem adjacent to the egg galleries

shortly before pupation by the larvae. Isolations from frass plugging

the egg niches and loose in the egg galleries consistently yielded

C. Ips. Isolations from 100 frass plugs from egg niches of Ips

avulsus, Ips calligraphus and Ips grandicollis showed 23, 100, and 97

of them, respectively, contained the fungus. Perlthecla were

frequently observed In the frass plugs shortly after the eggs hatched.






















































Figure 5. Peritheclum of Ceratocystis ips. Approx.
75X.
















14
W'
.*^


72

$3


Ascospores of Ceratocystis Ips.


S^q


0


*


Figure 6.
2000X.


Approx.


0








Isolations from each of the life stages of Ips avulsus, Ips

calligraphus, and Ips grandlcollis showed the fungus to be primarily

associated with the late larval and adult stages (Table 2). Eggs and

first-stage larvae of the three beetle species were Internally free

of the fungus, but C. Ips was Isolated from surface-sterilized second

and third instar larvae. Ips avulsus larvae showed the highest

Incidence of Internal contamination In both the second and third

Instars, 74 and 100 per cent, respectively. Surface-sterilized Ips

avulsus pupae were found to be free of C. Ips, but isolations from

100 Ips calligraphus and Ips grandicollis pupae yielded 23 and 4

isolates, respectively. Surfaco-sterilized general adults showed a

high Incidence of C. Ips isolation. No difference was observed In

the degree of Internal contamination of males and females of Ips

calligraphus and Ips grandlcollis, but 35 per cent of the Ips avulsus

males contained the fungus while only 8 per cent of the females were

Internally contaminated. C. Ips was Isolated from every non-

sterilized Ips calligraphus and Ips grandlcollis adult cultured.

Only Ips avulsus with 85 per cent of the males and 83 per cent of the

females showed less than 100 per cent incidence of contamination.

As previously mentioned, Ips avulsus larvae showed the highest

incidence of Internal contamination. This can possibly be explained

by the larval habits of this species. The larvae were usually con-

fined to within I centimeter of the egg gallery. They tunneled more

or less obliquely to the egg gallery and formed a "fan-shaped"

gallery. The phloem surrounding the larvae soon became stained with

C. fps after the eggs hatched, and as the larvae enlarged the gallery










Table 2. Relative frequency of Ceratocystis Ips isolation from Ips
avulsus, Ips callgraphus, and Ips grandlcollis.



Stage of Ips Ips Ips
Development avulsus calligraphus grandlcollls

Number % Number % Number %



SURFACE-STERILIZED:
Egg 100 0 100 0 100 0
Larva
Ist Instar 100 0 100 0 100 0
2nd Instar 100 74 100 16 100 33
3rd Instar 100 100 100 84 100 80
Pupa 100 0 100 23 100 4
Teneral adult
male 100 35 100 60 100 89
female 100 8 100 55 100 83
NON-STERILIZED:
Teneral adult
male 100 85 100 100 100 100
female 100 83 100 100 100 100




and fed, stained phloem was consumed. Ips calligraphus and Ips

grandicollis larvae, on the other hand, generally mined at a right

angle to the egg gallery. The larval galleries extended several

centimeters from the egg gallery and were not greatly enlarged.

C. Ips growth did not overtake the larvae until the late third Instar

or until after pupation. Some Ips calligraphus and Ips grandlcollls

larvae, however, mined more or less parallel to the egg gallery and

consumed stained phloem, thus accounting for a number of the larvae

from which the fungus was isolated.









The data (Table 2) Indicating Ips calligraphus and Ips grandl-

collls pupae were Internally Infected with C. Ips appear questionable.

Previous workers (Leach et al., 1934, and Grosmann, 1930) failed to

Isolate Ceratocystis species from surface-sterilized Ips species

pupae. In the present study all third instar Ips avulsus larvae

examined were found to be infected with C. Ips, yet none of the

pupae of this species yielded the fungus. Incomplete surface

sterilization of the Ips calllgraphus and Ips grandlcollis pupae

cultured would explain the results obtained since the pupal

chambers of both species frequently contained fruiting structures

of the fungus and the pupae were doubtlessly externally contaminated

with C. Ips spores. Grosmann (1930), however, found viable yeast

cells In the Ips typographus pupae; therefore, the possible validity

of the above data cannot be totally discounted.


Frequency of C. Ips Transmission


Both male and female Ips avulsus, Ips calligraphus and Ips

grandlcolls adults were found capable of transmitting Ceratocystis

ips (Table 3). Males of all three species, that successfully

attacked the bolts, were also successful In transmitting the fungus.

Similar observations were made with Ips calllgraphus and Ips grandl-

collis females, but Ips avulsus females showed less than 100 per cent

transmission. Of 92 successful attacks by Ips avulsus females, two

attacks were found that showed no sign of blue staining and from

which C. Ips was not Isolated. Since the males of this species










Table 3. Relative frequency of Ceratocystis Ips transmission by Ips
avulsus, Ips calllgraphus, and Ips grandicollis.



Beetle Species Successful Attacks Unsuccessful Attacks
C. fps C. ips C. Ips C. Ips C. Ips C. Ips
Present Absent Trans- Present Absent Trans-
mission mission



Ips avulsus
Male 83 0 100.0 8 9 47.1
Female 90 2 97.8 5 3 62.5

Ips calligraphus
Male 100 0 100.0 0 0 0.0
Female 95 0 100.0 5 0 100.0

Ips grandicollis
Male 97 0 100.0 3 0 100.0
Female 93 0 100.0 7 0 100.0

Check 0 0 0.0




demonstrated 100 per cent transmission, the probability of an Ips

avulsus gallery system being free of C. Ips appeared to be unlikely.

Even though some attacks by the beetles were unsuccessful, C. Ips

Infection frequently occurred (Table 3). An attack was considered

unsuccessful when a male failed to construct a nuptial chamber and a

female failed to excavate at least a centimeter of gallery. All

unsuccessful attacks by Ips calligraphus and Ips grandicollis adults

were stained by the fungus. Of 17 unsuccessful attacks by Ips avulsus

males, C. Ips was Isolated from 8, and 5 of 8 unsuccessful attacks by

females yielded C. Ips cultures. Even though the attacks were









considered unsuccessful, most of the beetles fed for a brief period,

and contacted the phloem and xylem.

Upon dissection, wood of successfully attacked bolts was visibly

stained by C. Ips. The stain was confined to the immediate vicinity

of the attack and did not extend deeply Into unscored areas of the

phloem. Perithecia were beginning to form In the nuptial chamber made

by the males and In the basal portion of the galleries constructed by

the females. Stain was not visible In the wood or phloem of bolts

where attacks were not successful unless the beetle had chewed through

the phloem to the wood. In any case, staining was very slight, and

Isolations were necessary to detect the fungus.


Larval and Pupal Development on Phloem-Based Rearing Medium


Ips avulsus, Ips calligraphus, and Ips grandlcollis larval and

pupal developmental rates on the phloem-based rearing medium were

similar at 300 C. (Table 4). The mean length of the larval period

was 8.4 days for Ips avulsus, 8.9 days for Ips calllgraphus and 9.2

days for Ips grandlcollis. With the exception of Ips avulsus, the

mean developmental time for the three larval Instars progressively

increased. Second instar Ips avulsus larvae required an average of

3.2 days to complete development while the third Instar required

2.8 days. Although no data were recorded, the pre-pupal stage for

the three species was approximately one day. The pupal period for

Ips avulsus, Ips calllgraphus and Ips grandlcollis averaged 2.8,

3.7, and 3.6 days, respectively.











Table 4. Mean larval and pupal development of Ips avuisus, Ips
calllgraphus, and Ips grandicollis on phloem-based,
semi-artificial rearing medium at 30 C.



Stage of ips avulsus Ips calligraphus Ips grandicollis
Development Mean No. Ob- Mean No. Ob- Mean No. Ob-
(days) served (days) served (days) served



Larva (total) 8.40.51 II 8.90.27 45 9.20.20 51

Ist instar 2.50.22 15 2.710.13 86 2.60.10 87
2nd instar 3.20.29 15 3.00.10 64 2.810.12 81
3rd Instar 2.80.30 13 3.70.22 49 3.60.18 71

Pupa 2.8+0.36 9 3.30.10 45 3.00.18 43




The Incubation period of newly deposited surface-sterilized eggs

was found to average 2.3, 2.7, and 2.8 days for Ips avulsus, and Ips

calligraphus, and ips grandicollls, respectively. With the mean

Incubation period added to the mean larval and pupal developmental

times, the average time required from egg to callow adult was 13.5

days for Ips avulsus,was 14.9 days for Ips calligraphus, and was

15.0 days for Ips grandicoills. After 7 days, general adults reared

on the medium were capable of Infesting pine bolts and establishing

broods. Thus, allowing a day for attack, mating and initiation of

oviposition, a complete life cycle, from egg to egg, averaged 21.5,

22.9, and 23.0 days for ips avulsus, Ips calligraphus, and Ips

grandicollis, respectively. These developmental rates are comparable

to generalized life cycles for the three species given by Thatcher

(1960).









Larval mortality on the medium was high. Of 100 newly hatched

larvae implanted at the beginning of the study, only 13 Ips avulsus,

50 Ips calligraphus, and 49 Ips grandlcollis larvae reached the pupal

stage. Most of the mortality was attributed to handling since it was

necessary to remove many of the larvae from the medium daily to

determine the stage of development. Additional observations on

several hundred larvae, handled only when Implanted in the medium,

showed larval survival to be 56.7 per cent for Ips avulsus, 80.6

per cent for Ips calligraphus, and 68.3 per cent for Ips grandicollis.

Ips avulsus pupae reared on the medium weighed significantly less

(p=.05) than wild (W) pupae; 1.66 and 2.20 milligrams, respectively.

Suggested reasons for this difference are discussed later. Ips

calligraphus and Ips grandlcollis pupae reared on the medium averaged

10.77 and 5.17 milligrams and were significantly larger (p=.05) than

wild (W) pupae (see Table II). The increased weight of medium reared

Ips calligraphus and Ips grandlcollls pupae was attributed to two

factors. First, the medium contained carbohydrates, proteins,

vitamins, and minerals In addition to phloem (see Table I) and was

no doubt richer In available nutrients than natural food. Secondly,

the pupae were reared from surface-sterilized eggs, and the larvae

were free of Internal parasites, particularly nematodes. Massey

(1957, 1960, 1962) and Nickle (1963a, b) found a high incidence of

nematode Infection in certain species of bark beetles and Indicated

the nematodes reduced the vigor of the beetles. Observations

Incidental to this study, showed Ips calligraphus and Ips grandlcollls

larvae, pupae, afd adults were frequently Infected with a number of










nematodes. The removal of these parasites by rearing the beetles

from surface-sterilized eggs, could account for the significant

Increase in weight. Beetles reared on the medium and transferred to

surface decontaminated pine bolts also produced pupae significantly

larger than wild (W) pupae (Table II).


Mass Attraction


A comparison of the attractiveness of wild (W) and blue stain-

free (BSF) Ips calllgraphus male infested bolts is presented In

Table 5. Although BSF bolts attracted more females, Chi-square

analysis of the data failed to show a significant difference in the

relative attractiveness of the two types of bolts. While one

observation Is usually not sufficient to draw a conclusion, the

conclusiveness of the attraction test clearly indicated that C. Ips

was not a factor in Ips calligraphus mass attraction.

Person (1931) suggested that the odor caused by yeast fermentation

influenced mass attraction of bark beetles. Subsequent work (Anderson,

1948; Vite' and Gara, 1961, 1962; Wood and Vite', 1961; and Wood,

1962) established that live male Ips bark beetles in freshly attacked

host material created the attraction. Wood and Vlte' (1961) noted that

for yeasts and/or other microorganisms to be responsible for the

attraction they would have to be specifically associated with the male

beetles. No such association has been reported In the literature.

Vite' and Gara (1962) treated logs with materials toxic to yeasts,

but the logs were still attractive to other beetles when attacked by

the pioneering male bark beetles. Anderson (1948) Innoculated pine












Table 5. Attractiveness of W and BSF Ips calllgraphus male Infested
slash pine bolts.



Number of Beetles Recovered New Attacks

Females Males* Total

BSF Bolts
I 3 4 7 3
2 9 5 14 3
3 2 I 3 0
4 5 3 8 I
5 7 3 10 I
6 7 3 10 I
Total 33 19 52 9

W Bolts
I 10 6 16 4
2 0 I I 0
3 4 2 6 I
4 3 4 7 2
5 4 2 6 0
6 6 3 9 I
Total 2 18 45 8



*Includes males Introduced In laboratory.


logs with yeast Isolated from Ips pint galleries, and found they were

not more attractive than the check bolts. Although the test presented

herein (Table 5) was designed to ascertain the effect of C. Ips on

attraction, BSF beetles used in the test were also free of yeasts.

Since bolts infested with males freed of yeasts and fungi attracted

more beetles than bolts with males contaminated with the micro-

organisms, the assumption that yeasts are not a factor In mass

attraction was again corroborated.








Ips Development In C. Ips Stained Bolts


Attempts to establish Ips avulsus, Ips calligraphus and Ips

grandicollis broods in bolts innoculated with C. Ips 8 days prior to

introduction of the beetles were unsuccessful. The males of all three

species constructed nuptial chambers, and females tunneled In the

stained phloem but, with two exceptions, deposited no eggs. An Ips

avulsus female laid two eggs shortly after initiating the egg gallery.

The eggs hatched and at dissection, 15 days, the larvae had pupated.

The pupae weighed 2.05 and 2.14 milligrams, respectively, and were

within the range of pupal weights given in Table II. An Ips calll-

graphus female laid one egg and the resultant pupa was recovered at

dissection. In handling, the pupa was crushed before it could be

weighed, but It appeared comparable in size to laboratory reared Ips

calligraphus pupae. The remainder of the females of all three beetle

species abandoned the bolts without ovipositing although some

constructed galleries the entire length of the bolts.

Females of the three Ips species produced apparently normal

broods in the check bolts, thus it appeared that C. Ips rendered the

Infected bolts unsuitable as breeding material for the beetles. The

Infected bolts appeared notably drier than the check bolts. Anderson

(1948) Induced Ips pini attack in logs dried for a year. He noted

that the females constructed egg galleries, but no brood was

produced; a situation very similar to that previously described for

the stained bolts. Mathlesen-KSgrlk (1960) found that certain Insect

associated Ceratocystis species utilized several amino acids, sugars,

and vitamins and Mathre (1964c) demonstrated that C. ips significantly








reduced the fructose content of ponderosa pine sapwood. Whether a

similar alteration of the chemical composition of the xylem and

phloem was sufficient to render the bolts unsuitable cannot be

answered here.

Even though the presence of C. Ips In the bolts apparently

created conditions not conducive to oviposition by the beetles, there

was no evidence that the fungus rendered the phloem unsuitable as

larval food. Although only three eggs were deposited, the resultant

larvae developed to the pupal stage. The developmental time and

size of the pupae did not appear to be retarded, although White

(1962) has shown that blue stain fungi reduce the nutritional value

of Scot's pine (Pinus sylvestris L.) sapwood as food for the larvae

of Hylotrupes bajulus L. Additional observations are necessary to

ascertain the effect C. Ips may have on the nutritional value of

pine phloem as food for Ips bark beetles.


Effect of C. Ips on Brood Development and Fecundity


Egg gallery construction


In brood development test bolts seeded with two males and two

females per male, the females usually excavated their egg galleries

In opposite directions; one extending upward and the other downward

from the nuptial chamber. Occasionally both females cut their egg

galleries side by side and the galleries were separated only by a

narrow strip of phloem (usually less than 0.5 centimeter). In

general egg gallery excavation ceased when the female reached the

end of the test bolt. Some females continued to burrow around the










end of the bolt and eventually turned back parallel with the grain

of the wood forming a "U" shaped gallery. Some Ips grandicollls and

Ips callIgraphus females burrowed out the end of the bolt and re-entered

at another site on the bolt.

'Egg gallery construction and oviposition by all three species were

completed 10 days after introduction of the females, and most of the

activity took place the first 5 days. Statistical analysis of gallery

length at 5, 10, 15, and 20 days failed to show a significant differ-

ence between mean lengths at any of the dissection dates. Thus, all

gallery length measurements, regardless of dissection date, were used

to determine mean gallery lengths for the W, BSF, and BS beetles of

each species. As shown in Table 6, W Ips avulsus females constructed

significantly (p=.05) longer galleries than BSF and BS females, while

BSF Ips calligraphus and Ips grandicollls females constructed

significantly (p=.05) longer galleries than W and BS females.

The eggs were deposited at Irregular Intervals in niches cut In

both sides of the gallery. When either Ips calligraphus or Ips

grandlcollls females constructed galleries side by side, most of the

eggs were deposited In the outer wall of their respective galleries,

and very few eggs were deposited In the narrow phloem strip

separating the galleries. Ips avulsus females also deposited more

eggs In the outer wall, when the galleries were side by side, but

several eggs were deposited In the adjacent gallery walls. Ips

avulsus females, particularly BSF and BS females, spaced their eggs

farther apart In the egg galleries than did the other two species

(Table 6). Ips grandicollls eggs were more closely spaced than Ips

calligraphus eggs.











Table 6. Mean egg gallery length and spacing between eggs of W, BSF,
and BS Ips avulsus, Ips calllgraphus, and Ips grandicollis
females In slash pine volts at 30 C.



Species Number of Galleries Mean Length Spacing Between
(cm.) Eggs (cm.)



Ips avulsus
W 46 13.51.4* 0.59
BSF 37 11.61.3 0.68
BS 30 12.211.I 0.86

Ips calligraphus
W 40 14.22.2 0.55
BSF 36 20.11.8* 0.48
BS 37 14.91.8 0.49

Ips grandicollIs
Sandcolll 73 11.51.7 0.39

BSF 61 13.92.2* 0.34
BS 33 11.91.8 0.31



*Significantly different (p=.05) from others within same species.
Duncan's New Multiple Range Test.


Although egg gallery excavation and oviposltlon were terminated

after approximately 10 days, the females remained In the galleries.

Most of the Ips avulsus females abandoned their galleries between the

15- and 20-day dissection dates, but some remained until the 20-day

dissection. Ips grandicollis and Ips calllgraphus females remained

in the gallery for the entire 20-day observation period. Approximately

10 days after introduction of the females, the males of all three

species vacated the nuptial chamber and re-attacked the bolts.










Ceratocystis Ips in brood development test bolts


Ceratocystis Ips was present In each W and BS bolt examined, but

careful examination of BSF bolts showed them to be free of the fungus.

Staining of the sapwood of BSF bolts was observed, but the stain did

not penetrate the sapwood as deeply as C. Ips and was somewhat darker

In color. Since Isolations from discolored sapwood did not yield

microbial growth and microscopic examination did not reveal fungus

hyphae or fruiting structures, It was assumed that the stein was

chemical In nature. An occasional BSF bolt became contaminated with

mold, and such bolts were discarded and the data were not Included In

the study.

Growth of C. ips through the gallery systems of all three Ips

species was somewhat faster In the BS bolts than the W bolts, but

the developmental pattern was similar to that described by Leach

et al. (1934). Staining was not visible in W bolts after 5 days,

but the fungus was easily Isolated from all sections of nuptial

chambers and egg galleries of the three beetle species. In most BS

bolts slight staining was visible in the nuptial chamber and basal

portion of the egg gallery. In a few bolts perlthecla were present

In frass plugging the first egg niches. After 10 days the nuptial

chambers and basal portion of the egg galleries In both W and BS

bolts were heavily stained and bore scattered perithecia. The

fungus had begun to spread perpendicularly to the egg galleries

through the larval tunnels, and the older members of Ips avulsus

broods were overtaken by C. Ips. Fifteen days after Introduction of








the females the entire length of the egg galleries was stained and

lined with perlthecla. Figure 7 shows C. Ips perithecla lining a

section of a 15-day old BS Ips grandicollls gallery 5 centimeters

from the nuptial chamber. Figure 8 shows a comparable section of

BSF Ips grandlcollls gallery. At 15 days the entire gallery system

of Ips avulsus broods, and most of the gallery system of Ips calll-

graphus and Ips grandicollis broods were covered by the fungus.

Perithecla and coremla were beginning to form In the older larval

tunnels, pupal chambers, and unconsumed areas of phloem. By 20

days the fungus had grown over the entire gallery system of all

three Ips species.


Brood size and mortality


Mean brood size (average number of eggs, larvae, pupae, and/or

adults per egg gallery) was determined using all observations since

statistical analysis failed to reveal any significant differences In

size of the broods at the different dissection dates. Wild (W) Ips

avulsus females produced significantly (p=.05) larger broods than BSF

and BS females (Table 7). Blue stain-free (BSF) Ips calllgraphus

broods were significantly larger than W and BS broods. Both BSF and

BS Ips grandicollis broods contained significantly (p-.05) more

individuals than W broods.

Brood mortality was based upon the difference between the number

of eggs niches cut by the females and the number of offspring

recovered at dissection of the bolts. As shown In Table 7, mortality

In all three species was highest In the W bolts, and overall mortality








the females the entire length of the egg galleries was stained and

lined with perlthecia. Figure 7 shows C. Ips perithecia lining a

section of a 15-day old BS Ips grandicollis gallery 5 centimeters

from the nuptial chamber. Figure 8 shows a comparable section of

BSF Ips grandicollis gallery. At 15 days the entire gallery system

of Ips avulsus broods, and most of the gallery system of Ips calIl-

graphus and Ips grandicollls broods were covered by the fungus.

Perlthecia and coremia were beginning to form In the older larval

tunnels, pupal chambers, and unconsumed areas of phloem. By 20

days the fungus had grown over the entire gallery system of all

three Ips species.


Brood size and mortality


Mean brood size (average number of eggs, larvae, pupae, and/or

adults per egg gallery) was determined using all observations since

statistical analysis failed to reveal any significant differences In

size of the broods at the different dissection dates. Wild (W) Ips

avulsus females produced significantly (p.05) larger broods than BSF

and BS females (Table 7). Blue stain-free (BSF) Ips calllgraphus

broods were significantly larger than W and BS broods. Both BSF and

BS Ips grandicollls broods contained significantly (p=.05) more

individuals than W broods.

Brood mortality was based upon the difference between the number

of eggs niches cut by the females and the number of offspring

recovered at dissection of the bolts. As shown in Table 7, mortality

In all three species was highest In the W bolts, and overall mortality















r
















Figure 7. Section of 15-day old BS Ips grandlcoll s
gallery 5 centimeters from nuptial chamber. Note perl-
thecia of Ceratocystis Ips lining gallery and masses of
ascospores at tips of perlthecla. Approx. 40X.

























































Figure 8. Section of 15-day old BSF Ips grandlcollls
gallery 5 centimeters from nuptial chamber. Note
absence of Ceratocystis Ips. Approx. IOX.


I I _, _











Table 7. Mean size and mortality of W, BSF, and BS Ips avulsus, Ips
calligraphus, and ips grandlcollls broods In slash pine
bolts at 300 C.



Species Number of Broods Brood Size Brood Mortality
Observed (No. Offspring) (%)



Ips avulsus
W 46 20.711.6 5.1
BSF 37 16.41.7 3.2
BS 30 14.02.0 2.7

Ips calllgraphus
W 40 28.5t3.8 16.1
BSF 36 35.7t3.9* 13.5
BS 37 31.83.9 8.6

Ips grandlcoll is
W 73 28.52.6 9.4
BSF 61 39.9t2.9* 4.6
BS 33 39.63.9* 6.5



*Slgnificantly larger (p=.05) from others within same species.
Duncan's New Multiple Range Test.


was greatest In Ips calllgraphus broods. The Increased mortality In

the W broods was attributed, In part, to parasites. A parasitic mite

was observed attacking the eggs of all three beetle species, and was

more common In Ips calllgraphus bolts. As previously mentioned, the

beetles In all stages, with the exception of the egg, were Infected

with nematode parasites. The possible pathogenic effect of these

nematodes has not been demonstrated, however. A fungus was also

found infecting the eggs of the three beetle species.








Brood composition


The composition of W, BSF, and BS Ips avulsus, Ips calllgraphus,

and Ips grandicollis broods at the different dissection dates is

depicted In Figures 9, 10, and II, respectively. The data are also

numerically presented In Table 8. As mentioned In the previous

section, there were significant differences in the size of W, BSF,

and BS broods within each beetle species. Such differences made

direct comparison of brood composition difficult. To obtain a clear

picture of the make-up of the broods and the shift in composition of

the broods with time, absolute brood composition data were converted

to relative values (Table 9). The relative composition of W, BSF,

and BS broods within species were compared at each dissection date

using Chl-square analysis (Table 10) to detect differences In rate

of development.

Although W Ips avulsus broods were significantly larger than BSF

and BS broods, the relative make-up of the three types of broods was

not significantly different after 5 days. At 10 and 15 days, however,

W Ips avulsus broods appeared to have developed faster, and the

relative composition of W broods was significantly different from BSF

and BS broods (Table 10). Wild (W) broods contained proportionally

fewer eggs and first Instar larvae and more pupae at 10 days (Table 9).

After 15 days W broods were predominately pupae and callow and general

adults while BSF and BS broods were composed of proportionally more

second and third Instar larvae and pupae. At 15 days, none of the

W Ips avulsus broods examined contained second Instar larvae. By

the final dissection date (20 days) all three ips avulsus broods were















o40 5 DAYS


I IS

20

10


10 DAYS
E 40 a 0.3
A
N 30

20
F
R rO In


S415 DAYS
40
E
N 30
C
20

ton

n- i p11- pn= |n=
20 DAYS
40

30

20
to I





egg 1 2 3 pupa adult
larval instar

STAGE OF DEVELOPMENT





Figure 9. Mean composition of W, BSF, and BS ips
avulsus broods In slash pine bolts at 5, 10, 15,
and 20 days at 300 C.












5 DAYS
so W
w
o BSF
30
BS
20




M 10 DAYS
E 40
A
N 30

20

S to10
E o In= il Ii

U 15 DAYS
E 40
N
C 30
Y
Y 20


I _
20 DAYS
40

30

20

10


egg i 2 3 pupa adult
larva I instar

STAGE OF DEVELOPMENT





Figure 10. Mean composition of W, BSF, and BS ips
calligraphus broods in slash pine bolts at 5, 10,
15, and 20 days at 300 C.

























M 10 DAYS
E 40
A
N 30

20
F
R oi


U 15 DAYS
E 40
N
C 30

20
Y I




20 DAYS
40

30

20

10


egg 1 2 3 pupa adult
larva I instar

STANCE OF DEVELOPMENT





Figure II. Mean composition of W, BSF, and BS Ips
grandicollis broods in slash pine bolts at 5, 10,
15, and 20 days at 300 C.






























"0
c-
-3





















-0

C




nO
C
I.0




O













0
o














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-





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0-



to 0
0

Cco















M-
u









(0


C) u






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0
Q,


I- -
I *
+1 +I
S\00
I *
-0
-N


N1*
I *
+1
IN *
NN-




I +I
I-
+1 +1
IN *
** -


co o\
* '
0 0
+1+1
C M





O -
0 0








-0
+ 1+1
+m -

0'0


UN 0 Un 0
--NC


0 00

+1+1
IN-
I *
I o"-


00
I *
50-







I +0
+1+1

I *
0 C--











0 -N4
+1 +1 +1
Sll noD
rn q fn


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+1 +1
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r,- CM I
++1



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+1 +4 +1
r co


+1
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+0'+'
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r-u-

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+ +1 +1
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+ +++3


OON
a a C14
* *


+1 +1 +1







CN4


I--




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+1 +t&








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+I
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r-N








+1 +1t+1
--0
\ in


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o CM +





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+ +d+1




N'ON




I I
.















*+ ,
+ 3+




+1 +1
*0 fn


'0-

44 +

on -

1 *













+1 +1 +1

O-N
3- ? CM
+it+it+i
'0,',C


+*+3+3
0 -N(
CMNC


%00

+1 +1
ooo

+ 4



* *
i-0
r^ ^;


om mo mmo
-- -N-- --









faU


'0-
I *
I I 0




3l3


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I ,I -C 11



N



I I -


I -








+ +l +I1
. .M g
**of.


u
0-
U
0.
U)












Table 9. Relative composition (percentage) of W, BSF, and BS Ips
avulsus, Ips calligraphus, and Ips grandicollls broods In
slash pine bolts at 5, 10, 15, and 20 days at 300 C.



Species Time Stage of Development
(days)
Larval Instar
Egg I 2 3 Pupa Adult


Ips avulsus
W




BSF




BS




Ips calligraphus
W




BSF




BS




Ips grandlcollls
W


43.2
3.2



40.6
12.5



50.0
6.1




61.1
18.3



50.4
15.2



33.8
11.7




50.5
22.3
1.5


42.8
3.5



27.8
11.3



32.6
18.5




28.1
15.2



34.7
15.7



39.4
13.0




44.1
19.4
12.0


13.9
29.9



31.6
27.2
16.0


17.4
32.9
15.8



10.1
26.8
8.6


14.8
28.8
13.6


26.7
34.9
13.6



5.3
41.2
54.7


54.3
8.9



47.1
56.6



40.2
36.4




39.6
62.5
10.5


40.1
57.2
3.9


39.8
34.4
6.6



17.1
31.6
15.3


9.1
17.1



1.7
11.7



2.0
23.3





23.2
8.4



16.4
8.6



21.3
7.8





17.8


73.8
100.0



15.5
100.0



24.2
100.0




5.5
80.9



12.6
87.4



30.6
85.6





66.8











Table 9. Cont.


Species Time Stage of Development
(days)
Larval Instar
Egg I 2 3 Pupa Adult



Ips grandicoll Is
BSF 5 57.3 37.6 4.9 --
10 12.4 13.1 31.3 43.0
15 4.3 11.8 46.7 18.7 18.6 -
20 - - 5.9 8.6 85.4

BS 5 57.2 35.4 7.2 - - -
10 8.2 18.4 49.7 23.6
15 - 8.9 30.3 45.2 15.3 -
20 -- -- -- 3.3 4.7 9.2




composed of adults. Most of the W adults were late tenerals, and some

had begun to emerge from the bolts. Most of the BSF and BS adults

were light In color with some still In the callow stage. Emergence

from BSF and BS bolts was not observed at 20 days.

A comparison of Ips calligraphus broods failed to show con-

sistent differences In the relative make-up of W, BSF, and BS broods.

At 5 days BS broods were composed of a significantly higher percentage

of second Instar larvae and relatively fewer eggs than W and BSF

broods (Tables 9 and 10). But, after 10 days the relative compositions

of the three brood types were not significantly different. Again, at

15 days BS broods appeared further developed than W and BSF broods and

contained proportionally more adults. At 20 days, however, no

significant difference was detected in the relative composition of the

three brood types.









significantly (Table 10) from the W broods. After 15 days, BSF and

BS broods contained pupae while W broods were composed of eggs and

larvae. Blue stain (BS) broods contained no eggs and proportionately

more third Instar larvae at 15 days and were significantly different

from BSF broods. After 20 days W, BSF, and BS broods were composed

of third instar larvae, pupae, and adults, but the relative number of

adults in BSF and BS broods was significantly greater than the W

broods.


Pupal weight


Pupal weight was used as an Indicator of the effect of the

presence or absence of Ceratocystis Ips on the nutritive qualities of

the phloem In the test bolts. As shown in Table II, W Ips avulsus

pupae were significantly (p=.05) heavier than BSF and BS pupae. The

average weights of BSF and BS Ips avulsus pupae were not significantly

different. Both Ips calligraphus and Ips grandicoilis BSF and BS

pupae weighed significantly more than W pupae. As with Ips avulsus,

there were no significant differences between the weight of BSF and

BS pupae of Ips calligraphus and Ips grandicollis.


Sex ratio


The sex ratio of adults recovered from the brood development

test bolts is given In Table 12. The ratio of males to females in

each species and each type within species appeared to be one to one.


Fecundity


in fecundity tests many eggs hatched before dissection of the










Table II. Mean weight (mg.) of 100 W, BSF, and BS Ips avulsus, ips calli-
graphus, and Ips grandicollls pupae reared In slash pine bolts
at 300 C.



Beetle Species Type of Pupa

W BSF BS



!ps avulsus 2.20*0.11* 1.71+0.23 1.76+0.11

Ips calligraphus 9.740.25 10.28+0.40* 10.17+0.48*
Ips grandlcollls 3.71+0.11 4.5410.14* 4.230.16*



Significantly different (p=0.5) from others within same species.
Duncan's New Multiple Range Test.


bolts, and actual egg counts were not feasible. Examination of

several hundred egg niches before the eggs hatched showed that each

niche, without exception, contained an egg. Consequently, the number

of egg niches cut per female was considered equal to the number of

eggs laid and was recorded as such.

Wild (W) Ips avulsus females constructed significantly (pa.01)

longer egg galleries and deposited significantly more eggs than BSF

and BS females (Table 13). The average number of eggs per centimeter

of egg gallery, however, was comparable for the three female types.

The number of eggs laid by both BSF and BS Ips calligraphus and Ips

grandlcollls females was significantly (p=.01) greater than that

laid by W females. Blue stain-free (BSF) Ips calligraphus females

also produced (p=.05) more eggs than BS females. The average length

of egg gallery cut by the three types of Ips calligraphus and Ips










Table 12. Sex ratio of W, BSF, and BS Ips avulsus, Ips calligraphus,
and Ips grandlcollis adults reared in slash pine bolts at
300 C.



Species Number Number Number Sex Ratio
Examined Males Females Males:Females



Ips avulsus
W 92 48 44 1:0.92
BSF 60 32 28 1:0.88
BS 70 37 33 1:0.89

Ips calligraphus
W 102 52 50 1:0.96
BSF 100 52 48 1:0.92
BS 99 52 47 1:0.90

Ips grandicollis
W 167 86 81 1:0.94
BSF 132 72 60 1:0.83
BS 115 56 59 1:1.05




grandicollis females was not significantly different, but BSF and BS

females of both species laid more eggs per centimeter of gallery than

W females.


Discussion of the Effect of C. Ips and Other Organisms on
Brood Development and Fecundity


The supposition is often presented that bark beetles and their

Ceratocystis associates are capable of Independent development, but

optimum development of both organisms takes place only when the two

occur In conjunction (Dixon and Osgood, 1961). Such seems to be the

case with C. Ips, but not necessarily with Ips avulsus, Ips calll-

graphus and Ips grandicollis. The fungus appears not only to be











Table 13. Mean length of egg gallery, number of eggs laid, and number
of eggs deposited per centimeter of gallery by W, BSF, and
BS Ips evulsus, Ips calllgraphus, and Ips grandicollis
females at 30 days In slash pine bolts at 30 C.



Number Mean Gallery Mean No. Mean No.
Females Length (cm.) Eggs Laid Eggs Per
Observed cm. Gal-
lery


Ips avulsus
W 10 51.25.75** 77.013.8* 1.5
BSF II 31.26.83 54.0 6.8 1.7
BS II 36.86.10 53.7t 9.3 1.4

Ips calligraphus
W 9 50.8-4.16 117.420.8 2.3
BSF 13 52.74.19 151.1110.6** 2.9
BS 12 47.815.65 139.610.5** 2.9

Ips grandicollis
W 15 37.35.42 108.219.2 2.9
BSF 16 44.66.21 148.516.1** 3.4
BS 14 40.15.77 147.3113.9** 3.7


* Significantly different (p=.05) from others within the
species. Duncan's New Multiple Range Test.
** Significantly different (p-.I0) from others within the
species. Duncan's New Multiple Range Test.


same

same


dependent upon the beetles for dissemination and Introduction Into

a suitable host, but also upon the activities of the beetles In the

inner bark to open avenues of spread once the fungus is In the tree.

When introduced Into host material over a small surface area as In

bolts unsuccessfully attacked In the transmission study (Table 3)

and pathogenicity trials similating bark beetle attacks (Mathre,

1964b and Nelson, 1934), the fungus may become established but does









not spread from the site of infection and sporulation does not occur.

Thus the action of adult Ips beetles in constructing galleries and

the feeding activities of the larvae are a requisite for optimum

development and spread of the fungus in the tree.

As previously mentioned, Leach et al. (1934) noted that until

a brood of beetles was reared in a fungus-free log, it could not be

safely concluded that blue stain fungi were not necessary for normal

development of the beetles. In this study it was clearly demonstrated

for the first time that all three Ips species were capable of develop-

ment through successive generations in pine bolts free of C. Ips.

Ips avulsus was reared through three successive generations without

observable effects when compared to beetles reared in the presence

of the fungus. Similar results were obtained with Ips calligraphus

and Ips grandicollis over four generations.

When brood development was examined more closely, it was found

that Ips avulsus broods free of C. Ips (BSF) were comparable in size

to broods reared in the presence of the fungus (BS). Additionally,

no differences were detected between length of egg gallery, brood

mortality, brood composition (rate of development), or pupal weight

of the two brood types. The number of eggs laid by BSF and BS

females after 30 days was not significantly different. Thus, it

is apparent that C. ips has no effect on brood development and

fecundity of Ips avulsus under laboratory conditions.

It was also apparent that C. ips had little or no effect on

brood development and fecundity of !ps calligraphus and Ips grandi-

collis although the data were not as conclusive as with Ips avulsus.










Ips calligraphus females free of C. Ips (BSF) constructed longer egg

galleries and produced significantly larger broods than females (BS)

In the presence of the fungus. Furthermore, BSF females laid

significantly more eggs than BS females after 30 days. Conversely,

however, BS Ips calligraphus broods appeared somewhat more advanced

than BSF broods at 5 and 15 days. This apparent increase In rate of

development was not consistent throughout the observational period,

and at 10 and 20 days the relative composition of the broods was

comparable. No explanation (other than random error) can be given

for the significant differences between brood size and fecundity of

BSF and BS beetles. It should be pointed out that In both instances

the differences barely met the minimum required for significance

and were possibly due to chance. Owing to the small number of

broods examined and the inconsistency of the data, It can neither be

assumed that the absence of C. Ips results In construction of longer

egg galleries and increased fecundity, nor the presence of the

fungus resulted In more rapid brood development. Considering all

measurements made on the various aspects of brood development and

fecundity, It was evident that the fungus has little or no effect on

the beetle.

With the exception of brood composition, no differences were

found between BSF and BS Ips grandicollis broods. At 5 and 20 days,

the composition of the broods was comparable, but at 10 days BSF

broods appeared somewhat more advanced than the BS brood. The

situation, however, was reversed at 15 days, and BS broods contained

more Individuals In the later stages of development. With this aspect









of brood development and fecundity being the only area in which

differences were detected and again due to the Inconsistency of the

differences, there is little reason to suspect that C. Ips is a

factor in brood development and fecundity of Ips grandicollis.

Although the results of this Investigation clearly Indicated

that C. Ips has little or no effect on brood development and fecundity

of the three ips species studied, the question still arises as to the

applicability of these findings to field conditions. It Is well

documented In the literature that Ceratocystis infection resulted in

reduction of the water content of the xylem and phloem. Shepard and

Watson (1959) further demonstrated the fungi indirectly reduced resin

production by causing collapse of the resin-secreting epithelial cells.

As previously mentioned, many authors believed reduced water content

and resin flow were necessary to create conditions favorable for

brood development by the beetles. The point in question, then, is

whether C. Ips is capable of lowering the moisture content and o.e.p.

of a tree, which at the time of attack was too high, to a level

suitable for brood development. Although no field studies were

conducted, observations in the laboratory indicated C. ips did not

become established rapidly enough to significantly Influence moisture

content or resin flow in the early stages of brood development. When

forced to attack bolts with a high phloem moisture content, beetles

of all three species, in the presence or absence of the fungus, were

equally "pitched out." In some bolts, the adults became established,

but the young larvae were "drowned" In resin soon after eclosion.

Under field conditions, therefore, It appears unlikely that C. Ips









would be a significant factor In the establishment of Ips broods. The

possibility that C. Ips or other Ceratocystis species would prevent,

as a result of excessive environmental moisture, a sudden Increase In

moisture content and o.e.p. In trees with well-established broods is

not excluded, however.

In addition to comparing brood development of the three Ips

species In the presence or absence of C. Ips, the maintenance of wild

(W) colonies of beetles afforded a comparison of BS and BSF broods

with broods enjoying their "normal" complement of associated organisms.

As previously noted, W broods of both Ips grandicollis and Ips calll-

graphus did not fair as well as the BS end BSF ones. These differences

were attributed to parasites; namely, parasitic mites and nematodes

and pathogenic fungi. Wild Ips avulsus broods, on the other hand,

appeared more vigorous than BS and BSF broods. The broods were larger

and development was more rapid. Wild Ips avulsus pupae weighed

significantly more than BS and BSF pupae, and the fecundity of W

females was greater. Since no basic differences between BS and BSF

Ips avulsus broods were exhibited, the apparent increased vigor of

the W broods did not appear to be correlated with the presence or

absence of C. Ips.

The consistency of the difference between W and BS and BSF Ips

avulsus beetles with respect to brood development, pupal weight, and

fecundity suggested that some factor or factors were missing from the

BS and BSF bolts. Although determinations were not made, two

organisms were consistently Isolated from the gallery systems and

adjacent tissues in W Ips avulsus bolts; a yeast and a fungus,








probably Tubercularlella Ips Leach, Orr, and Christensen. Callahan

and Shifrine (1960) suggested that yeasts played a role in the

nutrition of bark beetles. They noted that all species of bark beetles

have associated yeasts and that the yeasts were eaten by both larvae

and adults. Callahan and Shifrine added further that it has not been

demonstrated that "yeast-feeding" Is not a prerequisite for fertility

of the adults. The results of this study clearly refute Callahan and

Shifrlne's hypothesis. Brood development, fecundity, and fertility

of both Ips callIgraphus and Ips grandicollis were not adversely

affected when the beetles were reared In the absence of yeasts. Based

upon observations made during this study, however, the above statement

cannot be made for Ips avulsus since broods in the presence of yeast

as well as other organisms, exhibited more rapid development and larger

numbers. The yeast associated with Ips avulsus appeared to be the

same as that associated with the other Ips species; thus It Is most

unlikely that yeasts were responsible for the Increased vigor of W

Ips avulsus broods.

In addition to Ceratocystis Ips and the undetermined yeasts,

another fungus was consistently found associated with Ips avulsus.

The growth habits and structural characteristics of the fungi were

similar to those given by Leach et al. (1934) for Tubercularlella

ips. The fungus was most frequently observed In the pupal chambers

where fructification took place. By the time callow adults were

present the walls of the pupal chamber were lined with a white mass

of conldia (Figure 12). The fungus no doubt served as a food source

for Ips avulsus adults as newly emerged beetles were observed


















































Figure 12. Ips avulsus pupal chamber covered with
masses of conldla, probably Tubercularlella ips.
Approx. 5X.








"grazing" on the fungal matt. Most individuals did not stray from

the pupal chamber until emergence from the brood tree and apparently

restricted their pre-emergence feeding to the fungus.

Francke-Grosmann (1963) stated that among phloem-inhabiting

Scolytidae, some cases of association between fungi and beetles are

known which can be called ambrosia because they Indicate existence

of true symbiosis. As one example, she cited the association between

Trichosporlum tengens var. macrosporum Francke-Grosmann and Ips

acumlnatus Gyll. In this case the fungus possessed nutrient-rich,

globose conidia which completely cover the gallery walls. The young

beetles fed on the fungal mass which, according to Francke-Grosmann,

no doubt served as Important nutrients for the young beetles. Leach

et al. (1934) noted that Tubercularlella Ips resembled the so-called

"ambrosia" fungi by possessing large globose conldia rich in nutrients.

The relationship of T. Ips to Ips pini and Ips grandlcolils In the

United States (Leach et al., 1934) and Ips sexdentatus In Europe

(Slemaszko, 1939) then appears very similar to that described above.

The fungus in question In this study, probably T. Ips, was only

found in association with Ips avulsus. Whether Its presence was

responsible for the Increased vigor of Ips avulsus brood must remain

problematical. The dependence of Ips avulsus upon the fungus for

nutritive factors, if any, was not obligatory since beetles were

successfully reared In Its absence. Additional data are necessary

before the ecological Impact of the fungus on the development of

ips avulsus can be assessed.













CONCLUSIONS


Results of this study allow the following conclusions to be

drawn;

Ceratocystis ips was the only member of the genus consistently

associated with Ips bark beetles occurring In Florida. Another fungus,

probably Tubercularlella Ips, was frequently associated with Ips

avulsus, but not with Ips calllgraphus and Ips grandlcollls.

Ceratocystis Ips was transmitted by both males and females of

the three Ips species. Adults were both externally and Internally

contaminated with the fungus. Larvae, particularly the later Instars,

were also internally contaminated. Ips avulsus larvae demonstrated

a higher incidence of contamination than Ips calligraphus and Ips

grandlcollis.

Ceratocystis Ips had no influence on the mass attraction

phenomenon of Ips calligraphus males.

Pine bolts innoculated with Ceratocystis Ips prior to attack by

the three Ips species proved unsatisfactory as breeding material.

The presence, or absence, of Ceratocystis ips had little or no

effect on brood development and fecundity of Ips avulsus, Ips calll-

graphus and Ips grandlcollls. Larvae were reared on semi-artificial

medium without adverse effects when compared to broods reared in

bolts in the presence of Ceratocystis Ips. The three Ips species

were reared through successive generations In bolts free of








Ceratocystis ips without observable effects on vigor and fecundity

when compared to beetles in the presence of the fungus. Ips calli-

graphus and Ips grandicollis in the absence of Ceratocystis ips or

in the presence of Ceratocystis Ips alone produced larger brood with

lower mortality than wild beetles with the normal complement of

micro-organisms. These differences were attributed to parasites

present in wild populations only.

Wild Ips avulsus broods were larger and development was more

rapid than broods In the absence or presence of Ceratocystis Ips.

The presence of a fungus, probably Tubercularlella ips, with the

wild broods was suggested as a possible factor contributing to these

differences since the fungus resembled the "ambrosia" fungi. Ips

avulsus adults were observed feeding on the fungus, but their

dependence upon it for nutritive factors was not obligatory as

beetles were successfully reared in its absence.














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1965. Two larval rearing
media for Ips bark beetles. Fla. Entomol. 48: 25-7.















BIOGRAPHICAL SKETCH


William C. Yearlan, Jr., was born May 20, 1937, at Lake Village,

Arkansas. He attended elementary and secondary schools at Lake Village,

Arkansas, and was graduated from Lakeside High School in 1955.

He attended Arkansas A & M College, College Heights, Arkansas,

from September, 1955, until June, 1957. He transferred to the University

of Arkansas In January, 1958, where he received the degree of Bachelor of

Science in Agriculture In January, 1960. Upon 'receipt of the Crossett

Research Fellowship, he entered the Graduate School, University of

Arkansas in January, 1960, and was awarded the Master of Science Degree

with a major in Entomology In January, 1961.

On June 30, 1960, he married the former Marguerite LaVerne

Robertson of Eudora, Arkansas. They have no children.

He entered the University of Florida In February, 1961, and was

granted a research asslstantship with the Department of Entomology.

In April, 1962, he accepted a research asslstantship with the Depart-

ment of Entomology, Florida Agricultural Experiment Station. In the

summer of 1964, he was granted a research fellowship from the Southern

Forest Disease and Insect Research Council.






81


In May, 1965, he was appointed to the staff of the Department of

Entomology, University of Arkansas, as an Assistant Professor.

At present he Is a candidate for the Degree of Doctor of Philosophy.









This dissertation was prepared under the direction of the

chairman of the candidate's supervisory committee and has been

approved by all members of that committee. It was submitted to

the Dean of the College of Agriculture and to the Graduate Council,

and was approved as partial fulfillment of the requirements for

the degree of Doctor of Philosophy.





Date June 21, 1966






,,,Pean, College of Agriculture







Dean, Graduate School

Supervisory Committee:





Co-Cha rmen




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