RELATIONS OF THE BLUE STAIN FUNGUS,

CERA'lVCrSTIS IPS (RUMBOLD)

C. MOREAU, TO IPS BARK BEETLES

(COLEOPTERA: SCOLYTIDAE)

OCCURRING IN FLORIDA

By

WILLIAM C. YEARIAN, JR.

A DISSERTATION PRESENTED TO THE GRADUATE COUNQL 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-Chalmien 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 Or. K. R, Swinford for their criticism on the preparation of
the manuscript. The writer also wishes to express his appreciation
to Dr. L. C. Kultert 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-
cystls Ips (Rumbold) 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 uue 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.

It

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.

Ill

TABLE OF CONTENTS

PAGE

ACKNOWLEDGMENT H

LIST OF TABLES vl

LIST OF FIGURES vIM

INTRODUCTION I

LITERATURE REVIEW 3

Transmission of Ceratocystis Species 3

Specificity of Ceratocystis spp. - Bark Beetle

Association 6

Characteristics of Bark Beetle - Associated

Ceratocyst i s 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 15

Rearing Ips Species In Laboratory Colonies 19

Rearing Ips Species Free of C, Ips^ 21

Mass Attraction 24

Ips Development In C. Ips Stained Bolts 25

Brood Development and Fecundity 27

Iv

RESULTS AND DISCUSSION 32

Fungus Isolations 32

Frequency of C. Ips Transmission 37

Larval and Pupal Development on Phloem-Based

Rearing ^4edium 39

Mass Attraction 42

ips Development In C. ips Stained Bolts 44

Effect of C. ips on Brood Development and

Fecund i ty 45

Egg gallery construction 45

Ceratocystis Ips in brood development

test bolts 48

Brood size and mortality 49

Brood composition 33

Pupal weight 62

Sex ratio 62

Fecund I ty 62

Discussion of the Effect of C. ips and Other

Organisms on Brood Development and Fecundity 64

CONCLUS IONS 73

LITERATURE CITED 75

BIOGRAPHICAL SKETCH 80

LIST OF TABLES

PAGE

1. Phloem-based, semi -art! ft da I rearing medium for

Ips bark beetles. Amounts sufficient to make

100 grams 23

2. Relative frequency of Ceratocystis Ips

Isolation from Ips avulsus, Ips cal I Igraphus,

and Ips grandlcol lis. 36

3. Relative frequency of Ceratocystis Ips

transmission by Ips avulsus, Ips cal I Igraphus,

and Ips grandlcol lis. 38

4. Mean larval and pupal development of Ips avulsus»

Ips cal llgraphus, and Ips grandlcol I Is on phloem-
based seml-artif Iclal rearing meadlum at 30^ C 40

5. Attractiveness of W and BSF Ips cal llgraphus

male Infested slash pine bolts 43

6. Mean egg gallery length and spacing between

eggs of W, BSF, and BS Ips avulsus, Ips

cal llgraphus and \2&_ grandlcol lis females

In slash pine bolts at 30° C 47

7. Mean size and mortality of W, BSF, and BS Ips avulsus,

Ips cal llgraphus, and Ips grandlcol lis broods In

slash pine bolts at 30°T. 52

8. Mean composition of W, BSF, and BS Ips avulsus, Ips

cal llgraphus, and Ips grandlcol lis broods In slash

pine bolts at 5, 10, 15, and 20 days at 30° C 57

9. Relative composition (percentage) of W, BSF, and BS

Ips avulsus, Ips cal llgraphus, and Ips grandlcol lis

broods In slash pine bolts at 5, 10, 15, and 20

days at 30° C 59

10. Chl-square comparison of W, BSF, and BS Ips avulsus,
Ips cal I Igraphus, and Ips grandlcol I Is brood
composition at 5, 10, 15, and 20 days at 30° C 61

vl

11. Mean weight (mg.) of 100 W, BSF, and BS Ips avulsus,

Ips cal ilgraphuSy and Ips grand! col I Is pupae reared

In slash pfne bolts at 30" C 63

12. Sex ration of W, BSF, and BS Ips avulsus, Ips

cal I tgraphus, Ips grandlcolHs 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 cal llgraphus, and Ips grand I col Us females

at 30 days In slash pine bolts at 30" C 65

vlt

LIST OF FIGURES

PAGE

1. 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 "Duxseal** 20

3. Rotating turntable used to expose male Infested

pine bolts In attraction studies 26

4. Pine bolt rearing unit used in brood

deve I opment stud i es 30

5. Per I thee i urn of CeratocystIs Ips, Approx. 75X 33

6. Ascospores of CeratocystIs ips. Approx. 2000X 34

7. Section of 15-day old BSF Ips grandJcollls gallery

5 centimeters from nuptial chamber. Note

perlthecia of CeratocystIs l£s lining gallery and

masses of ascospores at tips of perlthecia.

Approx. 40X 50

8. Section of 15-day old BSF Ips grandlcoills gallery

5 centimeters from nuptial chamber. Note absence

of CeratocystIs Ips. Approx. lOX 51

9. Mean composition of W, BSF, and BS Ips avulsus

broods in slash pine bolts at 5, 10, 15, and

20 days at 50° C 54

10. Mean composition of W, BSF, and BS Ips ca 1 1 i -
graphus broods in slash pine bolts at 5, 10,
15, and 20 days at 30° C 55

Hi Mean composition of W, BSF, and BS Ips qrandicoilis
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 conldia, probably Tubercularlel la Ips.
Approx. 5X 71

Ix

INTRODUCTION

The association between blue stain fungi, particularly of the
genus Ceratocystis El lis and Halstead, and scolytid 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 resinosa Ait., to
prove that Ips grandicoliis (Eichh.) and Ips pini (Say) transmitted
the blue stain fungus, Ceratocystis ips (Rumbold) C. htoreau. 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. Craighead (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

I
I

action of the fungi on the wood and Inner bark. Hoist (1937) In the
United States and Grosroann (1930) In (sermany 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 frontalis Zlwn.,
In which the trees showed no signs of blue staining. Since beetle
brood developnusnt 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 cal iigraphus (Germ.), and Ips grandicol Ms (Elchh.), and their
blue stain fungus associate, (^ratocystls 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 (I960) 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 Find I ay
(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 Ceratocystls.
Species of Graph i urn and Leptographlum, many of which were shown to
be Imperfect forms of (Ceratocystls, and Tuberculariel la ips Leach,
Orr, and Chrlstensen also have been found to cause blue stain
(Mathre, i964a). The present study was restricted to the Ceratocystls
associates of Ips species bark beetles In Florida. Hunt (1956)
revised the genus Ceratocystls, and all previous citations from the
literature have been given In terms of his monograph.

Transmission of Ceratocystls Species

The bluing of timber was first described by Hartig In 1878
(Hartig, 1894). He recognized the fungal nature of the stain,
Ceratocyst is pill f era (Fries) C. Moreau, and noted that the fungus

commonly occurred In tr«es attacked by Insects. Von Schrenk (1903)
studied blue stain fungi In ponderosa pine, PInus ponderosa Laws,
attacked by Dendroctonus ponderosae Hopk. He isolated CeratocystIs
pill f era 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, I9S0; 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; Robinson-Jeffery and Grlnchenko, 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. pi I if era 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 (Hedge.) 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 solidification, 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, PInus resinosa Alt,,
and demonstrated transmission of Ceratocystis Ips (Rumbold) C. Moreau
by Ips grandicol I is (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 per I thee I a 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 et 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. VerraM (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 VerraM 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) BakshI, C. f loccosa
(Mathlesen) Hunt, C. pi ilfera, and C. plurlannulata (Hedge.) C.
Moreau; (2) those species associated with several bark beetles
usually In older material, but also spread by wind: e.g., C. minor,
C. minuta (Siem.) Hunt, C. piceae (Miinch) BakshI, and C. po I on I cum
(Siem.) C. Moreau; and (3) those always associated with certain
species of bark beetles: e.g., C. brunneo-cl I lata (Mathlesen-K.)
Hunt with Ips sexdentatus Boern., C. cana (Miinch) C. Moreau with
Blastophagus minor Hart, and C. clavata (Mathlesen) Hunt with Ips
acumlnatus Gyl I. in North America, Dendroctonus and Ips species

have been considered the n»st Important bark beetles of coniferous
trees. Several Ceratocystis species have been found associated with
Dendroctonus bark beetles (Bramble and Hoist, 1940; Davidson, 1955,
1958; Math re, 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 cal I Igraphus and Ips grandlcol Ms. The fungus was also found
In association with Ips avulsus (Rumbold, 1931), Ips confusus (Lee.)
(Mathre, 1964a), Ips emarglnatus (Lee.) (Mathre, 1964a; Rumbold, 1936),
Ips Integer (Elchh.) (Rumbold, 1936), Ips lecontel (Lee.) (Ellis,
1939), Ips oregoni (Elchh.) (Mathre, 1964a; Rumbold, 1936), Ips pinl
(Leach et al., 1934), Ips plastographus (Lee.) (Rumbold, 1936) and
Ips ponderosa Sw. (Mathre, 1964a). C. Ips was also found associated
with Orthotomleus eaelatus (Elchh.) (Verrall, 1941). Dendroctonus
valens Lee. 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), J£S^ species In Japan (Nislkado and
YamautI, 1933), and Orthotomleus proximus (Elchh.) In Sweden
(Mathlesen-Kaarik, I960). Certain J[££ 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-Kaarik (I960)
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. Mathleson-KSarlk (I960)
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. \£s^ In association
with j££ grandlcollls and j£S pinl. 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. Per I thee I a and

coremla were abundant In the old egg galleries, larval tunnels, and
pupal chambers. Perlthecla 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 endozolc dissemination since the
spores of embedded perlthecla 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 grandlcol I Is 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 (I960) 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 vltamln-autotrophlc. 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-ka'arik (i960) concluded that all these factors
together might produce optimal conditions for some particular fungus

10

or fungf 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-special I zed 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 commensal Ism 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 Ceratocyst I s 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
frontalis 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. Jjis and C. minor added further

•St. George, R. A. and J. A. Beal. 1927. Progress report on
the southern pine beetle (Dendroctonus frontalis ZImm.), U. S. Bur.
Entomol. Plant Ouar. DIv. Forest Insect Invest. Ashvllle, N. C,
(typewritten 40 pp.) (Cited from Olxon and Osgood, 1961, p. 18).

II

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 tangential ly

extended larval galleries In the phloem.

Benefits derived by the beetles from the association have not

been clearly defined. Craighead (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 frontalis.

Leach et al. (1934) stated:

... The b I ue-sta I n I ng 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 pinlperda L. to be Independent of their blue stain
associates. She noted that larvae of the beetles moved ahead of

12

the spread of the fungf 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)
aseptlcal ly reared Dendroctonus frontal Is, Ips cal Hgraphus and
Ips grand I CO 1 1 Is 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 frontalis 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 (^ratocystls Associates of Bark Beetles

Craighead (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 Craighead to suggest that the fungi played
a role In causing death of the tree. Subsequent pathogenicity trials
with certain Ceratocystis species proved Craighead's suggestion to
be correct. Nelson (1934) and Nelson and Beal (1929) were able to

13

kill small loblolly (PInus taeda L.), pitch (P. riglda Mil I.), short-
leaf (P. echlnata Ml II.), and Virginia (P. virglnlana, Mill.) pines
with C. minor. Bramble and Hoist (1940) and Caird (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. mon I ta (Rumbold) Hunt,
and C. schrenklana (Hedge.) 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. pi I If era (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 oleoresln 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.l. 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 b rev I com Is
Lee, was not clear. It was shown that the beetle preferred to

14

attack large trees with an o.e.p. below 60 p.s.l., but was able to
mass attack and kill large, high o.e.p. trees (Miller and Keen, I960
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. j£s
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 J££ species were completely stained by
+he 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. J£s 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. palustrls Mi I i.; pond,
P. scroti na Michx.; sand, P. clausa (Chapm.) Vasey; spruce, P^. glabra
Walt.; and typical slash, P. el llotti I var. el I iotti Engelm. pines
Infested with one or a combination of the three species of Ips bark
beetles occurring In Florida. Additional isolations 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 lO-day Incubation period at 24© C, a small piece
of water agar bearing fungus myceiia 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

15

16

In alcohol and flaming. Cultures from beetle galleries were obtained
by touching a small piece of sterile agar to the spore bearing tips
of per I thee i a 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 nonsteri I ized 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 ml I Milters 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 cal I igraphus and
Ips grandlcol Its 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)

17

cage (Figure I) located In a pine wcxxjs on the University of Florida
Agronomy Farm. A minimum of 100 wild Ips cal I Igraphus or Ips grand I -
col I Is 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 Marlon 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 sorb I c 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 30° C. and 40 to 50 per cent relative humidity. Following curing,
the ends of the bolts were again dipped for 30 seconds. After evapora-
tion of the alcohol, ends of the bolts were painted with a commercially
available, fungicidal 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
if«re artificially Infested with the collected beetles. "Starter

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

18

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

19

holes" large enough to admit a given Ips species, 1/16, 5/64, and
7/64 Inches for Ips avutsus, Ips grand I col I Is, and Ips cal I Igraphus,
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 30° C. Bolts
treated in the same manner, with the exception of introduction of
beetles, served as checks. Following the IO-<}ay 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-Mansvl I le Corporation, Chicago, Illinois.

20

Figure 2. Gelatin capsule used to hold b?srk beetles
prior to entrance into pine bolt. Capsule held In
place with "Duxseal."

21

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. Felted 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 30° 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 7° C.
In petrl 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

22

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-
phlo«w filtrate and heated until disolved; (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 petrl 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 grains. From
Year! an and Wilkinson (1965).

23

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
i.OO

0.50
1.00
1.00
0.10
0.20
0.15
O.Oi

(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 oviposltlon 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 30^ C. Ten newly-hatched larvae were
aseptlcally transferred to a rearing dish. Each larva was Inserted
Into a slit cut in the paper covering. The silt was then closed
and sealed with excess agar coated on the paper surface. The
dishes were held at 30<^ C. and 40 to 50 per cent relative humidity.

24

When the adults were approximately 7 days old, they were removed fro«
the medium in a sterile Isolation chamber and transferred to dishes
containing fresh medium, according to sex and species. The beetles
were held at 7° 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 \£s species reared on the phloem-based medium. Five
larvae were Implanted per dish. The dishes were examined dally 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
]£S calllgraphus and j^s grandlcollls 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. j£s with bolts Infested with wild males. Stash 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 calllgraphus males. Introduced 4 and 6 Inches, respectively,
from the top of the bolts. Six bolts were similarly Infested with two
Ips calllgraphus males reared pn the phloem-based rearing medium.

25

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 were recorded.

ips Development in C. Ips Stained Bolts

A study was conducted to ascertain (I) 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 xylew 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. ]££ 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

26

^r*j» •r«iB«^aegMR;lan>*."-^*'i

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

27

bolts were painted with an asphalt pruning compound* to reduce
desslcatlon. 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: (1)
Iramedlately 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
30® 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 21° C. until needed; and (5) immediately

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

28

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 add (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. i£S.

(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 F|
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 grandlcol I Is and bolts 6 to 8 Inches In diameter
were Infested with Ips cal I igraphus. "Starter holes" were drilled
In the Lark as previously described. Four holes were located mid-
way along the length of the bolts and equidistant from each other

29

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 30° C. and 40 to 50 per cent relative humidity.

The bolts were dissected either 20 days dps avulsus) or 25 days
dps calllgraphus and Ips grand I col lis) following Introduction of the
females. The teneral 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 jp£ species. Two
additional lots were Infested either with BSF or W Ips grandlcoHls
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.

50

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

31

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

Ceratocystis Ips (Rumbold) C. Moreau was the only member of
the genus found associated with Ips avulsus, Ips calllgraphus and
tps grand I CO 1 1 Is In Florida. A perltheclum and spores of C. Ips
are shown In Figures 5 and 6, respectively. Another fungus,
probably Tubercular lei la Ips Leach, Orr, and Chrlstensen, 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. Perlthecia of the fungus were abundant in
old egg galleries of all three Ips species. The fruiting structures
were commonly found In unlngasted 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 cal I Igraphus and Ips grand I col I Is showed 23, 100, and 97
of them, respectively, contained the fungus. Perlthecia were
frequently observed In the frass plugs shortly after the eggs hatched.

32

33

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

34

•n

^' 0

Figure 6. Ascospores of Ceratocystis tps. Approx,
2000X.

35

Isolations from each of the life stages of Ips avulsus, Ips
cal I Igraphus, and Ips grand I col I Is 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 cal Hgraphus and Ips grand I col lis pupae yielded 23 and 4
isolates, respectively. Surface-sterilized teneral 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
cal I Igraphus and Ips grand I col I Is, 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-
sterl I Ized Ips cal I Igraphus and Ips grandlcol I Is 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. ips after the eggs hatched, and as the larvae enlarged the gallery

36

T»hiP 2. Relative freauency of Ceratocystis Ips I sol at I
avulsus, Ips cal llgraphus, and Ips grand I col II

on from
s.

Ips

Stage of
Development

Ips

avulsus

Ips
cal 1 Igraphus

Ips

grand I CO

Ills

Number

%

Number

%

Number

%

SURFACE-STERILIZED:

Egg

100

0

100

0

100

0

Larva

1st Instar
2nd Instar
3rd Instar

Pupa

100
100
100
100

0
74

100
0

100
100
100
100

0
16
84
23

100
100
100
100

0
33
80

4

Teneral adult
male
female

100
100

35
8

100

100

60
55

100
100

89
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 cal 1 Igra

phus

and Ips

grandlcol lis 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 unti I after pupation. Some J£s cal I Igraphus and ]p£ grandlcol lis
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.

37

Tho data (Table 2) Indicating Ips cal I Igraphus and Ips grandl-
col I Is 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
sterl I Izatlon of the Ips cal I Igraphus and Ips grand I col I Is 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 cal I Igraphus and Ips
grand I CO 1 1 Is 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 cal I Igraphus and Ips grandt-
co I Ms 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

38

Table 3. Relative frequency of Ceratocystis Ips transmission by J!£S
avulsus, ]££ cal Hgraphus, and Jji£ grandlcol Us.

Beetle Species

Successful Attacks
9.' ips C. ips C. ips
Present Absent Trans-
mission

Ips avulsus

Male 83

Fema I e 90

Ips cal Hgraphus

Male 100

Fema I e 95

Ips grandlcol I Is

Male 97

Fema I e 93

Check 0

100.0
97.8

100.0
100.0

100.0
100.0

0.0

Unsuccessful Attacks
C . \ps_ C . J2£ C . 2ps_
Present Absent Trans-
mission

9

47.1

3

62.5

0

0.0

0

100.0

0

iOO.O

0

100.0

demonstrated 100 per cent transmission, the probability of an J££
avulsus gallery system being free of C. j£s appeared to be unlikely.

Even though some attacks by the beetles were unsuccessful, C. J[££
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 J_e£ cal I Igraphus and J_p£ grandlcol Us adults
were stained by the fungus. Of 17 unsuccessful attacks by Ips avulsus
males, C. j£S was Isolated from 8, and 5 of 8 unsuccessful attacks by
females yielded C. J££ cultures. Even though the attacks were

39

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. Perl thee la 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 cal I Igraphus, and Ips grandicol I Is larval and
pupal developmental rates on the phloem-based rearing medium were
similar at 30® C. (Table 4). The mean length of the larval period
was 8.4 days for Ips avulsus, 8.9 days for Ips cal I Igraphus and 9.2
days for Ips grandicol I is. 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 p re-pupa I stage for
the three species was approximately one day. The pupal period for
Ips avulsus, Ips cal I Igraphus and Ips grandicol I Is averaged 2.8,
3.7, and 3.6 days, respectively.

40

Table 4. Mean larval and pupal development of Ips avulsus, Ips
cal I Igraphus, and Ips grand I col I Is on phloem-based,
seml-arti f icial rearing medium at 30*^ C.

Stage of Ips avulsus Ips cal I Igraphus Ips grand I col I Is
Development Mean No. Ob- Mean No. Ob- Mean No. Ob-
(days) served (days) served (days) served

Larva (total) 8.4+0.51 II 8.9+0.27 45 9.2+0.20 51

1st Instar 2.5+0.22 15 2.7lO.I3 86 2.6+0.10 87

2nd Instar 3.2±0.29 15 3.0+0.10 64 2.8±0.I2 81

3rd Instar 2.8t0.30 13 3.7+0.22 49 3.6+0.18 71

Pupa 2.8+0.36 9 3.3t0.l0 45 3.0+0.18 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
cal I Igraphus, and Ips grandlcol I Is, 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 cal I Igraphus, and was
15.0 days for Ips grandlcol lis. After 7 days, teneral adults reared
on the medium were capable cf Infesting pine bolts and establishing
broods. Thus, allowing a day for attack, mating and Initiation of
oviposit Ion, a complete life cycle, from egg to egg, averaged 21.5,
22.9, and 23.0 days for Ips avulsus, Ips cal I Igraphus, and Ips
grandlcol lis, respectively. These developmental rates are comparable
to generalized life cycles for the three species given by Thatcher
(I960).

41

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 cal I Igraphus, and 49 Ips grand I col I Is 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 dally 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 cal I Igraphus, and 68.3 per cent for Ips grandlcol I Is.

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
cal I Igraphus and Ips grandlcol I Is 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 cal I Igraphus and Ips grandlcol I is 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, I960, 1962) and Nlckle (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 cal I igraphus and Ips grandlcol I Is
larvae, pupae, and adults were frequently Infected with a number of

42

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 3. 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 calllgraphus 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 Vite' (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

43

Table 5. Attractiveness of W and BSF Ips cal llgraphus male Infested
stash pine bolts.

Number of Beetles Recovered New Attacks
Fema I es Ma I es* Tota I

BSF Bolts

1

3

4

7

3

2

9

5

14

3

3

2

1

3

0

4

5

3

8

1

5

7

3

10

1

6

7

3

10

1

Total

33

19

52

9

W Bolts

1

10

6

16

4

2

0

1

1

0

3

4

2

6

1

4

3

4

7

2

5

4

2

6

0

6

6

3

9

1

Total

w

18

45

8

•Includes males Introduced In laboratory.

logs with yeast Isolated from Ips pini 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.

44

tps Development In C. fps Stained Bolts

Attempts to estab 1 1 sh Ips avuisus, Ips cal I tgraphus and Ips
grand I CO 1 1 Is 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 cal H-
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
cal I Igraphus pupae. The remainder of the females of all throe 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 pinl 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. Mathiesen-KMSrIk (I960) found that certain Insect
associated Ceratocystls species utilized several amino acids, sugars,
and vitamins and Mathre (1964c) demonstrated that C. j£S_ significantly

45

reduced the fructose content of ponderosa pine sapwood. Whether a
sfml lar 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 oviposit Ion 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 sylvestrls 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

46

end of the bolt and eventually turned back parallel with the grain
of the wood forming a "U" shaped gallery. Some Ips grand I col Us and
tps cal llgraphus females burrowed out the end of the bolt and re-entered
at anothier site on the bolt.

'^ Egg gallery construction and oviposltfon by all three species wore
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 cal I Igraphus and l£S grand I col I Is 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 cal I Igraphus or Ips
grand I CO 1 1 Is 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 grand I col lis eggs were more closely spaced than Ips
cal I Igraphus eggs.

47

Table 6. Mean egg gallery length and spacing between eggs of W, BSF,
and BS Ips avulsus, Ips cal I Igraphus, and Ips grand I col I Is
females In slash pine volts at 30° C.

Species Number of Galleries Mean Length Spacing Between

(cm.) Eggs (cm.)

Ips avulsus

W 46

BSF 37

BS 30

Ips cal I Igraphus

W 40

BSF 36

BS 37

Ips grandlcol I Is

W 73

BSF 61

BS 33

•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 grandlcol lis and Ips cal I Igraphus 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.

I3.5±l.4»
ll.6±l.3
12. 2+1.1

0.59
0.68
0.86

14.2+2.2
20.1+1.8*
14. 9+1. 8

0.55
0.48
0.49

1 1.5+1.7

13.9+2.2*

il.9tl.8

0.39
0.34
0.31

48

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. j£s 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 stain 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 2£s
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 perlthecla. 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. ]£$. Fifteen days after Introduction of

49

the females the entire length of the egg galleries was stained and
lined with perlthecia. Figure 7 shows C. Ips per I thee I a lining a
section of a 15-day old BS Ips grandlcollls gallery 5 centimeters
from the nuptial chamber. Figure 8 shows a comparable section of
^^ t ps grand I co 1 11 s ga 1 1 ery . At 15 days the entire gallery system
of Ips avulsus broods, and most of the gallery system of Ips cal 1 1-
graphus and Ips grand I col I Is broods were covered by the fungus.
Perlthecia 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 s I ze and morta 1 1 ty

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 staln.free (BSF) Ips cal I Igraphus
broods were significantly larger than W and BS broods. Both BSF and
^^ Ips grand I col I Is 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

50

Figure 7. Section of 15-day old BS Ips grand I col I Is
gallery 5 centimeters from nuptial chamberT Note perl-
thecla of Ceratocyst I s Ips lining gallery and masses of
ascospores at tips of perlthecla. Approx. 40X.

51

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

52

Table 7. Mean size and mortality of W, BSF, and BS Ips avulsus> Ips
cal llgraphus, and Ips grandlcoHis broods In slash pine
bolts at 30O C.

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

Ips avulsus

W 46

BSF 37

BS 30

Ips cal I Igraphus

W 40

BSF 36

BS 37

Ips grand I CO 1 1 Is

W 73

BSF 61

BS 33

20.7+1.6
I6.4±l.7
14.0+2.0

5.1
3.2
2.7

28.5t3.8

35.7+3.9*

3I.8±3.9

16.1

13.5

8.6

28.5+2.6

39.9+2.9*

39.6+3.9*

9.4
4.6
6.5

•Significantly larger (p«.05) from others within same species.
Duncan's New Multiple Range Test.

was greatest In Ips cal I Igraphus 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 cal I Igraphus 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.

53

Brood composition

The composition of W, BSF, and BS Ips avulsus^ Ips cal llgraphus,
and Ips grand I CO 1 1 Is broods at the different dissection dates Is
depicted In Figures 9, 10, and 11, 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 avuisus 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 avuisus 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 teneral
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 avuisus broods examined contained second Instar larvae. By
the final dissection date (20 days) ail three Ips avuisus broods were

54

5 DAYS

■ W
D BSF
= BS

ln= In^

^ w n ^

10 DAYS

-n- -r.= Ins |n5

15 DAYS

n =;

.5 W n = P n =

20 DAYS

1 2 3 pupa adult

larval Jnstar
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 30© C.

5 DAYS

■ W
D BSF
S BS

SJ

n

- Pi n =

10 DAYS

iHs 10=

ihS

15 DAYS

in5

5 pni pRJ

55

20 DAYS

i m q i n =

egg

2 3_

larva I instar

pupa

adult

STAGE OF DEVELOPMENT

Figure 10. Mean composition of W, BSF, and BS Ips
cai Hgraphus broods In slash pine bolts at 5, 10,
15, and 20 days at 30° C.

56

5 DAVS

■ w

D BSF
= BS

1

l|[

=

10 DAYS

Pn^ pni 1

s
S

1 1

=

15 DAYS

- Fn= 1

=

Hi iililll

iilll

20 DAYS

-

=

In=,

..-1

--m —

egg

1 2 3 pupa adult

larva I instar

STAGE OF DEVELOPMENT

Figure II. Mean coraposltlon of W, BSF, and BS Ips
grand I CO 1 1 Is broods In slash pine bolts at 5, 10,
15, and 20 days at 30° C.

57

H

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58

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59

Table 9. Relative composition (percentage) of W, BSF, and BS Ips

avulsus, Ips cal I Igraphus, and Ips grandlcolHs broods In
slash pine bolts at 5, 10, 15, and 20 days at 30° C.

Species Time Stage of Development

(days)

Larva I I nstar

Egg I 2 3 Pupa Adult

Ips avulsus

W 5 43.2 42.8 13.9

10 3.2 3.5 29.9 54.3 9.1

15 8.9 17.1 73.8

20 100.0

BSF 5 40.6 27.8 31.6

10 12.5 11.3 27.2 47.1 1.7

15 16.0 56.6 11.7 15.5

20 100.0

BS 5 50.0 32.6 17.4

10 6.1 18.5 32.9 40.2 2.0

15 15.8 36.4 23.3 24.2

20 100.0

Ips cal I igraphus

W 5 61.1 28.1 10. 1 -

10 18.3 15.2 26.8 39.6 --

15 8.6 62.5 23.2 5.5

20 10.5 8.4 80.9

BSF 5 50.4 34.7 14.8

10 15.2 15.7 28.8 40.1

15 13.6 57.2 16.4 12.6

20 3.9 8.6 87.4

BS 5 33.8 39.4 26.7

10 11.7 13.0 34.9 39.8

15 13.6 34.4 21.3 30.6

20 6.6 7.8 85.6

Ips grand I col I Is

W 5 50.5 44.1 5.3

10 22.3 19.4 41.2 17.1

15 1.5 12.0 54.7 31.6

20 15.3 17.8 66.8

60

Table 9. Cont.

Species Time

Stage

of Development

(days)

Egg

Larva 1 1 n

star

Pupa

1

2

3

Adult

ips grandlcoi i Is
BSF 5
in

57.3

i2.4

4.3

57.2
8.2

37.6
13.1
11.8

35.4
18.4

8.9

4.9
31.3
46.7

7.2
49.7
30.3

43.0

18.7

5.9

23.6

45.2
3.3

i5

20

BS 5

10

18.6

8.6

85.4

15
20

15.3
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 cal I igraphus 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.

61

Table 10. Chl-square comparison of W, BSF, and BS ips avulsus, ips
cal I fgraphus, and tps grand I col I is brood composition at
5, 10, 15, and 20 days at 30O C.

Dissection Date Brood Comparison

W vs. BSF BS vs. BSF W vs. BS

ips avulsus

5 NS NS NS

10 *• NS »»

15 *» NS *»

20 NS NS NS

Ips cal I Igraphus

»« ««

10 NS NS NS

15 NS ** *•

20 NS NS NS

Ips grand I CO 1 1 Is

^ l^g ^g ^g

««

10
15
20 ** NS

NS Not significantly different

** Significantly different (p«.0l) from others of the same species
on same dissection date.

The relative make-up of Ips grand! coll Is broods was not signifi-
cantly different at 5 days. At the 10-day dissection BSF broods
appeared more advanced than the W and BS broods with proportionately
more third Instar larvae (Table 9). On the basis of relatively
fewer eggs and first instar larvae and more second and third Instar
larvae, BS broods also appeared to be more advanced than W broods.
Both at the 15-and 20-day dissections BSF and BS broods differed

62

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 cal I igraphus and Ips grand I col I is 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 cal I Igraphus and Ips grandicol I is.

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

63

Table II. Mean weight (mg.) of 100 W, BSF, and BS Ips avulsus, I ps ca I II -
qraphus, and Ips grand 1 col I Is pupae reared In slash pine bolts
h l(P C.

Beetle Specfes Type of Pupa

W BSF BS

Ips avulsus 2.20*0.11* l.7l±0.23 l.76±0.ll
Ips calUgraphus 9. 74*0. 25 10. 28*0. 40* 10.17*0.48*
Ips grandlcollls 3.71*0.11 4.54+0.14* 4.23*0.16*

* 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 (p».OI)
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 cal I Igraphus and Ips
grandlcol I Is females was significantly (p«.OI) greater than that
laid by W females. Blue stain-free (BSF) Ips cal I Igraphus females
also produced (p».05) more eggs than BS females. The average length
of egg gal lery cut by the three types of jj^ cal I Igraphus and Ips

64

Table 12. Sex ratio of W, BSF, and BS Ips avulsus, Ips calUgraphus,
and Ips grandlcollls adults reared In slash pine bolts at
30° C.

Species

Number
Examined

Number
Males

Number
Fema I es

Sex Ratio
Ma I es : Fema I es

Ips avulsus

W 92

BSF 60

BS 70

Ips cal I Igraphus

W 102

BSF 100

BS 99

Ips qrandlcol I Is

W 167

BSF 132

BS 113

48
32
37

52
52
52

86
72
56

44
26
33

50
48
47

81
60
59

0.92
0.88
0.89

0.96
0.92
0.90

0.94
0.83
1.05

grandlcollls 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 OtJ-ier 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, I ps ca I H -
graphus and Ips grandlcol lis. The fungus appears not only to be

65

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 avuisus, Ips cal I Igraphus, and Ips grand I col I Is
females at 56 days In sfash pine bolts at 30° C.

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

lery

Ips avuisus

W 10 51.2*5.75** 77.0t|3.8» 1.5

BSF 11 31.2*6.83 54.0*6.8 1.7

BS It 36.8*6.10 53.7* 9.3 1.4

Ips cal I Igraphus

_ g 50.8-4.16 117.4*20.8 2.3

BSF 13 52.7*4.19 151.1*10.6** 2.9

BS 12 47.8*5.65 139.6*10,5** 2.9

Ips grand I CO 1 1 Is

W 15 37.3*5.42 108.2*19.2 2.9

BSF 16 44.6*6.21 148.5*16.1** 3.4

BS 14 40.1*5.77 147.3*13.9** 3.7

* Significantly different (p=.05) from others within the same
species. Duncan's New Multiple Range Test.
** Significantly different (p=.0l) from others within the same
species. Duncan's New Multiple Range Test.

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 simllating bark beetle attacks (Mathre,
1964b and Nelson, 1934), the fungus may become established but does

66

not spread from the site of Infection and sporulatlon 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 call Igraphus
and Ips grand I CO 1 1 Is 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 Ips call Igraphus and Ips grand I -
CO 1 1 1 s although the data were not as conclusive as with Ips avulsus.

67

Ips cal llgraphus females free of C. |££ (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 cal I Igraphus 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. J[££ 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 grandlcol lis 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

68

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. l^ Is a
factor In brood development and fecundity of Ips grandlcol lis.

Although the results of this Investigation clearly Indicated
that C. j£s has little or no effect on brood development and fecundity
of the three J^s species studied, the question still arises as to the
applicability of these findings to field conditions. It Is well
documented In the I Iterature 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 res In-secret I ng 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. j£s 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. J[£s 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 ecloslon.
Under field conditions, therefore. It appears unlikely that C. ]£s

69

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 grand I col Ms and Ips cal II-
graphus did not fair as well as the BS and 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.

70

probably Tubercularlel la tps Leach, Orr, and Chrlstensen. Callahan
and Shifrlne (I960) suggested that yeasts played a role In the
nutrition of bark beetles. They noted that all soecles of bark beetles
have associated yeasts and that the yeasts were eaten by both larvae
and adults. Callahan and Shifrlne added further that ft has not been
demonstrated that "yeast-feed tng" Is not a prerequisite for fertility
of the adults. The results of this study clearly refute Callahan and
Shlfrlne's hypothesis. Brood development, fecundity, and fertility
of both Ips call Igraphus and Ips grand I col I Is 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
simitar to those given by Leach et al. (1934) for Tubercularlel la
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 conidia (Figure 121. The fungus no doubt served as a food source
for Ips avulsus adults as newly emerged beetles were observed

71

Figure 12. Ips avulsus pupal chamber covered with
masses of contdla, probably Tuberculariel la Ips.
Approx. 5X.

72

"grazing" on the fungal matt. Most Individuals did not stray frm
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
Scolytldae, some cases of association bet><een 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 J£S
acumlnatus Gyll. In this case the fungus possessed nutrlent-rlch,
globose conldia 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 Tubercularlel la Ij)s resembled the so-called
"ambrosia" fungi by possessing large globose conldia rich In nutrients.
The relationship of T . jps to jps plnl and jps grandlcollls In the
United States (Leach et al., 1934) and jps sexdentatus In Europe
(Slemaszko, 1939) then appears very similar to that described above.
The fungus In question In this study, probably T. 2ps, 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 t

Ceratocystis Ips was the only member of the genus consistently
associated with j£s bark beetles occurring In Florida. Another fungus,
probably Tubercular lei la Ips, was frequently associated with Ips
avulsus, but not with J^s cal I Igraphus and j£s grandlcol lis.

Ceratocystis Ips was transmitted by both males and females of
the three 2£S species. Adults were both externally and Internally
contaminated with the fungus. Larvae, particularly the later Instars,
were a I so I nterna 1 1 y contam I nated . Ips avulsus I arvae demonstrated
a higher Incidence of contamination than Ips call Igraphus and Ips
grandlcol lis.

Ceratocystis Ips had no Influence on the mass attraction
phenomenon of Ips cal I Igraphus 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 cal 1 1-
graphus and Ips grandlcol I Is. Larvae were reared on semi -artificial
medium without adverse effects when compared to broods reared In
bolts fn the presence of Ceratocyst I s Ips. The three Ips species
were reared through successive generations In bolts free of

73

74

Ceratocystis Ips without observable effects on vigor and fecundity
when compared to beetles In the presence of the fungus. Ips cal 1 1-
graphus and Ips grand I col I Is 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 Tubercular lei la 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

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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, I960. Upon receipt of the Crossett
Research Fellowship, he entered the Graduate School, University of
Arkansas in January, I960, and was awarded the Master of Science Degree
with a major in Entomology in January, 1961.

On June 30, I960, 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.

80

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

Co-Chalrmen

^^^.^Pean, College of Agriculture

Supervisory Committee:

Dean, Graduate School

S323 ,4;^