SEASONAL BEGSHEMICAL CHANGES
EN THE RED PENE WEE,
FEMS RESWDSA MR, AND THEER
{NFLUEE‘ECE 0% THE LARVAE OF TBE
EUROPEAN PENE SHOOT MQTHf
REYACEOMA BEEOUANA (SCHEF)

Thesis fer the Degree of M. S,
§¢iSCHEGAN STATE UNWERSITY
R. SITARAMAN
1972

ABSTRACT

SEASONAL BIOCHEMICAL CHANGES IN THE RED PINE TREE,
PINUS RESINOSA AIT., AND THEIR INFLUENCE ON THE
LARVAE OF THE EUROPEAN PINE SHOOT MOTH,
RHYACIONIA BUOLIANA (SCHIFF)

 

 

BY

R. Sitaraman

The needle, shoot and the bud of the red pine

tree, Pinus resinosa were chemically analyzed for total

 

lipids, total protein, starch and three sugars: sucrose,
glucose and fructose. Quantitative determination of the
above mentioned cellular substituents was also made. It
was observed that the amounts of total lipids, total pro-
tein, starch and the sugars changed during the different
seasons. The incidence of the larvae of the European pine
shoot moth on the red pine was also studied. No cor-
relation was observed between the changes in amounts of
the cellular substituents considered and the incidence

of the larvae of the Eur0pean pine shoot moth, 3. buoliana.

SEASONAL BIOCHEMICAL CHANGES IN THE RED PINE TREE,

PINUS RESINOSA AIT., AND THEIR INFLUENCE ON THE

 

LARVAE OF THE EUROPEAN PINE SHOOT MOTH,

RHYACIONIA BUOLIANA (SCHIFF)

 

BY

RC—Sitaraman

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Entomology

1972

n~
0))

‘3%} ACKNOWLEDGMENTS
u?

The author records his deep sense of gratitude
to Dr. Matthew J. Zabik, Associate Professor of Entomology,
for having served as the chairman of the guidance com-
mittee and also for the valuable guidance, constant
encouragement and help rendered in the execution of this
work and in the preparation of this thesis.

The author wishes to express his sincere thanks
to Drs. James W. Butcher, Professor of Entomology and
Associate Dean, Ronald E. Monroe, Associate Professor
of Entomology, Roger A. Hoopingarner, Associate Pro-
fessor of Entomology, Stanley G. Wellso, Associate Pro-
fessor of Entomology and Loran L. Bieber, Associate
Professor of Biochemistry for having served as guidance
committee members and for their valuable suggestions and
constructive criticisms offered throughout the course of
this investigation.

The author's thanks are also due to Mrs. Viola
Wert for the help rendered in the analysis of plant
materials for protein content; and to Mr. Steven K. Derr
for the assistance provided in the collection of plant

materials.

ii

This study was supported by funds from the
Department of Forestry and from the Agricultural Experi-
ment Station.

The author also wishes to express his sincere
appreciation to his wife, Kasthuri, for the inspiration

she provided during the period of this study.

iii

TABLE OF CONTENTS

ACKNOWLEDGMENTS . . .
LIST OF TABLES . . .
INTRODUCTION . . . .
REVIEW OF LITERATURE .
MATERIALS AND METHODS .

Materials . .

Preparation of the Plant Samples for

Chemical Analysis.
Methods. . . . .

Extraction and Estimation of the
Extraction and Quantitative Determination of

Sucrose, Glucose and Fructose. . . .

Extraction and Quantitative Determination of

Starch . . . .

Total Lipids

Quantitative Determination of Total Protein.
Statistical Analysis of the Chemical Data

and the Incidence.

RESULTS AND DISCUSSION.

Seasonal Changes in the Amounts of Total
Lipids in the Needle, Shoot and the
Bud of the Red Pine Tree . .

Seasonal Changes in the Amounts of Total Pro-

tein in the Needle,
the Red Pine Tree.

Seasonal Changes in the Amounts of Starch in

Shoot and the Bud of

the Needle, Shoot and the Bud of the Red

Pine Tree . . .

Seasonal Changes in the Amounts of Sugars in
the Needle, Shoot and the Bud of the Red

Pine Tree . . .

iv

Page
ii

vi

10
10
10
11
11
12

14
16

17

18

18

18

22

22

Page

sucrose 0 O O O O O O O O O O O O 22
Glucose . . . . . . . . . . . . . 23
Fructose. . . . . . . . . . . . . 23

Seasonal Changes in the Incidence of the Larvae

of the European Pine Shoot Moth, R. buoliana . 23
Discussion . . . . . . . . . . . . 25
CONCLUSIONS. . . . . . . . . . . . . . 27
Suggestions for Future Investigation . . . . 29
LITERATURE CITED . . . . . . . . . . . . 30

LIST OF TABLES

Table Page

1. The Chemical Composition of the Needles of the
Red Pine Tree Sampled at Three Different
Times 0 O O O O I O O O O O O O 19

2. The Chemical Composition of the Shoot of the
Red Pine Tree Sampled at Three Different
Times . . . . . . . . . . . . . 20

3. The Chemical Composition of the Bud of the
Red Pine Tree Sampled at Three Different
Times . . . . . . . . . . . . . 21

4. The Incidence of the Larvae of the European

Pine Shoot Moth in the Red Pine Samples
Collected at Three Different Times . . . 24

vi

INTRODUCTION

The European pine shoot moth, Rhyacionia buoliana

 

(Schiff) (Olethreutidae: Lepidoptera) (Heinrich, 1923)

is a major pest of a number of species of pines in many
parts of the U.S.A. Although a number of species of pines
are attacked by R. buoliana, the most common hosts are

the red pine,Pinus resinosa Ait., and Scotch pine,

 

Pinus sylvestris L., (Miller, 1967).

 

Siminovitch §E_§l, (1953) have investigated the
chemistry of living bark of the black locust, Robinia

pseudo-acacia L., in relation to its frost hardiness.

 

Parker (1957) worked on the seasonal changes in some
chemical and physical properties of living cells of

Pinus ponderosa and their relation to freezing resistance.

 

Pomeroy 25 21. (1970) studied the seasonal biochemical
changes in the living bark and needles of red pine,
Pinus resinosa, in relation to adaptation to freezing.
While working on artificial diets for the larvae
of the European pine shoot moth, Chawla and Harwood
(1968) observed that the addition of pine tissue to the
artificial diet did not enhance the growth of the larvae

or the pupation. The objectives of this investigation

were to find out whether the amounts of total lipids,
total protein, starch, sucrose, glucose and fructose

in the needle, shoot and bud vary during the different
seasons of the year and if they do change, how it affects
the incidence of the larvae of the European pine shoot
moth. It was hoped that such an investigation would help
in obtaining a better understanding of the nutritional
requirements of the larvae of this pest and thus enable
one to utilize such knowledge for developing a non-
insecticidal method of control.

In this investigation the red pine was chosen
because it is the most extensively planted tree in the
Lake States as well as parts of the Northeast, and it is
planted primarily for the production of saw timber
(Slabaugh, 1957). A smaller number of red pine trees
are also used as Christmas trees (James, 1958). Another
reason for choosing red pine was that it is a common host

of the larvae of the European pine shoot moth.

REVIEW OF LITERATURE

The first infestation of the EurOpean pine shoot
moth in the U.S.A. was recorded by Busck (1914) in Long
Island, New York, and since then this pest has spread to
many other states of this country. Heinrich (1926) found
that the European pine shoot moth was well established
in New York, Connecticut, New Jersey and Rhode Island.
The incidence of the European pine shoot moth was also
reported by west (1936) and Friend gt 31. (1938) in
Connecticut. Benjamin st 31. (1959) recorded that the
European pine shoot moth was a serious threat to the
growing of hard pines in southeastern Wisconsin. Beique
(1960) noted that in Quebec city and vicinity the European
pine shoot moth was infesting the mugho pine, Ping§_mughg
Turra. Pointing and Green (1962) found that in southern
Ontario the infestation of the European pine shoot moth
was detected as early as 1926. In Michigan, the first
infestation of this pest was reported by McDaniel (1930)
near Detroit and subsequently it has spread to many
counties of the state of Michigan.

Many workers have studied the nature of the

injury caused by the larvae of R. buoliana and its

effect on the host tree. Friend and West (1933) reported
that in the fall the larvae bore into buds and into the
deveIOping shoots in the spring; and these attacks con-
stitute the principal injury to the host. The death of
the buds and shoots lead to the deveIOpment of lateral
buds just below the tip of the twig with a consequent
bushy growth. When the infestation is severe, the ter-
minal growth is inhibited and because little new growth
survives to produce needle tissue, a progressive defoli-
ation of the tree may take place. When the attacked
growing shoot is killed, the terminal shoot of the tree
is badly distorted. The main stem becomes permanently
curved, and this condition is the so-called "post-horn"
or "bayonet," (Friend and west, 1933). Benjamin (1959)
recorded that the larvae of the European pine shoot moth
distorts and stunts red pine and other hard pine species.
European pine shoot moth larvae seldom kill the trees
but often cause serious injury to both lateral and ter-
minal buds and as a result, the trees may be so stunted
as to be of no value for Christmas trees, pulp, lumber
or ornamentals. Talerico and Heikkenen (1962) noted
that the vertical growth of trees is not retarded,
furthermore their study in four plantations in Lower
Michigan indicated that of all the samples examined,

3 to 17% of the trees have never been attacked by the

larvae of the EurOpean pine shoot moth. Of the remaining

83 to 97% of the trees, 60 to 90% had injuries which
were correctible by pruning and 7 to 15% had injuries
that were permanent.

Fox (1955) found that since its detection in 1933
near Detroit, Michigan the European pine shoot moth had
spread throughout the southern half of the Lower peninsula,
and each year its distribution extended northward, due
in part to the warming influence of the Great Lakes; thus,
Fox considered this pest as a serious threat to many
thousands of acres of plantings of susceptible pines
throughout the state. Mead and Barnes (1970) in evaluating
the performance of ponderosa pine in southeastern Michigan
have reported that the European pine shoot moth has
damaged most of the stands planted during the last 20
years, killing the terminal shoots and causing post-horns.
They also reported that the damage of R. buoliana seemed
to be less severe in ponderosa plantations than in red
pine plantations.

The biology and behavior of the European pine
shoot moth have been studied by many workers (Haynes and
Butcher, 1962; Haynes, 1960; Pointing, 1961; and 1963).
Pointing (1961) noted that in southern Ontario the
incubation period in field cages lasted for 8 to 14 days
and there are 6 larval instars and the third instar
larvae undergo diapause. The first and second instar

larvae feed on the needle tissue enclosed within the

needle sheath. The third instar larvae hollow and destroy
one to several buds during the late summer before passing
the winter in or on a bud concealed under the mass of
hardened pitch.

In Michigan, the adult moths emerge in the early
part of June and oviposition begins within a few days.
The eggs are laid on the bark and needles in single or
in small groups. The eggs hatch in about two weeks after
oviposition. The newly hatched larvae feed at the base
of the current year's needle and later their summer
activity is confined to terminal and lateral buds. The
larvae burrow into the bud and remain there until the
following spring. The over-wintering larvae resume
activity again about April 20 causing most severe injury,
deforming and killing. Pupation takes place within the
buds some time late in May to early June. The adults
begin to emerge in about 16 days after pupation (Haynes,
1960). Friend 22 El. (1938) studying the distribution
of the larval population of the European pine shoot moth
noted that the terminal part of the tree had higher con-
centrations of larvae while the concentration decreased
in the lower parts of the tree. Green and Pointing (1965)
working on the natural dispersal of egg-laden female pine
shoot moths recorded that the adult female population
appeared to be comprised of at least two major types in

terms of activity. One segment seemed to include females

of relatively low activity responsible for spreading
infestation through contiguous areas by numerous tree
to tree flights and females of relatively greater
activity participating in longer range dispersal of
the species.

Many workers have contributed to the knowledge of
the chemical control of European pine shoot moth, its
natural parasites and also the influence of climatic
conditions on this pest. Butcher and Carlson (1961)
observed that an excellent control of European pine shoot
moth was obtained with 0.5, 1.0, 2.0 pounds of dimethoate
in spring and summer treatments on red pine tree,

g. resinosa. Connola 25 21! (1954) obtained favorable
results against this pest with an aerial spraying of 4
pounds of DDT in 4 gallons of emulsion per acre in 1951,
but there was no reduction in the injury by this insect
pest in the tests conducted in 1952, and suggested that
unknown factors were involved in the timing of the
sprayings. Haynes gt 2l° (1958) used six systemic
insecticides against the EurOpean pine shoot moth in tests
conducted in two red pine plantations in wexford and
Ottawa counties in Lower peninsula of Michigan, found
that foliar sprayings applied in August and April gave
highest initial control of European pine shoot moth.
Thimet, Phosdrin and American Cyanamid 12008 all gave

approximately the same degree of control. They also

observed evidence of systemic activity in the following
spring in several of the summer treatments. Butcher and
Haynes (1958) reported that Phosdrin sprays at the rate
of 1 pound actual per 100 gallons of water were superior
to Guthion, DDT and BHC sprays at the dosages and formu-
lations used in these experiments for the fall control of
the European pine shoot moth in 3-year-old red pine.
Miller (1959) studying the European pine shoot
moth parasitism in Lower Michigan observed that Tricho-
gramma minutum, an egg parasite parasitized about 4% of

the eggs, and Itoplectis conquisitor (Say) was found as

 

a solitary internal pupal parasite and Calliephialtes

 

comstockii (Cresson) and Eurytoma pini Bugbee as a
solitary external larval parasite. Beique (1960)
reported that in Quebec city and vicinity a braconid,

Orgilus obscurator (Nees) was a major parasite of 5.

 

buoliana larvae. It was also observed that in a study
over a five-year period in Quebec city, more than 60%
of the larvae were parasitized by this parasite.
Schmiege (1963) reported that the dauer larvae of a
neoaplectanid nematode were able to infect the third
instar larvae of the European pine shoot moth, hidden
in the buds and pitch blisters.

Batzer and Benjamin (1954) studying the cold
temperature tolerance of European pine shoot moth in

Lower Michigan observed that low winter temperatures

play an important role in the northward distribution of
this insect; but where the winter temperatures fail to
attain a lethal level, this insect was a serious menace.
West (1936) reported that in Connecticut the overwintering

larvae could not withstand winter temperatures below -18°F.

MATERIALS AND METHODS

Materials

 

The pine samples used in this investigation were
collected monthly from a red pine plantation in Grand
Traverse county, Michigan. Ten trees were randomly
selected for sampling. These were growing under similar
conditions and were of the same size. All the collections
were made in the morning between 9 and 11 A.M. Only the
new growth from the periphery of the tree was collected
from a height of 4 to 5 feet above the ground. Fifteen
branches, each being about 7 to 8 inches long, were col-
lected from each of the selected trees and the samples
were stored in a cold chest and transported to the
laboratory and then stored in a freezer.

In order to determine the incidence of the larvae
of the European pine shoot moth, 25 infested branches,
about 8 inches long, were collected from each of the
selected trees and kept separately. ‘

mfléfi’éhé’iiiii 3225.???“
For extraction and estimation of the total

lipids the fresh material was used. The needle, shoot

10

11

and the bud were cut into small pieces, about a quarter
inch long, and then used for the extraction of total
lipids.

For the extraction and estimation of starch,
total protein and the sugars, the plant samples stored
in the freezer, subsequent to collection, were lyophil-
ized for 15 hrs; ground in a Wiley mill and then stored

in small vials in the freezer.

Methods

To determine the incidence of the larvae of the
European pine shoot moth, five infested branches were
randomly selected out of the 25 infested branches col-
lected and then dissected and examined for larvae. An
estimate of the number of larvae found in the 25 branches
was obtained by multiplying the mean number of larvae in
one branch by 25.

Extraction_and Estimation of
the Total Lipids

 

 

One 9 of the fresh plant material was extracted
with 20 ml of a mixture of chloroform and methanol (1:1)
for 5 min in a omni-mixer at a high speed. The extract
was filtered into a flask and the residue was again
extracted with another 20 ml of the mixture for 5 min
and filtered. The two extracts were combined and mixed

well. The combined extract was then washed 3 times with

12

a 10% Sodium chloride solution. An aliquot of 10 ml was
pipetted out into an aluminum weighing boat, already
dried for 3 min in a desiccator, weighed again and thus
the amount of total lipids in one 9 of the fresh plant
material was obtained and then the percentage of total
lipids was calculated on a dry weight basis.

Extraction and Quantitative Determination
of Sucrose, Glucose and Fructose

 

 

The extraction of the sugars was done following
the procedure of Hassid and Neufeld (1964) and the
separation and the quantitative determination were done
by following the procedures of mehof and Tucker (1965)
and Hassid and Neufeld (1964), respectively. Five hundred
mg of the lyophilized powder was extracted with 50 m1 of
80% ethanol at 70°C in an omni-mixer for 30 min. The
extract was then filtered through a sintered glass
funnel, the residue was air-dried and stored in a freezer.
The extract containing the sugars was boiled to remove
the alcohol with addition of small amounts of water.

After the alcohol was completely removed, the sugar
extract was clarified using the procedure of Siminovitch
22 31. (1953) as follows: To the sugar extract were
added 2 ml of lead acetate, 4 m1 of disodium phosphate
and 1.2 g of bone charcoal in succession and mixed well.
The treated extract was then filtered in_yagug through

a millipore filter on a Buchner funnel. The clarified

13

extract was then evaporated and redissolved in 3 ml of
70% ethanol. One hundred pl of this extract were spotted
on thin layer plates coated with MN 300 Cellulose, and
the sugars (sucrose, glucose and fructose) separated by
running twice to a length of 15 cm. The solvent system
used was made up of t-butanol-methyl ethyl ketone-formic
acid-water (40:30:15:15). Pure analytical grade sugars
were used as standards and the spots containing the
sugars, corresponding to the respective standards were
scraped off and eluted with 4 ml of water. Test tubes
containing 1 ml of the sugar extract and 2 ml of water
were kept immersed in cold water and then 10 ml of 0.2%
Anthrone reagent were added and mixed well. After the
contents of the tubes were cooled for about 5 min, they
were transferred to a boiling water bath.and heated for
16 min. The contents of the tubes were then cooled and
the intensities of the bluish green color developed were
measured against a reagent blank in a Beckman DB-G Spec-
trophotometer at 620 nm and the amount of the various
sugars in 1 m1 of the extract were obtained by referring
to the respective standard curves. All samples were run
in triplicate. From the values obtained from the standard
curves, the percentages of sucrose, glucose and fructose

were calculated on a dry weight basis.

14

Extraction and Quantitative Determination
of*Star6h

 

 

The residue obtained from the extraction of sugars
was washed with ether, air-dried and then used for the
extraction of starch following the procedure of Pucher
23 21. (1948). To the residue in a centrifuge tube 4 ml
of water were added and the residue was mixed well after
which the tube with the contents was kept in a boiling
water bath for 15 min in order to gelatize the starch.
The contents of the tube were cooled down to about 22°C
by immersing in cold water. Then 3 ml of 72% perchloric
acid were added to the residue and the contents of the
tube were mixed well by vigorous shaking. After all the
samples were treated with perchloric acid, the starch
was extracted. For extracting the starch, the contents
of the tube were transferred to a mortar and 200 mg of
fine sand were added. The mixture was then ground with
a pestle for 5 min and the contents of the mortar trans-
ferred to a centrifuge tube. The mortar and pestle were
washed with 5 ml of water and the washing was also trans-
ferred to the centrifuge tube. After all the samples
were extracted the tubes along with the contents were
centrifuged and the extract filtered through a sintered
glass funnel into a flask. The residue was again extracted
following the same procedure as before except that the
residue was not heated with 4 ml of water. The extracts

were combined and mixed well and the volume was made up

15

to 40 ml with water. To precipitate the starch: to an
aliquot of 10 m1 of the starch extract were added 5 ml
of 20% sodium chloride solution and 2 ml of Iodine-
Potassium iodide, mixed well and the contents of the
tube were allowed to sit for 25 min. Then the contents
of the tube were centrifuged, the supernant liquid was
decanted and discarded, taking care to see no precipitate
was lost. The precipitate was washed by suspending it in
5 ml of alcoholic sodium chloride solution; after the
precipitate was thoroughly washed, the contents of the
centrifuge tubes were centrifuged and the wash solution
was decanted. To decompose the starch—iodine complex,
and in order to liberate the starch, 2 ml of 0.25 N.
alcoholic sodium hydroxide were added, the contents were
shaken gently till the bluish color of the precipitate
disappeared, then centrifuged and the alcoholic sodium
hydroxide was discarded. The liberated starch was then
washed with 5 ml of alcoholic sodium chloride solution,
centrifuged and then the wash solution was discarded.
The starch was dissolved in hot water and the volume was
made up to 40 ml with warm water. Quantitative determi-
nation of starch was done according to the procedure of
Hassid and Neufeld (1964). To an aliquot of 1 ml of
starch extract were added 10 ml of 0.2% Anthrone reagent
while the tubes were kept immersed in cold water, mixed

well by vigorous shaking and after cooling the contents

16

for about 5 min the tubes along with the contents were
heated for 16 min in a boiling water bath. The contents
of the tubes were then cooled, and the intensities of the
bluish green color developed were measured in a DB—G
Spectrophotometer at 620 nm against the reagent blank.
By referring the reading obtained from the spectrOphoto-
meter to a standard curve of glucose, the amount of glu-
cose in 1 ml of the extract could be calculated. By
multiplying the amount of glucose obtained by a factor,
0.9, the amount of starch in 1 ml was calculated; then
the percentage of starch was calculated on a dry weight
basis.

Quantitative Determination of
o a Protein

 

 

The quantitative determination of total protein
involves the digestion of 40 mg of lyophilized powder
by using the Technicon continuous digestor and a
digestion mixture made up of 3 g of Selenium dioxide,
900 ml of concentrated sulfuric acid and 20 ml of
perchloric acid diluted to 1000 ml with distilled water.
Ammonium sulfate produced reacts with sodium hydroxide,
thus giving ammonia. The quantitation of ammonia is
achieved by utilizing the Berthlot reaction in which the
formation of a blue indophenol complex occurs when
ammonia is reacted with sodiumphenate followed by the

addition of sodium hypochlorite; the intensity of blue

17

color produced is measured by a colorimeter. A standard
curve was obtained by running different amounts of wheat
seed samples and determined the total nitrogen in those
samples. The amount of total nitrogen in 40 mg of the
pine sample was obtained by referring to the standard
curve. From the value of the total nitrogen obtained
the total protein was calculated using a factor, 6.25,
then the percentage of total nitrogen was calculated on
a dry weight basis.

Statistical Analysis of the Chemical
Data and the Incidence

 

 

The incidence of the larvae of the European pine
shoot moth and the chemical data were statistically
analyzed using variance of analysis. Variation between
the trees was tested using the F test; but time could

not be tested as it cannot be repeated.

RESULTS AND DISCUSSION

Seasonal Changes in the Amounts of Total

Li ids in the Needle, Shogt and the
Bud of the RedRPine Tree

In the needle the amount of total lipids increased

 

 

from August to October; it increased from 4.6% in August
to 6.4% in October (Table 1). In the case of the shoot
the same trend was also observed, in the month of August
the amount of total lipids was 4.7% and it increased to
5.3% in October (Table 2). The amount of total lipids
in the bud increased from 10.0% in August to 12.3% in
September and then decreased to 10.4% in the month of
October (Table 3).
Seasonal Changes in the Amounts of Total
__Protein in the Needle, Shoot, and the
Bu 0 t e e Pine ree

The amount of total protein in the needle decreased
from 9.0% in August to 7.3% in October (Table 1). In the
case of the shoot it decreased from 4.3% in August to
3.2% in October (Table 2). Though the amount of total
protein in the bud decreased from 6.4% in August to 5.1%
in September, it increased to 5.3% in the month of

October (Table 3).

18

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22

Seasonal Changes in the Amounts of Starch
in the Needle, Shoot and the’Bud’
of the Red Pine Tree

 

 

 

In the month of August there was no measurable
amount of starch in the needle, shoot and the bud
(Tables 1, 2 and 3). This phenomenon of disappearance
of starch in autumn conifers has been reported by many
workers (Mer, 1876; Schluz, 1888; Lidfross, 1896; Lewis
and Tuttlt, 1923; and Guttenberg, 1927).

The amount of starch increased from 0% in August
to 0.59% in September and then decreased to 0.09% in
October (Table 1). In the case of the shoot there was
an increase in the amount of starch from 0% in August
to 0.78% in October (Table 2). In the bud it also
increased from 0% in August to 0.66% in October (Table 3).

Seasonal Changes in the Amounts of Sugars

in the Needle, Shoot and the Bud of
the Red Pine Tree

 

 

 

 

Sucrose

In the needle the amount of sucrose decreased
from 0.18% in August to 0.16% in October, in the needle
(Table 1). In the case of the shoot the amount of
sucrose was 0.16% in the month of August and it increased
to 0.31% in October (Table 2). In the bud the amount of
sucrose decreased from 0.24% in August to 0.13% in Sep-
tember and then it increased to 0.19% in the month of

October (Table 3).

23

Glucose

There was a progressive increase in the amount of
glucose in the needle from 0.25% in August to 0.40% in
October (Table 1). In the shoot, the amount of glucose
increased from 0.23% in August to 0.58% in September and
then decreased to 0.43% in October (Table 2). A steady
decrease in the amount of glucose was observed in the
bud; it decreased from 0.34% in August to 0.23% in

October (Table 3).

Fructose

The amount of fructose in the needle increased
from 0.35% in August to 0.49% in October (Table l); but
in the case of the shoot, though the amount of fructose
increased from 0.49% in August to 0.96% in September,
it decreased to 0.60% in the month of October (Table 2).
In the bud the same trend was also observed; the amount
of fructose increased from 0.27% in August to 0.40% in
September then it decreased to 0.25% in October (Table 3).

Seasonal Changes in the Incidence of

the Larvae of the European Pine
Shoot Moth, R. buoliana

 

 

 

The incidence of the larvae of the European pine
shoot moth decreased from August to October; it decreased

from a mean of 23.5 in August to 16.0 in October (Table 4).

Table 4.

24

The Incidence of the Larvae of the European
Pine Shoot Moth in the Red Pine Samples
Collected at Three Different Times

 

 

Sampling . a
Time Mean Inc1dence
August 23.5
September 16.5
October 16.0
Coefficient
of variation (%) 91.0

 

aEach mean is an average of 10 trees.

25

Discussion

 

Many workers have studied the influence of the
addition of the host tissue to the artificial diets on
the growth of the larvae. Beck (1953) found that the
addition of aqueous plant extracts to the artificial
diets results in the optimal growth of the larvae of

the European corn borer, Pyrausta nubilalis (an).

 

Ishii (1952) and Matsumoto (1954) enhanced the growth
of rice stem borer and oriental fruit moth, respectively,
by the addition of plant tissues to the diets. On the
contrary, Chawla and Harwood (1968) working on the
artificial diets for the larvae of the European pine
shoot moth, R. buoliana, observed that though the
European pine shoot moth feed only on pine tissue, the
freshly hatched larvae can be reared on diets lacking
pine tissue; they also observed that the addition of
pine tissue to the diets did not raise the percentage
of pupation. This is in contrast to the influence of
the plant extracts and plant tissues on the growth of
larvae of the European corn borer, rice stem borer,

and the oriental fruit moth, respectively. However,
the same phenomenon as in the European pine shoot moth
was also observed in the cotton bollworm; in which case
Vanderzant andeeiser (1956) found that addition of
cotton leaf meal to the diet did not stimulate the

growth of the larvae. In the case of the larvae of the

26

European pine shoot moth satisfactory growth was observed
when the other coniferous tissues were added; so Chawla
and Harwood (1968) suggested that pine tissue is not a

requisite for the larvae of the European pine shoot moth.

CON CLUS I ONS

In this investigation it was observed that the
amounts of total lipids, total protein, starch and the
sugars in the needle, shoot and the bud of the red pine
tree do change during the different seasons of the year.

In the case of total lipids, the amount of these
cellular substituents increased steadily from August to
October in the needle and in the shoot as against the
steady decrease in the incidence of the larvae of the
European pine shoot moth during the same period. In the
case of the bud, variation in the amount of total lipids
did not follow the variation in the incidence of the
larvae. This seems to indicate the absence of any
relation between the amount of total lipids and the
incidence of the larvae of the European pine shoot moth.
The coefficient of variation for the total lipids in the
needle was 20%, for the shoot 23.5% and for the bud 25.7%,
thus showing that the technique used for extraction and
estimation was good.

As far as the total protein is concerned, it was

noted that the variation in the amount of these cellular

27

28

substituents followed the same trend as that of the
incidence in the needle and the shoot; the amount of
total protein as well as the incidence decreased from
August to October. However, when the correlation coef-
ficient was worked out it was observed that there was no
significant statistical correlation between the amount
of the total protein and the incidence of the larvae of
the European pine shoot moth. The coefficient of vari-
ation for the total protein in the needle was 8.4%, for
the shoot 15.9% and for the bud 16.4%.

Starch was found to increase in the shoot and the
bud from August to October with a decreasing incidence
of the larvae during the same period, thus ruling out
the possibility of any relation between the amount of
starch and the incidence of the larvae of the European
pine shoot moth. The coefficient of variation values
for the starch in the needle, shoot and the bud were
high thus indicating the role of an extraneous factor
in the analytical technique used. The coefficient of
variation for the starch in the needle was 44.0%, for
the shoot 46.9% and for the bud 45.7%.

Of the three sugars considered, only glucose in
the bud followed the same decreasing pattern as that of
the incidence of the larvae of the European pine shoot
moth; but it was found that there was no statistically

significant correlation. Moreover, the values of the

29

coefficient of variation were also high for these sugars
in the needle, shoot and the bud thus indicating extraneous
error in the analytical technique.

In the present investigation though, it is diffi-
cult to come to a definite conclusion, due to the low
population level of the larvae of the European pine shoot
moth found in the field.during the period of this investi-
gation it can be suggested from the data obtained that
the changes in the amounts of total lipids, total protein,
starch and the sugars did not seem to have an influence
on the incidence of the larvae of the European pine shoot

moth.

Suggestions for Future Investigation

1. The seasonal changes in the amounts of other
cellular substituents of the red pine could be
studied and should be correlated to the incidence

of the larvae of the European pine shoot moth.

2. As time cannot be repeated, it would be better to
conduct the investigation in a few different
locations with similar soil and climatic con—
ditions so as to get better information about
the change in the incidence of the larvae during

the different seasons of the year.

LITERATURE CITED

LITERATURE CITED

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32

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33

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34

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