Some experiments on the freezing and hardening of the adults of the Colorado potato beetle,
Leptinotarsa decemlineata say
by Reginald Wilson Salt
A THESIS Submitted to the Graduate Committee in partial fulfillment of the requirements for the
Degree of Master of Science In Entomology
Montana State University
© Copyright by Reginald Wilson Salt (1933)
Abstract:
no abstract found in this volume SOMS EXPERIMENTS ON THE EEEEXm ASD HARDENING- 07 THE
AOTLTS 07 THE COLORADO POTATO BEETLE,
Lfrptlnotarga decemllneata Say.
by
REGINALD V. SALT
A THESIS
Submitted to the Graduate Committee la
p a r tia l fu lfillm en t of the requirements
fo r the Degree o f Master of Science
In Entomology a t Montana
State College
Approved;
a c .J ti^ 4 .
In Charge o f Major Soxfe
nlng Committee
Graduate Committee
Bozeman, Montana
June, 1933•
t-v a r?
TJBLS Of COKTUITS
ISTH0W8T1OK .................................. ............................................................ ...
Page
2
JBKmwLmmmT . . .................................. ... . . . ...................................... . 2
BSTISW Of LI TSRATOTG..............................................
. . . . . . . . .
3
APPARATUS ASfD ..................................................................................................... 21
BXPSBIMHHTS
LOW TaiPBBATOHS .......... ...............................................
2b
TBSBZIBG CUBTSS...................................... ... . . ........................... ...
29
KULTIPLS TBESZIBG....................... ' .................... 35
COESSLATIOH OT TBSSZIBG POINTS AND BATS OT COOLING . . . . . .
17
MULTIPLE BBBOUiroS ...............................................................................
*&
HAHEBIEG OT WTISOTABSA AJUOLTS . . . . . . . . . . . . .
^
. .
DISCUSSION OT TOB !ITERATORS AND SUBJECT , ........................... ...
5*
SUMMABT . . . . . . . .
64
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BIBLIOGRAPHY .................................................................................................. .
4454L
66
-2 SOMS EXP3EIMSUTS OH THE FHSS2IHG AED HABDSSIHO 0? TES
ATOLTS 0? TIS COLORADO POTATO BSSTLSt
Lentlnotarsa decemllneata Say.
IHTBOTOCTIOH
Only during the past few years have entomologists devoted much
time to the d e ta ils o f the freezing and hardening o f Insects, with one notable
exception.
Beamur (1736)* published an ac(fount o f h is experiments on the
freezing of In sects, using h is newly Invented thermometer.
The tran slatio n
o f th is account is included fo r reference in th is paper in order to dhow the
remarkable clearness, accuracy and value o f Bsamur1s experiments. Bis con­
clusions, two centuries old, are now considered much nearer the tru th than
many theories which have been proposed only recently.
The lite r a tu r e on the subject i s not voluminous, and only a email
p a rt of i t i s the re su lt o f fundamental research.
The p a ra lle l subject o f
the freezing and cold-hardening o f plants is much o ld er and more advanced.
The w rite r’s in te re s t in the subject developed from a consideration o f the
measurements of water-binding in in se c ts.
This subject appears to be founded
on so many unstable assumptions th at i t was thought necessary to go back and
tr y to find out ju s t shat physical and physiological processes are Involved
in the freezing and hardening o f in se cts.
The following work Is the re su lt
o f th is attempt to learn anything a t a l l about th is extremely complicated
subject.
The w rite r wishes to acknowledge h is gratitude to Dr. A. L. S trm d
♦-Reference i s made hy author and year to L iteratu re Cited.
-3 fo r the proposal o f the problem and fo r constant help and advice during I t s
development; end to Dr. W. McK. Martin, Prof. 0. A. Mall, Dr. R. M. Melaven,
Dr. A. J . M, Johnson, and P rof. W. D. Tallaan. fo r helpful criticism s end
assistance.
acrra# o? tlTBRATOEK
%e subject o f cold-hardiness and the freezing o f Insects has
developed from a th eo retical and p ractica l study o f Insect hibernation.
The
h isto ry o f these studies Ie presented by Peyne (1926) In a short a r tic le In
which she mention* such Important developments as Reanrar1S recording (1736)
o f the fa ta l temperature o f wood-boring larvae with Me newly Invented
thermometer; Blrby and Spence’ s work (ISIS) on the hibernation o f bees;
Vaudoner1s discovery (1827) th at some Insects displayed p e rio d ic ity end could
hibernate In the presence o f h i # temperature and abundant food; lo b ll l and
M ellonl’e (1831) use o f the thermocouple to determine the temperature o f
in sects; and Seudder1S discussion (1887) o f the subject in h ie "B u tterflies
o f Eastern Dnited S tates and Canada*. Sesmur1S contributions are eo Important
th at a tran slatio n I s included here,
"IiaaaBjLiTP
4k
% K,a&
(Translation o f pages I W-Iby, Vol.2:1736)
'
*Vhile the larvae are very email, in sp ite o f the various
layers which make up th e ir n ests, they remain quite exposed to the
rigors o f w inter. Por a f te r a l l , a neat attached to branches vfeich
no longer have leaves, and about which the a i r c irc u la te s fre e ly
on a l l sid es, o u # t not to be long in acquiring In i t s In te rio r
the same degree o f coldness of the a i r surrounding I t . These
extremely small larvae, then, which thereby see® to be v e ry d elica te, must therefore be strong enou# to re s is t the cold. I
-Uhave been curious to find out what degree they could r e s is t, .
and above a l l , what degree o f cold was capable o f k illin g them.
There was a t le a s t a small consolation, while winter makes us
feel a very severe cold, o f !mowing th at i t saves us from insects
which are m ultiplying too f a s t, and which would have defo liated
our tre e s in the spring and to the end o f the summer. But the
experiences th at I have had have taught me th at we have nothing
to hope in th is country fo r the destruction o f th is kind of
c a te r p illa r by the cold o f our worst w inters, since they are in
a sta te of re sis tin g a g reater cold than th a t of 1709.
*fe know how £o make ice in any season, Tby surrounding with
lee mixed with s a lt the th in vessel in which i s the water one
wltiies to freeze. Physicians know also th a t the degree o f cold
th a t one can produce by su itab le mixtures o f ice and c e rta in s a lts ,
i s much superior to the degree of cold o f water which Ie beginning
to freeze. The thermometer o f which I described the construction
in the Metolres o f the Acadeuy in 1730 ou^tit to go down as f a r as
the greatest cold o f 1709, about lU l / 4 degrees below the point
where the freezing of water begins (-1 7 .9 * 0 .). Toward the end of
February and during the f i r s t lays o f March, I placed a thermometer
in the middle of a mixture o f crushed ic e and se a-salt; the liq u id
o f the thermometer dropped to 15 degrees (-15.8*0), i . e . , about
3M of a degree below the point where the g reatest cold o f 170$
would have made i t drop. At the same time th a t I sank my
thermometer in to th is mixture o f s a lt and ic e , I Immersed there
a email g lass tube in which I had placed seven or eight o f our
small larvae; i t was closed a t the lower end, and i t s upper end
which was above the ic e , was open; I l e f t i t there nearly h a lf an
hour. Ihen I took tb& small larvae out o f the tube in Which they
had suffered excessive cold, they appeared dead. I warmed them
l i t t l e by l i t t l e , beginning Ty placing them in ordinary ice; in a
quarter o f an hour they were in such a s ta te th at I could see th at
they were aliv e: they s tirr e d and walked.
"The next day I put them to a s t i l l more rigorous te s t; I
surrounded the glass tube in which I had put them with a mixture
o f ice and todks-salt which made the liq u id In the thermometer drop
to more than 17 degrees below freezing (-2 1 .2 5 % .). In th is
second t r i a l , the larvae had then to r e s is t a degree o f cold
nearly three degrees g reater than th a t o f 1709; i t k ille d none
o f them. The sudden passage o f a i r su ffic ie n tly tempered ( fo r
when I ca rrie d on these experiments the liq u id o f the thermometer
was about e i # t o r nine degrees above freezing (10-11*0.), the
passage, I eay, o f air,tem pered by a i r o f such excessive coldness,
should be fo r them a much more rigorous te s t than th a t o f the
same coldness o f longer duration, which would become such only
by B a c e e e s lv e eceoaralatlone made during a great number o f days,
aa happen# In winter. Also I h a v e made these la rrm sustain
a cold o f 19 degrees without having them perish.
"L ister has already remarked th a t c a te rp illa rs are In a sta te
o f resistance to very great cold; he reports that he has found
them s t i f f with ic e , and eo rig id th a t in dropping them in a
glass they made a noise lik e th at which would be made by a small
stone, o r a small stic k which i s dropped; th at la th is s ta te ,
however, they were a liv e , and th at they had given Incontestable
proof when he had warned them, that they had walked. This was
a great astonishment; I f aa insect whose blood, o f Which a l l the
liq u id s had been fro sen, easts back to l i f e , th is was a true
resurrection; fo r since a l l circ u latio n , a l l movement of the
liq u id s are stopped, the animal I s a dead animal; a t le a s t, we
have no oth er conception o f the sta te o f death. I believed It
o u # t to be proved i f the c a te rp illa rs Whose liq u id s have
actu ally been frozen, come back to l i f e , as I t were, Our
common c a te rp illa rs are not the only ones on which I have made
these te s ts . I wished to know i f those o f o th er species had the
a b ility to r e s is t such a great cold. One o f those Wiose resistance
against cold I wished to ts e t was the Fine c a te rp illa r, of which
we sh a ll speak soon; and o f those Which were hatched and raised
on th is species o f tree in the v ic in ity o f Bordeaux. I put
several of them in a g lass tube and'made them su ffer, lik e the
common ones, a cold o f 15 degrees below freezing (-18.8*0.). Ihen
I took them out o f the tube they were s t i f f , hard as a stone, o r
Ilbw harder Ice. I broke several o f them as one breaks a so ft
stone; th e ir Whole Inside was completely fro sen; also I re-heated
those which I l e f t Whole; they did not com# back to l i f e ; they were
too well.dead.
"A degree o f cold much le ss than th at Which a ffe c ts the common
ones I s s u ffic ie n t, therefore, to M il those o f the Fine. In other
experiments, a degree o f 10-11 degrees o f cold (-12.5*0 -IU iO.) was
su ffic ie n t fo r the l a t t e r ones. I have taken from the tube Which
had attain ed 8 o r $ degrees o f cold (-10 to -1 2 % .), some Which
were already quite hard, which upon fa llin g Into a porcelain cup,
made quite a noise; and Wileh a f te r having been held fo r some time In
a temperate atmosphere, gave signs o f l i f e , and soon regained th e ir
fe w e r v ita lit y . But these larvae had not heen frozen completely.
Al th o u # they had a c e rta in amount o f rig id ity When taken from the
tube, they s t i l l had a degree o f e la s tic ity . Places pressed pore
way under the finger, Wiich did not happen in the ease o f those
Whldh were completely fro sen, and which died. FeAape even, that
the l i t t l e stiffn e s s Wileh they had, only came from vapor Wileh
was frozen around them; a vapor similar to th a t Wiieh freezes on
the outside surface o f the vessel Wilch contains the mixture o f
s u it and Ice.
•th a t 1® ce rta in , 1® th at I have never seen larvae Which
were re a lly frozen, those liquid® had turned to ic e , Shich were,
not k ille d . S tartin g when a l l movement o f th e ir liq u id s ceased,
they were p erfectly dead c a te r p illa r s , ju s t as any other animal
la a sim ilar case would be a dead animal. But there remain
always these pecu liar fa c ts , th at in sp ite o f the small amount
o f heat in the body o f c e rta in species o f c a te rp illa rs , however
delicate they o i ^ t seem, because they aye extremely email, the
Hquida Which f i l l th e ir bodies cannot be fro sen by a degree o f
cold considerably more than th a t o f our hardest w inters. That
there are specie# o f c a te rp illa rs much la rg e r, end in appearance
much stronger. Whose liq u id s can be frozen by a degree o f cold­
ness much le s s than th at which does not a ffe c t the liq u id s o f
others. The Mnd o f blood, the liq u id s Whitih circ u late In the
vessels of different species o f c a te rp illa rs , are therefore in
comparison to others as with alcohol; o r a very strong brandy
Compared to a very weak brandy. The l a t t e r w ill be hardened,
reduced to ic e , by a degree o f cold much le ss than m other
degree o f cold, under which a very strong brandy w ill a l l remain
as a liq u id .
" I t i s known that movement of water Is an obstacle to freez­
ing; quiet water, th a t o f a ditch o r pond, freezes. While the
water of a riv e r remains a liq u id ; the more rapid the Current,
the le ss chance o f so lid ify in g . I f the c irc u latio n o f the liq u id s
o f our small consaon larvae were more rapid than the circu latio n
o f the pine c a te rp illa rs , from th at alone, i t mast take more cold
to f ix the f i r s t ones in th e ir canals than to f ix the second ones
in th e irs; but th is consideration has l i t t l e o r no p a rt in the
effect we are considering. I cut o ff the head o f three o f our
n a i l c a te rp illa rs ; I put them in a glass tube with others of
th e ir kind Which were a liv e and healthy; I lo n r e d the tube into
a M ature o f ic e and s a lt which made the liq u id o f the thermometer
drop to 15 degrees below freezing (-1818^0.), When I took the
c a te rp illa rs out o f the tube, those Which had had th e ir heads cut
o ff were p lia b le and so ft lik e the others; th e ir liq u id s had not
been frozen. PreaAshtie I t follows that these liq u id s do not
need to be in the movement o f a rapid circ u la tio n in o rd er to
conserve th e ir f lu id ity against a degree o f cold o f 15 degrees
belew freezing (-18.8*0.). I e are not surprised th at o f the
inflammable o r spirituous liq u id s, and of the liquid# charged
w ith ,sa lts A ic h r e s is t very great cold without freezing, we have
hundreds and hundreds o f examples; but i t o u # t to appear to us very
peculiar th a t a liq u id Which i s not a t a l l inflammable, A itih seems
to us very In sip id and quite watery, th at such a l i q uid, I say, a#
the blood o f some species o f c a te rp illa rs , can preserve i t s
flu id ity In sp ite of great cold* That liq u id la not, then, so
simple th a t we judge i t by the same standards we usually use to
discover the nature o f liq u id s.
"The blood o f large animals, b ird s, quadrupeds, and ourselves,
easily coagulates; besides, they are more e a sily frozen than the
blood o f in se cts. The blood o f a pigeon, Which was made to flow
warn Into a glass tube, was reduced to hard ic e by a degree o f
cold of 7 o r 8 degrees below freezing (-9 to *10% .), and could
have been frozen by a le s s cold. The blood o f a lamb sustained
three degrees o f cold (-3.75*3.) without freezing, but a cold of .
5 degrees (-6.2*0.) converted i t Into ic e. Large animals have in
th e ir bodies a heat and a p rin cip le of beat which i s not found in
those o f In sects. Mg animals, then, have no need o f having a
blood which freezes as d if f ic u ltly as th at o f Insects.
't
"Whoever made Insects seems also to have co n stitu ted th e ir
blood d iffe re n tly according as they are exposed to endure g reater
o r le ss cold. We have seen, besides, th at numbers o f species of
in se cts, a f te r having liv e d in the form o f c a te rp illa rs , pass the
Whole w inter In the fora o f c h iy sa lld if, and th at there are
chrysalid s' which during th is harsh season are attached to w alls,
eaves o f houses, and leaves o f trees; and Which are 'awaraed*
there, l . e . , they are not covered by a cocoon, be I t o f s ilk o r
some other m aterial. Such i s the ch ry salis o f the most handsome
o f the cabbage c a te r p illa r s , and such are nenbere o f other
chrysalids o f the kind Which have the Industry to suspend them­
selves by means o f a band o f s ilk threads. I have subjected
several o f these chrysalids to very great degree# o f cold, cold
o f more than 15 to 16 degrees below freezing (-18 to -2 0 % .),
without th e ir freezing. We know th at o th er chrysalids pass the
winter down In the ground; th ere, they are not exposed to as
great a cold as they are In any p art of the a ir . I have subjected
to a cold o f 7 o r 8 degrees below freezing (-9 to -1 0 % .) several
o f those Wtilch stay underground; i t was su ffic ie n t to make then
perish. Thus the Insects Which remain exposed to great cold are
in & positio n to withstand i t . Hiose which are more sen sitiv e
to the Impressions of cold act as I f they foresaw What would take
place during the winter on the surface o f the ground, and Which
they could not r e s is t. I say th at they act as th o u # they foresaw,
because i t i s not the approach o f w inter o r the actual cold s&ich
causes than to en ter the ground; we have seen th at there are
c a te rp illa rs which burrow in Ju ly and August, and s t i l l others in
the early spring. A short time a f te r having entered the ground,
they transform in to chrysalids; and I t I s not u n til the following
year th a t the b u tte rfly leaves I t s ch ry salis."
;
TJvarov (1931) gives an excellent review o f the subject, In Which Ie
Included a tab le lis tin g the effects o f extreme low temperatures on about
th ir ty Insects o f various orders and stages, with the references.
In h is
discussion of the theories o f cold resistance In Insects, Uvarov s ta r ts with
Beanmr1S explanation In 1736 o f the resistance o f an Insect to apparently
complete freezing.
Bem ir explained that While an Insect may appear to be
completely frozen, there remain some flu id s in the tody Which freeze a t a
much lower temperature, and th at death occurs only When a l l o f the flu id s
are frozen.
This viewpoint, considerably older than th at o f EachnaetJew, i s
nevertheless much more accurate.
Although BachmetJew (1901) made a few technical erro rs In h is work
which le d to fa lse conclusions, h ie work Ie considered monumental. He based
h is theozy o f cold resistance In Insects on observations o f th e ir body
temperature by means o f the therm oelectric method. Hle observations showed
th a t an Insect can be cooled gradually to a low " c ritic a l po in t", about
-1 0 eG. At th is p o in t, Which I s ca lle d the under-coolIng point by presentday workers, ice begins to fora in the tissu es and the temperature rise s
due to the lib e ra tio n o f heat o f fusion.
Hie point to Which the temperature
ris e s is not designated by any p a rtic u la r name by Bachmetjew, but I t I s , o f
course, below 0*0.
"rebound point*.
This point w ill be referred to In th is paper as the
I t has been erroneously referred to ae the "freezing
point" by several authors, but th is nomenclature w ill la te r be shown to be
wrong.
hard.
Further gradual cooling causes the Insect to become frozen quite
In th is la te n t o r anablotlc sta te no metabolic processes are possible
I
but the Insect can be reanimated by warning.
I t Is only When fhe Insect I s
cooled to a c e rta in " fa ta l point* th a t I t I s k ille d , and Bachmetjew places
-9 th le point a t the same temperature level as the " c ritic a l point".
The
theory I s based on the purely physical conception o f the supercooling of
flu id s, hut i t has no hearing on the free sing o f in sects, a fa c t which has
been demonstrated not only by l a t e r workers, but by much of Baehraetjew*s
own data.
A graphical representation of BachmetJew1s conception i s given
In Fig. I .
He Considered that the " c ritic a l point" depended on several con- •
dltlons!
( I ) , the velocity o f cooling; (2 ), the development and sex o f the
specimen; (3 ), the n u tritio n a l s ta te of the in sect; (U) the rep etitio n o f
cooling; (5 ), the time o f exposure, and (6 ), the "sap co efficien t".
Bachmetjew1s theory was severely c r itic iz e d soon a f te r i t s
appearance by Kodls (1902), according to whom the super-cooling o f body
flu id s has nothing to do with the freezing o f water in the protoplasm, with
which the fa ta l e ffe c t i t connected. Unfortunately th is c ritic ism escaped
notice and was ignored by BachmetJew in l a t e r works in which he repeated and
developed h is views.
Maximov (1913) offered the following serious c riticism o f Bachmetjew1S
theory.
By determining the quantity o f ice formed in the pupae o f Celerto
euphorbia# L .. BachraetJew found th a t a l l the flu id s in them froze completely
a t -U. 5°3. while the c r iti c a l point was -IO 0C.
the theory.
These figures seen to support
However, the sp ecific heat o f the frozen pupae within the lim its
-6 .7 to -1 6.3 was found by BachraetJew to be 0.$17, very l i t t l e lower than th a t
o f the body flu id s, (1 .01).
Since the sp ecific heat o f ice i s only h a lf th a t
o f water, Maximov concluded th at freezing was got complete a t -U .5°C.
The
same conclusion I s reached by considering the fact th at o f the U$ s a lt content
-
10 -
Br DcakK
7$E.T-mancnt Iitutt j h u f fo f
Ternj^orahy
a Hu
/V = Uccjinnincj oj- Keak sh j^ o r
«5u ^>rd - Oj>K m a I ZLone
k.2 *
O j> h m v r n
Su b -oj>hmal zone
= 3 e c |in n i'n ( j
c o l d jKjjx**-
~Fej-riporary c o ld 3Kj ^or-
-su^ g lcd
1—
- ;
■
"Temporary cold 3hujx>r
-n »
Fig. I .
Zones of v i t a li t y and death of an in se c t.
(Re-drawn a fte r Bachmetjew1 IQOJ)
1
-Ilof Insect blood, about 80$ Is sodium chloride, which has a e u tectic point
o f about -22% .
Thus, even I f no other s a lts were present, the body flu id s
could not possibly freeze completely a t -U.5*C.
Eecent experiments by
Sacharov (1928, 1930) show th a t I t Is Impossible to freeze completely the
body fluids o f ce rtain Insects even a t -21.2% .
Payne (192?) found th at com­
p le te freezing occurs only In the neighborhood o f -bO* o r lower.
TJvarov points out th at Bachmetjew's p rincipal mistake la y In M s re­
garding the "body flu id s of an Insect as a homogeneous liq u id which follows the
re la tiv e ly simple physical laws o f supercooling and freezing.
This viewpoint
I s obviously Incorrect since apart from dissolved e le c tro ly tic substances,
there are Insoluble fa te , p ro tein s, end other colloids which are bound to
» -
'
a ffe c t the physical properties o f the flu id s.
Bachmetjew'e theories, however,
though superseded, are widely known, and are s t i l l repeated by many w riters.
More recent work along these lin e s has c la r ifie d the subject In
many d e ta ils, althou^i the actual physiological phenomena, Shlch a f te r a l l
form the true b asis of the problem, are as yet very vague.
P erio d icity la
cold resistance has been one o f the main points of attack , and In th is connect­
ion more work has been done with p lan ts than with !a s s e ts .
Chandler (1913)
and Bosa (1921), showed th at c e rta in p lan ts ex h ib it p e rio d ic ity gad th a t cold
hardiness can be Induced in them. Harvey (1918) Induced hardiness In plants
by exposing them to moderately low temperatures.
Gueylard and P o rtle r (1916)
were the f i r s t to point out a seasonal v ariatio n In the cold resistance o f
In sects.
They observed th at larvae of Cossas Coewus I . survived repeated
freezing a t -20* In w inter, but larvae o f the same species taken la spring
succumbed a t -I? * .
Knight (1Q22) supercooled P e rlllu e M eculatus in w inter
-1 2 .
to -17% and la one case even to -26* without freezing, yet In MastSh and
l a t e r a temperature o f -10* caused freezing and death.
Bodine (192L, 1923)
found that the to ta l water content of ce rtain grasshoppers decreased during
hibernation and was la te r restored to normal proportions.
Probably the most extensive studies along th is lin e are these of
Payne.
In one o f h e r f i r s t works (1926a) she found that In the ease o f oak
borer larvae, which are normally subjected to extremes o f temperatures, freezing
points vary with individuals and seasons.
She found an excellent correlation
o f freezing and undercooling p o in ts with average monthly temperatures.
In the
f a l l , the undercooling point f a lls ahead o f the outside temperature, a c tin g
as a facto r o f safety .
In the spring, the hardiness i s lo s t with risin g
temperatures and i t i s a t th is time th at a sudden cold snap I s most fa ta l.
Following the idea o f previous authors as already discussed, Payne attempted
to Induce cold hardiness a r t i f i c i a l l y by holding non-hardy in se cts a t a
moderately low temperature, and to break up hardiness ty holding hardy Insects
a t a moderately h l£ i temperature.
The attempt was very successful.
She also
Induced hardiness ty dehydration, showing the effect of free-w ater content
on hardiness.
Payne measured hardiness in these experiments In terms of
undercooling and freezing points; low undercooling and free sing points
indicated a h i# i cold resistan ce, and vice versa,
("low* and "high* are
used In th is paper in a geometrical sense, not arithm etical; l . e . the sign
is considered.)
The points were determined by means o f a thermocouple and
a pyrovolter, but the author does not s ta te the method o f freezing.
J t may
be pointed out here th at Payne1S use of the term "freezing point* i s not
exactly co rre c t, the true freezing point being slig h tly higher than the
-13"retioxmd point11, as w ill be pointed ont la te r .
Payne (1926b) soon afterward* selected fo r comparison thrw
ecological groi^si ( l) the oak borers, normally exposed to extremes o f temper,
atnre; (2) the aquatic in sects, never exposed to temperatures below G*C,, and
(3) stored product Insects, representing, supposedly, a tro p ica l o r a sub.
tro p ical group.
In these experiments the oak-borer larvae showed marked
p erio d ic ity , as already stated .
!Rie aquatic in sects showed no p e rio d ic ity ,
nor was there any sig n ifican t difference among Individuals, species, orders,
o r stages of development.
The mean undercooling o f a l l the specimens used,
representing lU genera in U orders, was 1 .5 2 * t0 .3 e, and th& mean freezing
point 0.57"+ 0.03*.
In the case o f the th ird group, the stored product p e sts, Payne
found no p erio d ic ity , but found more v ariatio n in undercooling and freezing
points than In the aquatic group.
Bobinson (1926) working on the granary
weevil, Sltonihllug CTanarius. and the rice weevil, Sitonhllua orsrsa. trie d
to harden them by a moderate lowering of the temperature over a long period
o f time, but the re su lt was death.
!Rie n atu ral conclusion o f these workers was th at those in sects which
are normally subjected to temperature extremes acquire a cold resist#**# In
the f a l l and lose i t In the spring. While those which are never subjected to
extremes are incapable o f adapting themselves when a r t i f i c i a l l y exposed, even
when th is exposure Is made gradual.
TW hardening of ce rtain lneeete in the f a l l , o r when placed
a r t i f i c i a l l y a t moderately low temperatures, as evidenced by a drop in th e ir
undercooling and freezing p o in ts, le d Boblnson to apply to In sects the *bound-
water" theory already developed hy Hewton and Oortner fo r p la n ts.
According
to th is theory, m der ce rtain stim u li, ( la th is case low temperatures), the
hydrophyllc co llo id s present In the Insect body are capable o f adsorbing o r
•binding* water.
% e water, on being "bound*, loees most o f the typical
physical properties of water.
Ib r example, the freezing point o f bound water
Is greatly depressed, and indeed i t is on the assumption th at a t -20% . none
o f the bound water but f ill o f the free water Is frozen, th a t Ibblneon1S (1931a)
method of determining the bound water content o f a system i s based.
Bie method
as applied to an In sec t, I s b rie fly as follows: The Insect, o f known w el#it.
I s frozen a t a constant temperature o f -20% . fo r several hours and then
transferred quickly to a calorim eter, where & determination Ie made of the
number o f calo ries required to melt the ice formed within the tissu e s,
th is
determination Ie based on the fa c t th a t to melt on# gram o f lee without raisin g
i t s temperature requires 80 c a lo rie s o f h eat.
By calcu latio n , the amount o f
free water p er gram o f so lid i s determined. The fin a l step is to dry the
m aterial to constant weight, (100%. o r in a vacuum even a t 60-65*), as a
measure of to ta l water content.
Qie difference between the to ta l and free
water values Is a measure o f the bound water in the specimen.
Bie theory o f water binding i s o f great Importance in w inter harden­
ing.
The adsorption o f the water occurs on the surface o f the co llo id al
p a r tic le s .
Because of th e ir email else, (O.l-O.OOLu), these p a rtic le s present
a re la tiv e ly larg e surface.
Under a fa llin g temperature, the p a rtic le s a ttr a c t
water and adsorb i t as *films* around themselves, the Helbholts "double layer*.
The thickness of the film may increase u n til i t i s g reater than the diameter
o f the p a rtic le .
Bte water on the Inner lay ers i s held by Inconceivably blg&
-15pressures due to surface energy, often running Into thousands o f atmospheres.
Many of the physical properties of th is water are changed In the process,
e.g . I t w ill not conduct e le c tric ity ; I t w ill not dissolve such substances
as sugars; I t can be considerably compressed; and i t s freezing p o in t le g reatly
lowered.
With fa llin g temperatures of autumn and adsorption o f water by the
colloids In the insect tissu e . I t i s obvious th a t the remaining aqueous
solution w ill be more concentrated and the freezing point w ill drop. A c e rta in
degree o f protection against cold weather Is thereby established.
Hewton and Gortoer (1922), working with hardy v a rie tie s of w inter
^aeat, established the fact th a t fo r plants there is a d irec t co rrelatio n
between w inter hardiness and percent o f bound water.
Boblnson (1927), was
the f i r s t to show that the same h eld fo r ce rtain In sects. He te sted the hardy
&
the moderately hardy
* uramethea. end the non-hardy
granary weevil, SltooM lus granarlus. In arriv in g a t the same conclusions as
Hewton and Gortoer.
He hardened the f i r s t two species both n atu rally , out­
doors, and a r t i f i c i a l l y in a constant temperature cabinet held a t -1 3 % .,
Ju st above th e ir freezing temperature.
In a non-hardy condition In which the
f i r s t two species started , only 9-10# o f the water was bound.
during the experiment 6e* *12-52#.
This Increased
I t Ie In te re stin g to note th a t the to ta l
water content remained the same.
-
BoMneon stresses the Importance o f the p er cent o f water botmd
before equilibrium Ie reached.
I f am Insect In a non-hardy rummer condition
Ie placed In a refrig e ra tin g cabinet representing winter conditions, i t is
exposed to an unnaturally abrupt -change and may be k ille d before i t can begin
to protect i t s e l f .
He suggests, therefore, a study o f ( I ) watejwMnding
capacity, to show the percentage o f water adsorbed ahd how quickly; (2) waterholding capacity, to Whow the a b ility to re ta in bound water under conditions
o f rapid rise s in temperature, which Ie especially Important in spring
m ortality.
I t has ju s t been sta te d th a t Eoblneon (1927) in M s experimental
hardening o f Telea nolybbenme and Callcsamla oromethea found th at the to ta l
water content remained the same. Payne (1926b) s ta te s th a t the most pronounced
feature of hardening was the low mole tu re content.
The oak-borer larvae in
fu lly hardened condition had a low moisture content, but in a non-hardy condition
they had a high moisture content.
content.
The to ta l water content o f Smchroa n m e tata varied from 31*1$ In
Pebruary to
to 73*53.
P erio d icity was thus exhibited In moisture
In August.
That o f Dandreidss canadensis varied from 57.1#
The larvae were haked fo r four hours a t $0%.
The adequacy o f th is
method o f desiccation w ill he questioned.
In the same paper Payne describes a m ultiple freezing experiment.
Repeated freezing o f the same in sect o r tissu e exhibited no h y ste re sis, and
the rebound and undercooling points remained the same.
Samples o f blood from
the aortas showed d efin ite c ry s ta ls , while transparent larvae were also seen
to have cry sta ls within at the time the freezing point was recorded.. The
process o f freezing In th is group was Interpreted as c ry s ta llo ld a l, the f i r s t
o r primary freezing point being th at o f the blood.
Payne found fa rth e r th a t the hardened oak-borer larvae survived
freezing, and th at on lowering the temperature s t i l l mere, second undercooling
and freezing po in ts were recorded.
The secondary freezing point occurred
n«ar
fo r the oak-horer gro-up and was always f a ta l.
The tissu e ft©#*.
!»€ a t th is temperature was not d efin itely Iso lated , W t the nervous tissu e
and fa t were suspected, Payne went so fa r as to sta te th at
Insects are k ille d when the primary freezing point Is reached, while fu lly
hardened Insects are net k ille d u n til the secondary freezing occurs.
Experiments were run Tty the same author to determine the relationship between
the freezing point and the survival o f Insects When exposed to low temperatures
fo r as long as twelve hours.
I t was found th at In sects with h i # freezing
points were never able to withstand long exposures to low temperatures. However,
in se cts with low freezing p o in ts could be k ille d by long exposure When a short
exposure would not be fa ta l.
This author also dissected out the central
nervous systems of 90 oak-borer larvae and froze them.
point recorded w as-hS•; the rebound point - h $ \
The undercooling
,
Apart from the seasonal v aria tio n In cold hardiness, there e x ist
variatio n s during the individual development o f an Insect.
Ludwig (192S)
found considerable difference in the a b ility to withstand low temperatures
among the various in sta rs o f Japanese beetle larv ae.
Their hardiness In­
creased a t f i r s t , then decreased to a minimum which occurred Just after the
f i r s t molt. Hardiness Increased considerably during the second and th ird
in s ta rs in which stages the w inter Is usually passed.
Payne (1927a) c a lls atten tio n to the fa c t that two facto rs o f heat
eaorgy are involved in the study o f cold hardiness: ( I) the Quantity factors
(2) the In ten sity Factor.
Cold hardiness may thus be e ith e r the a b ility to
withstand long periods o f moderately low temperature, (the quantity fa c to r).
-IS o r the a b ility to w lthatm i short periods o f Intensely low temperatures,
(the Intensity fa c to r).
In h er experimente, aquatic In sects, considered
highly specialised along the quantity facto r, endured long periods a t 0*,
hut none survived freezing, even though the freezing point was only about
I e lower.
Another group th at may be specialized along the quantity fa c to r
i s that group o f so il lneecte normally liv in g below the fro s t lin e .
the stored product Insects cannot withstand doraacy.
Most o f
TM oak-borers develop
a b ility to survive doraacy In September and October, but a t th a t time are
• t i l l non-hardy to the in te n sity facto r and are k ille d by freezing.
Upon
fu rth e r low temperature exposure, o r else dehydration, they become hardy to
the In ten sity facto r.
In considering cold hardiness to the in te n sity fa c to r only, Payne
*
(1927c) brings out the Importance o f the water content.
She fnr m
d 4
i■
«ad g ta c rlsla y lrg ln lc a to be self-dehydrating In the f a l l . Whereas P o n lllla
japonlca did not exhibit th is phenomemn.
le s t a ll, o f th e ir free water.
She considers th a t the f i r s t two species
Boblnson (1927) found th at In hardening Telea
Polyphemus and G alleseala nromethea. the to ta l water content remained the
same.
I t therefore appears th at ce rtain Insects are dehydrated In the
hardening process while others are ro t,
Payne reports a drop In to ta l water
content from August to December o f 5# for P on lllla laoonlaa. 1]# A r Dsmdraldea
Bk* for Synchroa wunctata. and 20# for
mfulnm. Tha oak
borars are e e lf - dehydrating, she sta te s, hut never Ioee a l l o f th e ir fre e water.
Payne In the same paper p lo tte d blood conductivity readings against
survival tmqysTeture# !Toir P o p illla japonloa. Pl#*Tl#ia vlrelnlca aaad Demdrais^
-19GanatSmwl*.. and found an excellent co rrelatio n .
I t Ie extremely unfortunate
th a t she does not sta te her method of determining survival beyond the f@@%
th a t the in te n sity fa c to r only was considered.
The method o f free sing end
thawing and o f handling m e t c e rta in ly affect the re su lts.
In a l a t e r paper, Payne (1928) discusses the various facto rs a ffe c t­
ing the hardiness of the Japanese beetle to the In te n sity facto r.
They are
( I ) dehydration; (2) disease; (3) n u tritio n a l s ta te , and (U) temperature a t
which kept.
In her experiments, dehydration was accompanied by a high death
ra te so that the process was considered selectiv e.
hardy.
Hte survivors were cold-
larvae kept a t 20%. and 100$ rela tiv e humidity fo r one month lo s t
h a lf th e ir weight and were a l l k ille d When subjected to the free sing process.
Kept a t 10%, and 100$ re la tiv e humidity fo r one month, they also lo s t h a lf
th e ir weight, but 25$ survived freezing.
The freezing p oints were high.
Some very in te re stin g points are brought out In the earn# paper in
regard to starvation and cold hardiness.
"In general", s ta te s Payne, "early
stages o f starvation are accompanied hy an Increase in cold hardiness, l a t e r
stages ty a decrease.
The point o f decrease of hardiness comes when the
digestive tra c t cle a rs,
fre sh ly molted larvae cannot withstand freezing
w t l l they have eaten.
Pre-pupae with c le a r digestive tr a c ts are not cold
hardy*.
This refers to both the quantity and In ten sity fa c to rs.
Larvae o f the Japanese beetle frequently exuded a flu id upon thaw­
ing.
When th is gave a te s t fo r amino acids and p ro tein s, the larvae always
died; i f not, they usually survived, and the exudate was considered to he only
water.
Occasional blackening of larvae a f te r freezing was th o o # t to be due
to oxidative enzymes lib e ra te d by a change In c e ll perm eability.
-20No change in pH o r re Bplratory quotient mas associated with hardiness
to e ith e r facto r, hat the respiratory rate in hardened larvae was much lamer
than in non-hardy ones,
Also, as the length o f time the larvae mere kept a t
10%. increased, the percent survival a f te r f reelin g decreased.
Fayse con­
cluded th at the two types o f hardiness are inversely rela ted a f te r a c e rta in
point has been reached.
I t cannot he in terp reted as a lo se o f v ita lity , since
larvae kept under such conditions can complete th e ir development with a normal
death rate i f removed to room temperature.
In fa c t, the development i s
accelerated ty th is doraacy.
Sacharov (1930) studied changes in the f reelin g point o f c a te rp illa rs
of the Brown-tail moth, Hramia sbaeorzhaea Don., taken stra ig h t from Mhen- •
nation, and again a f te r feeding fo r three to four days in a mama room.
Feed­
ing resulted in an Increase in to ta l mater content, Shlle the quantity o f f a t
decreased.
o f feeding.
The cold hardiness mat greatly reduced hy the three to four days
Comparing hardy wood-holing larvae o f Plaaionotue arcuatne L.
and the non-hardy Mney bee, t e l l a e lllf e r a . he found th a t the hardy in sect
contained only 5 ^ o f w ater, h at lU.^S o f f a t , Shereaa the non-hardy insect
contained
o f mater and only 2.7$ of f a t .
Warav (1931) considers th at
these data prove convincingly th at cold heel stance depends on the balance of
"freesable* mater and o f f a t in the Insect body.
.21.
APPARATUS ARD M3TH0D
The apparatus used In th is work was quite often changed In minor
respects to su it the Individual experiment.
The changes made were In the
free sing mechanism, while the system o f recording temperatures was l e f t un­
changed.
The general set-up is shown in H g. 2.
Temperatures were read hy
means o f thermocouples and a sen sitiv e galvanometer.
A description o f the
galvanometer I s as follows: D1Arsonval Type, Leeds and Northrop, Type B,
( h i s e n s i t i v i t y ) ; Sensitiveness 0.005 micro-ampere* per scale division;
Period 5 seconds; Resistance IjO ohms; C ritic a l external damping resistance
300 ohms.
Further resistance of ho and 100 ohms were connected In series with
»
1
the galvanometer, giving two temperature scales.
The scale consisted o f a
6 Inch x I Inch hoard, l6 fe e t long, Txmt into the form o f an arc of a c irc le
whose center was represented by the galvanometer m irror.
3 fe e t.
The radius ~ was
A 3-v o lt galvanometer lig h t bulb was mounted In fro n t o f and s lig h tly
below the galvanometer m irror, so th a t i t s re fle c tio n was thrown by the m irror
onto to the scale.
The K a le was calib rated from+ 1*0. to -1 3 % ., represent-
ing an external resistance o f ho ohms, smd from +2%. to •2 9 % ., representing
a resistance o f 100 ohms. The degrees were marked o ff by means o f black
adhesive tape.
The ca lib ratio n was effected by placing a thermocouple In
contact with the bulb of Bureau o f Standards thermometer graduated in tenths
o f a degree, wrapping with adhesive tape, and placing In about 10 to 15 cc. o f
d is tille d r a te r in a v ia l.
The v ia l was lowered into a s a lt and lee solution
and cooled very slowly. In order to eliminate the la g o f the thermometer
mercury behind the thermocouple.
The ca lib ratio n s were checked several times
-
22-
CONSTANTAN
C O PPE D
Fig. 2.
v
Diagram o f apparatus used. A, Galvanometer;
B, Galvanometer lig h t bulb; C, Scale, calib rated
as shown in Degrees Centigrade; D, Resistances o f
to and 100 Ohms; E, Dewar fla sk known junctions,
containing ic e and water; F, Thermocouple used to
record in sect temperature; J, Thermocouple used
to record environmental temperature; K , Calcium
chloride bath; L, Refrigeration c o ils .
-23durlng the course of the work.
Since the "known" o r "cold" junction was a t
OeC., obtained by a mixture o f water and Ice In a Dewar fla sk , the 0*6. mark
wws the same on both scales.
I t w ill be seen th a t with such a larg e scale as th at presented by a
16 foot board, and with the se n sitiv e but well-damped galvanometer used, g reat
aacumey was obtainable.
Bot merely that* but In fyeealag am Insect* a am*,
p is te time-temperature record was thrown on the scale, each temperature reaction
being exactly duplicated on the eeale as i t occurred.
Dslng a s top watch, the
w riter was able to record with ease, the temperature accurate to 0.1* and the
time accurate to one second, and take readings every few seconds.
Thus an
accurate time temperature curve could be graphed fo r subsequent analysis and
comparison.
In some cases temperatures were read accurately to 0.0$*, but
usually I f the Insect i s cooling a t a ra te o f %* o r more p er minute and the
observer Is recording the time, an accuracy o f 6.1* Is a l l th at can be
expected, unless an automatic time-recording device i s used, o r two observers
are present.
Of course, the galvanometer i s much more accurate than the
record obtained, but the e rro r due to the personal element i s always present,
yet is m a ll enough to make readings quite accurate to 0.1**
The refrig e ra tio n cabinet used was la I t s e l f e n tire ly Inadequate fo r
work In which a constant low temperature was required fo r more than a few
hours.
I t consisted o f a cork-insulated cabinet having re frig e ra tio n c e lls
running around the Inner sides.
The In tern al dimensions rare 23 Inches x
11 inches x 20 inches in depth.
The co il# ra re cooled by.the expansion o f
ammonia, regulated by an expansion valve situ ate d ju s t above the cabinet.
The re frig e ra tio n plant consisted of an ammonia compressor end a 2 H.P, motor
Which rare used to cool an adjoining sto re room.
Dy closing the valve to the
coil* in th is room, the valve above the refrig e ra tio n box eonld be opened
and the l a t t e r cooled. Many d lf flc n ltie e were met with here, - elm## the w i t
va* not automatic and the expansion valve m e Inadeqw te. However^ temperatures
ss low as -30% . were sometimes obtained.
In order to secure a constant low temperature, the cabinet was
equipped with a fan to provide c irc u latio n and a nlchrome wire heating w i t
operating through an e le c tric relay from a mercury toluene thermo regulator.
Ih is type o f thermo regulator hae come in to common use so i t w ill net be daeerlbed here.
With th is apparatus, the temperature as read by a thermometer
in serted th ro u # a cork into a v ia l, was constant to w ithin+ 0.$40.
The
thermometer bulb was placed In a v ia l to duplicate the position o f the in sects,
Which were frozen In a sim ilar r i a l and sim ilar p o sition.
Due to the un­
r e lia b ility of the ammonia refrig e ra tio n apparatus, constant atten tio n was
needed to keep the cabinet a t a constant temperature, so th a t 10 hours was
about the maximum time of running.
L ater in the work a constant temperature hath was su b stitu ted , and
found to be much more convenient and re lia b le .
A bucket o f eWlelw chloride
solution wittx a f reelin g point o f •$ $ • was placed in the re frig e ra tio n cabinet,
and in i t were arranged a motor s t i r r e r , a k nife h ea ter, and a Micmry toluene
thermo regulator.
A quart s i l k b o ttle was also Immersed nearly to the top in
the solution and was made a permanent fix tu re .
A h a lf Inch hole in the eofk
in the top o f the milk b o ttle was su fficien t to allow the passage o f the
insect to he fro sen.
Bie temperature o f the a i r in the s i l k b o ttle wee
recorded by means o f e thermocouple situ ated about h a lf an inch away from the
suspended in se c t.
By throwing a switch, e ith e r the temperature o f th e-in sect
= -25e r th at of the a i r near I t could be read from the scale.
Since the bath liq u id conducted heat very eloviy. I t was found
convenient to cool the cabinet the day before the experiment to a temperature
below m * required.
When the experiment was ready, the bath was heated to the
required temperature In a short time by means o f the knife h eater.
Twm then
on, the bath cooled a t a ra te o f about 0.1* p er hour, so th at an experiment
could be run a t a v irtu a lly constant temperature fo r two o r three hours with­
out the use o f a thermostat.
Occasional use o f the knife h eater would keep
the temperature constant to w ithin 0,1* o r 0.2*.
The thermocouple used In recording the temperature o f the Insect was
designed to f i t under the e ly tra o f the adult potato b eetles, on Which
p ra c tic a lly a l l the experiments were run.
Since the length and resistance of the
copper and Constantsa wires used are important in a set-up such as described
above, standard lengths of c e rta in copper and Constantsa w ires were used.
Only two se ts o f thermocouples were used In th is work, and they were id e n tic a l.
They were made by melting the end o f the copper wire In a hot flame u n til a
small bead was formed, when the end o f the constants* wire was fused Into
it.
The bead was then file d u n til i t was f l a t , and the contact o f the two
wires could be p la in ly seen.
A 12 cm. length o f 3 am. glass tubing was then
slipped over the end o f the thermocouple and cemented Into such a po sitio n
that the thermo junction extended about h a lf an Inch beyond the edge o f the
glass tubing.
The connected wires are o f course Insulated, and fo r fre e s-
Ing adult potato beetles they are heat Into an arc sim ilar to th a t presented
by the e ly tra o f the b eetles.
The thermocouple then f i t s snugly between the
e ly tra and the so ft body and forme an excellent sem i-internal contact.
—26—
LOW T5MPEBATOB3 SUBTTTiT. SXPBmTWBaT:
in experiment was undertaken to determine the time-temperature
m ortality relationships of L eotinotarsa decealln eata M o lts.
This experiment
was performed during the la s t p a rt o f AogAst and the f i r s t p a rt o f September,
on Insects taken d irec tly from the fie ld .
Tea se rie s o f fiv e b eetles each, In ten eosfced v ia ls , were placed in
the refrig eratio n cabinet which was held a t a constant temperature fo r ten
hours,
At the end o f each hour one v ial was removed from the cabinet and
allowed to warm a t room temperature fo r 2U hours.
the m ortality was determined.
At the end of th is time,
Due to the h ab it o f these beetles of feigning
death, they were subjected to a heat of 30* to 35*0. f o r about h a lf an hour,
and i f no eigne of l i f e were apparent by that time, they were subjected to a
gradually Increasing heat from an e le c tric lig h t bulb held close by.
Any
movement except a very slow muscular contraction was considered to indicate
life .
The constant temperatures used were -5 ° to -12* In one degree in te r ­
v als, and most o f these were duplicated.
The experiment required one day fo r
each temperature, since only one cold cabinet wae availab le.
graphically the resu lts o f these experiments.
H g . 3 shows
A temperature o f -5* produced
no m ortality in ten hours, while temperatures o f - ll* and-12* produced 100$
m ortality during the f i r s t hour.
-10* are intergrades, as shown.
The curves a t temperatures o f -8 , -9 and
The curves fo r -6* and -7* are not shown,
fo r although the experiment was duplicated several times a t these temperatures
absolutely no uniform ity o f re su lts could be obtained.
The explanation
-
-ig - ?•
27-
Time-mortali t y carve of non-hardy adults o f
Leptlnotarsa decemlineata.
-
25-
probably lie * la the fact th at th is temperature range I* th a t o f the under,
cooling temperatures.
The l a t t e r vary quite a l i t t l e , and therefore I f c e rta in
o f the Insects are frozen by exposure to -6® o r -7®, e h lle others do not reach
th e ir undercooling points a t such temperatures, the amount o f m ortality w ill be
affected.
I t l s assumed th at an Insect which Is held unfrozen a t a temperature
"7* for 10 hours has a much t e t t e r chance o f survival than one which I s held,
frozen, a t -7* fo r the same time.
This facto r w ill be seen to a ffe c t the -8®
curve to some extent, making I t Irreg u lar.
Tho Insects used In these experiments were In a summer, non-hardy
condition.
Although no p a ra lle l te s ts were made o f hardened Insects, I t Ie to
be expected th at In such a case the m ortality would be decreased along with the
drop In undercooling and rebound points which accompanies hardening.
An attempt was made to Induce the rebound a t a temperature above the
noxaal undercooling point of the In sect.
With the cabinet a t a temperature of
-9®, no rebound could be Induced,' even thou^i the v ia l and thermocouple were
vigorously tapped and shaken.
Two o r three b eetles were trea ted In the same
way a t -6®, without the rebound taking place, althou#i th is temperature I*
below the normal undercooling p o in ts o f many o f these In sects.
Since the
l a t t e r were In a non-hardy condition, th e ir rebound points had undoubtedly
X
been passed even a t -5% since another experiment using b eetles o f sim ilar
■
!
condition showed th at only U b eetles out o f 120 had a freezing point o f lower
than -5®. ,The beetle# were, then. In an undercooled condition when they were
subjected to a g ita tio n , yet th is was not su ffic ie n t to s ta r t the c ry s ta llis a tio n
o f Ice.
-29*
FB2EZIRG CUMFS
The apparatus used la much th at shea a sample I s being frozen, the
complete time-temperature reactions from Oe down Is presented on the scale in
fro n t of the observer1e eye.
In order to preserve th is v isu al data, the idea
was early conceived o f recording time as well as temperature, and p lo ttin g
the two as a time-temperature curve.
This idea has proven extremely useful,'
since I t enables one to In terp ret what happened to the Insect as the temperature
dropped.
A sample curve o f d is tille d water is shown in Fig. U. Water, being
a homogeneous system o f inown p ro p erties, shows very well the various stages
o f which the curves are composed.
F ir s t, the curve from 0* to the under­
cooling point is a very smooth curve in a l l cases, the time increasing as the
temperature decreases.
Second, the rebound, from the undercooling point to th e
rebound point, u sually s ta r ts fa s t and ends slowly.
Third, from the rebound
p o in t, the temperature begins to drop again, but more slowly, since the heat of
fusion o f the freezing ice must be dissipated in to the environment. As more
and more ice is formed, the sp ecific heat i s lowered, since th a t o f ice i s only
about h a lf th at o f water.
be seen in the fig u re.
Thus the curve gradually becomes steeper, as may
As the temperature approaches that o f the environment,
however, the temperature d iffe re n tia l becomes le s s and le ss and the curve
approaches the environmental temperature assyoptotically.
In the figure shown,
0*3253 grams o f water were frozen in a small v ia l, with the cabinet a t a
temperature o f exactly -IO0C.
For comparison, a time-temperature curve is given fo r a potato b eetle
30 -
T E M P E R A T U R E IN
-
TIME I N MINUTES
Fig. U.
Freezing curve o f O.3253 grams o f d is t ille d
water. The lower curve represents the
temperature o f the refrigeration cabinet.
adult wiggling 0.1U60 grams. This Is shoim In H g . $.
cabinet
temperature was constant a t -12, the c a lc lm chloride hath being used.
The
fin a l stage Is not represented In th is curve since the beetle was needed
aliv e fo r fu rth e r freezing.
I t w ill be noticed th at the temperature Srsps
from the rebound point almost In a straig h t lin e , whereas in the ease o f water,
■
,
the temperature remains at the rebound point fo r tome time, and then f a l l s In
a gradually accelerating d eclin e.
The difference i s probably due to the lew
water content of the b eetle.
A Tijralld larv a weighing 1.1S20 grams, and the same w elgit of
d is tille d water, were therefore frozen under Id en tical conditions. In the same
v ia l, with the cabinet temperature constant a t -18*.
The thermocouple was
Immersed In the water, and was placed In very good contact with the so ft body
o f the Tlpolld la rv a .
freezing.
The outside o f the larv a was carefu lly dried befbre
Both freezing curves are given In H g . 6.
I t w ill be seen th at th e
Tlpulld larv a remained almost a t the rebound temperature even longer than the
water.
A h lg i water content is therefore assumed, In co n trast to the potato
b eetle o f H g . 5*
-
'
32-
500
+00
TIME IN SECONDS
F ig .5.
Freezing curve o f a Lentinotarsa adult
showing only one rebound.
-
33-
-O DunuOMMirc* • !.isao
a
0—0 TlPUllOAt iA«V4-/./a*P C.
TIME IN MINUTES
Fig. 6. ''Freezing curves o f a Tipulid larva
and the same weight of d is t ille d
water under almost id en tica l
conditions. The lower curves
represent the temperature of the
refrigeratin g cabinet.
-3L_
MtiiTiPLi
ramtim
Payne (1926b) describes a m oltlple fre e sln e e ^ e rle e n t.
Bepested
freezing o f the same In s is t o r tissu e eA IM ted no h y steresis, she found, end
the Tmdercooling and rebound jo in ts remained the came.
Two th ird -ln e ta r Wclanorlue dlfferem t^alls nymphs were used la a
sim ilar experiment by the w riter.
One was fro sen 10 tim es/ every 10 to 12
minutes, and gave corrected freezing points as given In Table I .
The temperature
o f the Insect a t the time th at I t was removed from the cabinet to W warmed a t
room temperature i s given.
The cabinet temperature varied from .17.2* to -20.3*0.
TJLBLS I .
I
T rial
Corrected
freezing
-6 .7
uoint
Temp, a t
A lc h
~9.0
removed I i
%
5
— 5—
-7 .4
-6 .6
-5 .5
Bone
-9 .0
-9 .0
-11.5
—20.0 —10.0 -19.0 -17.0 -17-0 -17.0
2
5
b
-6 .7
g
9
10
Sone
None
Son#
7
- # .l
f,
The o th er Insect was also frozen 10 times, but each time was removed
when I t had reached a temperature o f -10.0*.
I t was warned a t room temperature
fo r the same time as I t took to cool to —10* the previous tints.
are lis te d in Table I I .
Hte re su lts
The cabinet temperature varied from -12.5* to -24.2*0.
TJLBLS I I .
x
T rial
I
2
1S
4
9
6
7
I
Q
10
Corrected
'
freezing
- 3 .5 -4 .4 -3 .9 -4 .5 -4 .7 -4 .2 -4 .0 . # 5 .4 .3 -4 .7
ue Int
Temp, a t
A ic h
-S0.0 -10.0 -1 0 .0 «10.0 -1 0 .0 -1 0 .0 -1 0 .0 -1 0 .0 -1 0 .0 -1 0 .0
removed
Time to
cool to -10*5. 12 11
Il
IU
19
13
10
9
g
g
ihSb »
f
i
-35ia o th er experiment was carried on to determine the e ffe c ts o f re­
peated freezing a t Interval# o f I to 3 days.
In th is experiment, 9 Leptlnetarsa
decemllneata adults were frozen in a v ia l placed In the temperature cabinet
held a t a constant temperature o f -IOeOt O.
A thermometer In serted th ro n g a
cork into a sim ilar v ia l and in a sim ilar p o sitio n , was used to record the
cabinet temperature.
A strong a i r circu latio n was provided by a fan, and heat
ms# obtained from c o ils of nlchrome wire connected th ro u # a relay to a mercurytoluene thermo regulator.
by about 1.0% .
The temperature as recorded by the thermometer varied
In freezing the In sects, the time was recorded a t 0% - I* ,
-2 * , and at I % Intervals down to the undercooling point, the time o f the l a t t e r
and o f the rebound point also being taken.
As soon as the rebound point had
been d e fin ite ly reached, the in sect was removed from the cabinet and allowed to
:
,
' '
warm a t room temperature. The ra te o f cooling varied from about 1 .5* to 2.5*
p er minute, calculated from 0* to the undercooling point.
Table J lI gives
the corrected freezing points o f the individuals, the average freezing point#,
the date# o f freezing, and the average magnitude o f the rebound, i . e . the
difference between the undercooling end the rebound points.
I t w ill be seen from Table I I I th a t there is no d e fin ite trend in
the changes o f the freezing p o in ts.
The in sects used were taken from the
fie ld just before use, and were kept in v ia ls a t room temperature and fed potato
leaves during the experiment.
They were, o f course, in a non-hardy condition,
fo u r out o f nine survived freezing 17 times.
This"freezing? however,
represent* ju s t the beginning o f the freezing process, very l i t t l e ice being
firmed in the body.
TABLS I I I ,
freezing Pointe and the Average Magnitude of the Rebound, in Degrees Centigrade
Beetle
S b , . , ..
I
I
7
8/26
S /a
-2.3 -1 .4
-4 .6 -2 .5
-2 .4 -3 .5 -4 .0
- 1.8
—2.8
-3 .6
-5.3
S
-3.1
A l -4 .1 -4.3
—2.8 -2 .9 -3 .2
_ z M -3 .7 —4.0
- 3.3 -3 .1 -3 *4
8
9
10
Aver.
Averege
aagntture -3.8
o f the
I|eb22-3yl»™.
3.7
3.9
8/19
-2 .3
-3 .3
-3 .5
-3 .6
-5 .0
-3 .4
-4 .8
-2 .5
- 4 .0
% 3%
3.7
-1 .9
—3.8
—2.5
-# .2
-3 .7
—2.8
-4 .3
-2 .7
■-5.2
-3 .3
9/1___ 9/1
1/6
q /7
<l/g
q/*
- 2 .1
- 2 .0
- 2.8
-3 .9
-3 .8
-4 .1
-4 .4
-3 .4
-3 .9
-3 .8
- 5*6
-3 .9
-4 .1 -3 .1
-3 .1 -3 .0
-4 .3
-4 .8 : U
-2 .7 - 2.8
-3 .3 -3 .2
-2.4»
-4 .5
-3 .0
-3.U
-4 .1
-3 .0
-3 .3
-2 .8
-3 .5
—2.9
-3 .7
-4.8
-2 .4
-2 .2
-2 .2 - l . g
-2 .9 -4 .0
-1 .2 -2 .9
4.5 -4 .2
q /lf
-4 .9 -4 .7
-
2.8
-
2.8
—2.4 —3.2
- 4,
-5 .7
-3 .6 -3 .5 - 3 .4 -3 .2 -3 .2 -3 2
4.1
3,9
3.6
3.8
3.8 3.4
-37CQRESLATI0% 0 ? TBBZlNG PODfTS AM) RlTK PT CXKKJga
C arter (1925) co rrelated degrees o f tmdereoollng and ra te a t sblch
the Insects (lry h u g obtectus ad u lts, la rra a and pupae) were cooled, meaaared
la degrees p er minute. He found no sig n ifican t relatio n sh ip .
He gives M s
correlation charts In h ie paper, and i t Is a t once noticeable th at they are
not at a l l representative.
For Instance, in the chart fo r the a d u lts, 22 .
points are close to a rate o f 0,5 degrees p er minute, and one so lita ry point
a t 1.8* p er minute.
0.1-0.5* per minute.
For the pupae, a l l 23 o f the points l i t in the range
For the larv ae, 18 points l i e between O.25# and 0.7*
per minute, and G points between 1.75 and 2.3* p er minute.
recorded was 2,3* p er minute.
The fa s te s t rate
Such groupings are c e rta in ly not representative,
although I t w ill be seen from the following experiment th a t C arter’s conclusions
were correct within the range used.
In the present experiment, 20 Leptlnotarsa decaniln eata M o lts were
frosen seven times each as fa r as the rebound p o in t.
The following data were
taken each time; the undercooling and rebound points; the time to cool from
0*0. to each o f these points; and the cabinet temperature a t the time of
freesing.
Eleven cases o f m ultiple rebounds occurred, but these are not !in­
cluded in the present analysis since the f i r s t rebound o f these i s not compa­
rable to the single rebound o f the o th er 129.
In order to allow fo r the
v a ria tio n in the else o f the in se c ts, the rate o f cooling i s expressed in
degrees per minute, calculated from the time required to cool from O^C. to
the undercooling point, rather than on terms o f the cabinet temperatures re­
corded.
The experiment was planned so as to secure as uniform a d istrib u tio n
o f rates as possible.
The rehound points were corrected to give f reeling p o in ts, and these
wsre p lotted against the average ra te of cooling from OeG. to the undercooling
point In degrees p er minute.
Fig. 7 shows the resu ltin g co rrelatio n chart.
The correlation co efficien t, 0.2125, was calculated ty the Pearsonlm method.
S-rnsh a low co efficien t Is not enou$i to estab lish even a hare relationship.
It
Is therefore concluded th a t, within the time-temperature lim its used, there i s
no relationship between the freezing points of potato beetle adults and the
rate o f cooling.
The lim itin g of th is conclusion is very necessary.
I t Is obvious
th at In the case o f a very m a ll Insect being cooled a t a very fa st ra te , the
small amount o f heat of fusion lib e ra te d by freezing would be dissipated so
fa s t th at the rebound point would hot be reached before cooling again s ta rte d , '
I f Indeed the rehound would be noticeable a t a l l .
For Instance, la freezing
an aphid close to the previously cooled c o ils o f a COg expansion apparatus,
the rebound would probably not be noticed, even when using such a sensitive
recording apparatus as has been described.
Such rapid freezing never occurs in nature, and I s therefore of
l i t t l e consequence.
This also applies to the occurrence o f m ultiple rebounds.
I t has been stated above th at more than one rebound has never been observed
by the w riter When a slow rate of cooling was used.
Since even th is slow rate
Is much f a s te r than occurs In nature, esp ecially in the case o f Insects Which
hibernate underground, i t seems reasonable to conclude th at potato beetle
adults freezing under natural conditions exhibit but one rebound.
must also be placed on th is statement.
Brt lim its
In most o f the experiments described
the beetles were not subjected to temperatures much below th e ir freezing
33-
-
-
8'
CORRELATION
COEFFICIENT
0 .2 I2 S
o
-V
• O
°
I
>
O
O O
°O
O
Ot O ° °
%
N
LU
LU
Qi .
U-
O0
° uO
0O0O
OO
° O1 ° 0O ' O °n 00
%
0
t
«0° f o
’
O
Ov O
OO
O
°
00 O
°
°
R A T E OF C O OLIN G IN D E G R E E S P E R
Fig. 7 .
°
M INU TE
Correlation table showing the rela tio n between
freezing points and rate o f coolin g of
L e n t i n o t a r s a adu lts.
-Io polnts so that they could be kept aliv e fo r re-free zing.
For th is reason,
any statements made in th is paper regarding the occurrence o f only one
rebound must be lim ited to the temperature range used.
Below th is temper-
atu re, fa rth e r rebounds ml^ it occur, bat th is has not been determined as yet
fo r Ieotln p tarsa decemllneata.
-U l-
ITOLTOPIg BSBOTma
Dartng the course o f various experiments on the freezing of
Leptlnotarsa ^ecemllneata ad u lts, there were frequent cases In which more
than one rebound occurred.
The same phenomenon has been observed In the f re e s -
Ing o f grasshopper nymphs o f a hibernating species.
In such cases, however,
the maximum number o f rebounds recorded has been two. Potato b eetle larvae
have been given as many as five d is tin c t rebounds In a time in te rv a l o f le s s
than three minutes and a temperature range o f -4 .9 to -6.3*5.
Pig. 8 shows
the p a rtic u la r case referred to , In which four rebounds are d e fin ite , and the
h e sita tio n a t -4 .9 * i s considered as a rebound.
The free sing apparatus used
was such th at the minor rebounds were recorded accurately, since the galvanometer
was quite sen sitiv e and well-damped, the thermocouple was small, and the
environmental temperature constant.
There was no chance o f acquiring heat from
the constant temperature heating u n it, since the c irc u latio n o f a i r in the
cabinet was adequate and the b eetle was frozen Inside a corked v ia l.
In most
o f the experiments, boreover, constant temperature was maintained by the
calcium chloride bath already described. In which there was no p o s s ib ility o f
environmental heat producing these rebounds.
The heat represented by these secondary rebounds, then, was heat o f
fusion of ice being formed In the In se c t's body, exactly as In the case of the
major rebound.
Before each o f these rebounds, necessarily, there was under-
cooling, and since an aqueous solution cannot possibly undercool In the presence
o f ic e, each rebound must represent the independent freezing of separated
systems.
While m ultiple rebounds have have been o f frequent occurrence during
-
42 -
ZOO
TIM E
F ig .8.
IN S E C O N D S
Freezing curve of a non-hardy Lentlnotarsa
adult, showing m ultiple rebounds.
th is freezing work with potato beetle adults, no reg u larity has been observed,
even with‘the same individuals.
One factor seeming to a ffe c t the occurrence
o f more than one rebound Is the rate o f cooling.
In running the experiment
lis te d under "Multiple Freezing", the cabinet temperature used was -1 0 e.
In
th is experiment i t was noticed th at there were no cases In which more than one
rebound occurred.
These Insects were removed When the f i r s t rebound point was
reached, but the w riter feels confident th at he can t e l l by the magnitude and
behavior of the rebound I t s e l f whether o r not subsequent rebounds w ill occur.
I f the rebound I s the only rebound, I t Is of f a i r magnitude; i t begins with
a fa st rise in temperature, gradually slowing as i t approaches the rebound
point, Where i t h e s ita te s a t le a s t a few seconds before a slow drop in
temperature begins.
I f more than one rebound Ie to occur, the f i r s t rebound
i s of email magnitude, and the ris e in temperature to the rebound point is very
h esitan t and Irreg u la r.
Moreover, in such cases the rebound point Ie no sooner
reached than the temperature begins to drop again quite f a s t.
When the la s t
rebound point i s reached, the temperature drop i s very slow,
These phenomena
are Illu s tra te d in Figs. 10 to lU.
The w riter therefore fe e ls confident th at
in the above experiment. Where the cabinet temperature was constant a t -IOtO. 5*0.
extremely few i f any secondary rebounds would have taken place i f the in sects
had been allowed to cool fu rth e r.
This seemed to be the case only when
cabinet temperatures o f -IO e o r h l^ ie r were used.
In a subsequent experiment,
where a cabinet temperature o f -12® was used, 32 out o f 50, o r 6U$ o f the
in sects had more than one rebound.
For comparison, the occurrences o f
Multiple rebounds under varying cooling conditions is given in Table IV.
TABLB I V .
Cabinet temp,
used
-1010.5*0.
- 7.5 to -13.5*0.
-1210.2*0.
-1 3 .5 to -25*0.
—20 to —30 eC.
Ho, cases Multiple
Rebounds
0
0
32
11
IS
Total Ho.
frozen
117
Ug
50
92
107
Percentage of
0
0
6U.0
12.0
lU.O
I t Is suggested th a t In the case o f the loimr ra te o f cooling there
Is le es lag in the temperature difference between the Inside and outside of
the Insect than In the case o f fa s t cooling.
In the l a t t e r case, the Irre g u la r
cooling ml#it lead to the freezing of the outer tissu es sooner than the Inner
ones. However, a change o f two degrees, between -10* and -12* seemed to mmV*
a l l the difference between one rebound, o r more.
Variation In the size of th e
Insects would produce a g reater difference In the rate o f cooling than would
th is two degree difference in cabinet temperature, so th a t the above suggestion
seems to be u n ju stifie d .
taken here.
Further analysis Is needed, but w ill not be under­
. HARDENING 0? I3PTIB0TABS1 ADOLTS
Ia order to determine the effect o f moderately low temperatures
©a Laotlnotarsa o r the quantity fa c to r of cold-hardiness, to use Payne's
nomenclature, twenty adults In a non-hardy condition were selected and from a.
The undercooling and rebound points were recorded, and each b eetle removed a s
soon as the rebound point had been d efin itely reached.
The beetles were
divided Into four groups of fiv e , one group being placed a t 2*0., one a t 5*»
one a t 8*, and the other a t 11*0.
They were stored In corked v ia ls o f about
20 ce. capacity, the ones a t 8* and 11* being fed potato tuber.
bo
L ittle o r
feeding was done a t these temperatures. Each series was removed and frozen
as above a t weekly In terv als fo r six weeks.
The refrig e ra tin g cabinet varied
la temperature from -20* to -30*, these temperatures being recorded by means
o f a thermometer placed In a v ia l sim ilar to the v ia l in which the Insects
were frozen.
In fifte e n cases out of one hundred and seven, o r lW , more
than one rebound occurred.
Table 7 gives the corrected freezing p o in ts of those Shlch exhibited
only one rebound, with the average fo r each se rie s .
The freezing points of
thpse with more than one rebound are not given, since i t I s not known Whether
these m ultiple points are comparable to the single ones.
Troa observatlpn
o f the table, I t w ill be seen th a t no hardening occurred a t 8* o r a t 11*,
and th at hardening was g reater a t 5® than a t 2*.
In th is experiment, as In others th at the w rite r ca rrie d on,
humidity was not considered.
Undoubtedly humidity affects th is subject to a
considerable extent, as has already been demonstrated by Payne (1929).
The
-UG.
TABLE V.
M R Z IfG POINTS 0 7 SERIES 0 ? LBPTIWTAMA TKCawr.Tmg^ CORBBCTBD ACGOHDIM
TO fOBfOLA.
Bo.
I
2
2"C.
U
5
0 iteeki
-3.99
-U.81
-5.22
Awr. -U.5U
6
7
5"Q.
-3.02
(Cab, T«mm. "Bmm m -20» to -1SOtO.)
I week 2
3 weeks 4 week# 5 weak# 6 week#
-5.it2
-u.55 -7.36 , Dead
-U.72
-K 58 -7.54
-9.31
Dead
-7.97
-U.05 -6.00
Dead
-7.37
-6.33
4M»
-4.36
-4.08
-3.60
Dead
-7.07
Dead
-U.U7 -6/59
-5.76
-
-U.38
-U.Ul
-6.96
- U.35
-5.92
-3.86
-4.37
-7.38
—6.13
-7.83
-7.79
-8.77
-7.64
-9.36
-9.19
Dead
Dead
-7.75
-10.31
Dead
-7.99
-9.62
52.18
Dead
-6.50
"
-5.39
-5.95
-3.78
-6.91
—6.81
-3.9U
Ayer. -U.27
-5*60
-5.1?
-3.65
-6.35
-U.U6
-5.03
-4.62
-5.05
-5.27
-3.70
-2.20
-2.19
-2.97
-2.69
-2.19
-3.58
—
1.06
-1.35
-3.14
-3.15
-2.24
-3.43
g
9
10
-6.35
-3.U3
-
-7.28
-6.21
—6.82
-4.93
-3.89
-5.95
-2.92
-U.27
-U.13
-3.83
-U.07
Aver. ^ . 9 9
-5. Ul
-3.8U
-U.98
16 -5.59
17 -3.97
I l eC. 18
-3.57
19 . -5.1U
20 -3.21
-U.07
-U.18
-3.18
-3.62
—
-1.15
-3.94
-2.U2
-3.75
-1.64
-3.15
Aver,, —^.30
-3.76
-2.58
11
12
s«e. 13
IU
15
_
-5.2U
-
Dead
-3.36
-3.21
—
1.86
Dead
Dead
Dead
Dead
•
-4.18
Dead
Dead
;
-2.90
-2.10
-1.80
-3.28
above result*, therefore, are v alid only from a temperature standpoint.
The
fe e t that the v ia ls were corked and th a t potato tuber was placed with the
se rie s a t 8e and 11* would produce a M^hi humidity In the se rie s which m t^it
a c t against hardening.
Another experimental hardening o f Ieu tln o tarsa was carried on during
the winter.
The Insects used were removed from an outdoor s o il hibernation
cage la te In November,
They were kept a t room temperature over moist sand
fo r two to three weeks and were fed potato tuber.
Many o f them were then
frozen, and 20 were selected as having a high freezing point and only one
rebound.
In a few days, 10 o f these Insects were weighed and then frozen
under Identical conditions, with the refrig e ra tin g cabinet a t -12*.
Time m s
recorded, correct to I second, so th at time-temperature curves might be con­
stru cted ahd compared.
The Insects were allowed to cool beyond the f i r s t re­
bound point In order that any successive rebounds would he recorded, and also
to determine the nature o f the curve beyond th is point.
The In sects were
removed When the rate of cooling became uniform and the temperature was hot
low enough to he le th a l.
Most were removed a t -6 .5 to -7.5*.
Having undergone th is I n itia l freezing, the eerie# o f 20 beetles
were placed a t 5*0* ln corked v ia ls of about 20 cc. capacity and without food.
The freezing was repeated each week fo r four weeks under almost Id en tical
conditions, the Insects being weighed each time before freezing.
v ariatio n s In weight are shown In Table VI.
The
TjfflLS TI
Bamomur#. In ##«*#
0
I
2
3
U
W f I A t . le » i
0.1277 grams
0.1090
"
0.1006
0.1092
O.O976
T *
I It. 7
21.3
1U.5
23.6
The irre g u la rity In the flgaree fb r three seeks1 exposure Is
probably due to the fact th at between the second and th ird week seven o f the
se rie s'd ie d and were substituted from the "other IO beetles A lc h had been
subjected to the sane hardening conditions, but were not fro sen a f te r I and 2
'
■ ■
. ,
weeks* exposure. However, the figures fo r the f i r s t and second weeks are
sig n ific a n t.
Here again, however, humidity probably has it# e ffe c t and the
re su lts must be considered from a temperature standpoint only.
The object o f th is experiment when I t was sta rte d warn two-fold.
Tl r e t, i t was thought that by making a Correctioh fo r the v ariatio n in weights
o f the in sects used and bringing them a l l to a common wel^xt b asis, the timetemperature curves would be enouA alik e to s trik e an average of the 10 curves
each week.
Second, I t was thought th at I f the in se cts lo s t o r bound water in
the hardening process, thereby decreasing the percentage o f fre e water In the
*
body, the slope o f the cu rv e,p articu larly th a t portion following the la s t re­
bound, would become steeper as hardening progressed.
The f i r s t object was soon seen to be th e o re tica l only, o r a t le a s t
to apply ohly to a homogeneous system lik e water.
Obviously, the freezing
curves of d ifferen t weights o f water, other conditions being equal, could be
mm** to coincide by introducing a weiAh correction.
Ib le i s not the case
with swh complex systems as those of Insects, as w ill he read ily seen by
examination of figures $ to lU.
indicated hy th e ir nxsnbere.
The individual in sects correspond as
fig u re 9 shows the I n i t i a l free sing curves of
the 10 |.ia tl« n ta rs a adults, before any hardening had been Induced, idien
b ro u # t to a common welgit b asis o f 0.1 gran.
This correction waa made by
m ultiplying the time by O .l/x , Where x Ie the welgit o f the Insect In grams,
figure 10 chowe the sane curves, uncorrected fo r w ei^it.
SSlnce the weight
correction seems to complicate m atters rath er than to sim plify them. I t was
abes&doned.
The curves o f the 10 beetles a f te r hardening fo r I , 2, 3» =®,d U weeks
*
are Shown In figures 11 to lU.
As already mentioned, only three o f the o rig in al
ten b eetles are Included In the three weeks' exposure curves, and only two In
the h weeks' exposure curves.
In comparing these ch arts, the main v ariatio n
seems to come during the f i r s t week o f exposure.
points dropped about one o r two degrees.
The undercooling and rebound
Also, the elope o f the curves a f te r
the la s t rebound has been reached Ie much steeper.
Bie curves representing
2, 3 , and U weeks' exposure do not vary appreciably from those o f the f i r s t
week.
I t Ie noticeable, however, th a t the ten Insects vary considerably among
themselves and th at i t is u se le ss 'to attempt an average curve.
Single cases
w ill be noticed In figures 12 , 13 and lU where the rebound points were
abmereally hid&.
Bie w riter recognizes th at a f te r examination o f these curves, a
c e rta in c ritic ism may be ju s tifie d .
I t w ill be noticed th a t the rebound p o in t
In p ra c tic a lly every case does not seem to be a point of equilibrium a t which
the temperature remains constant fo r some time while freezing progresses, but
-
50-
T IM E .
F ig .9-
IN
SECONDS
Freezing curves o f 10 non-hardy Leutinotarsa
adults, with time corrected to a weight b asis
o f 0.1 gram.
-
51-
T I M E IN S E C O N D S
F ig .10.
Freezing curves of the same 10 in sec ts as shown
in Fig. 9» without weight correction.
F ig .11.
Freezing curves a fte r an exposure to + 5*0. for
one week.
-
53-
300
T IM E :
Fig. 12.
450
IN SELCO N D 5
Freezing curves a fter an exposure to 4 5°5. for
two weeks.
-
5% -
t
i
300
TIME
F ig.13 .
450
IN
SECO N D S.
Freezing curves a fter an exposure to +50C. for
three weeks.
-
55-
300
T IM E .
F ig . lU.
450
IN
SECONDS
Freezing curves a f te r an exposure to -AR0G. for
four weeks.
-56i» merely the point ehere the temperature eeaeee to rise and s ta r ts to fa ll
I t might he charged th at the rate of cooling i s so fa s t th at the
attainment o f equillhrlxsa i s not secured, so th a t the rebound points lis te d
are meaningless.
Xt i s admitted that the rate o f cooling used i s much g reater
than th at Which would ever be obtained in nature. However, the co rrelatio n
o f freezing points and the ra te of cooling has already been shown to be too
m a ll to estab lish even a bare relationship.
The w riter therefore feels
ju s tifie d in concluding th at since a v ariatio n in the rate o f cooling between
'
0#7 and 8.6 degrees p er minute does not affect the freezing p o in t, a v ariation
between 0.0 and 0.7 degree• p e r minute would not a ffe c t i t e ith e r.
I f such,"
I* the case, the rebound points Illu s tra te d are quite accurate, and the rate
o f cooling a f te r th is point has been obtained is dependent on the weight,
surface area, fre e and to ta l water content, and specific heat# o f the various
tissu es of the in se c t.
In sp ite o f the fact th at th is se rie s m s selected fo r having high
rebound points and only one rebound, a l l the ch arts show a f a ir ly large
proportion o f m ultiple rebounds, reaching a maxima on the f i r s t week and
decreasing again,
In order to determine the effect of hardening on to ta l moisture
content, IjQ ad u lts in U se rie s o f 10 each were placed under the same hardenla g conditions as the insects in the experiment ju s t described. Eaeh se rie s
was w elded in i t i a l l y , and each week one se rie s was removed from the 5*
hardening cabinet, weighed, desiccated a t 105* fo r six hours, and re-weighed.
The re su lts are given in Table TXX,
-57im a m
1.745 *
1.3W0
1.552
I . USlg
1.352»
1.2675
0.»79 «
0.537
0.3975
0.U595
T rm these data Ijr Is seem that the percentage to ta l water content
remained practically constant.
Tkt the f reccing points dropped during each
treatm ent, esp ecially during the f i r s t week.
solutions had to become more concentrated.
To secure th is result, the body
Two explanation# teem plausible.
W r e t, the conclusion to Which IoMneen came, th a t water was bound by the
hydrophilic co llo id s, being removed thereby from the role of solvent, with a
resulting depression o f the f rooming p oint.
Secondly, i t i s seen from Tables
TI and TII that although the percentage to ta l water content remained the same,
the absolute weight dropped approximately Vjj$ during the f i r s t week o f harden­
ing.
Since the electro ly tes in the body solutions are p ra c tic a lly a l l non­
v o la tile , being mostly dissociated aa ions, they do not en ter appreciably i f
a t a l l into th is If# weight decrease. But I t i s these e le c tro ly te s which
a ffe c t the freezing point depression to the g reatest extent.
Therefore, with
the same amount o f electro ly tes and a decreased absolute water content, a mere
concentrated solution re su lts and the f rooming point Ie depressed.
theories are not contradictory, o r even mutually exclusive.
These two
They are given
as possible explanations and i t may be th at both operate to some extent,
lobinson has presented data In support o f Me explanation, but th is does not
invalidate the other theory.
5^
DTBOmSTrm Cr? v m x.T,*pn„Tp* a m o r T g , m m m cT %* (
A
In rerlewliig the lite r a tu r e ©ae cannot help being lapreeeed with
a ce rtain weakneee. P ra c tic a lly a l l of the knowledge ©f the subject td date
I s the re su lt of experimental work, but very few workers have described th e ir
methods and apparatus such th at others could get comparable re su lts by using
the same set-up.
The importance o f the experimental procedure In th is woxk
can hardly be over-eaphas!sed.
Hand In hand with th is weakness I s the use
o f new terms without adequate d efin itio n .
- 1
-
One thing i s ce rta in ; i f previous
authors had described th e ir experimental method
'
bo
that l a t e r ones could
c r itic is e and make Improvements, the subject would be much more advanced today.
The tern 11f reeslng-point* has been much misused.
Most authors
have used i t to designate the equilibrium point to which the temperature rises
a f te r the f i r s t ic e formed In the bo^r has llh e ra te d i t s heat o f fusion,
forgetting that they are dealing with a solution.
This “observed depression
of the freezing point" must be corr&cted in order to obtain the true depression,
due to the fact th at when the f i r s t ice c ry sta ls are formed the remaining
solution is more concentrated.
Thus the observed depression o f the freezing v
point Is the freezin g point o f th is more saturated solution.
Gortner (1929)
gives a very good explanation o f th is physical phenomenon In M s discussion
o f freezing point depression.
I f a gram o f pure water is uniereooled to
- I eC. before Ice c ry sta lliz a tio n begins, one-elghtlsth o f the water w ill
separate In the form o f le e.
Since the la te n t heat of fusion o f water Ie
SO c a lo rie s, the o n e - e l^ tle th of a gram o f ice formed lib e ra te s one ca lo rie
o f heat.
Also since the sp ecific heat o f water i s 1.0, the temperature o f
th# g tm o f water I s raised one degree, o r from - I * to 0^8. Had the water
ropercooled to «»3*0. before c iy e td llts a tle a began, three»el^htlethe o f the gram
e f water would have f rosea, lib e ra tin g three c a lo rie s of heat which would raise
the temperature o f the water three degrees, o r from «»3e to 0*.
Using these
values, one can e a sily correct the observed depression o f the f reelin g point
%y means of the fommUa,
^
or
A
(V - '%j)
V
= A ' - O. o i z s a * ^ '
shere T ■ w el^it o f the water, (solvent).
A = corrected depression o f the f reeling p o in t.
A = observed depression o f the freeslng p o in t.
M s degrees o f undercooling before Ice-separatlon begins.
The true freeslng p o in t, therefore, cannot be recorded by a th ermometer, e le c tric a l o r otherwise, M t must be calculated from o th er data.
The
"observed depression o f the fre esln g point* Is termed in th is ps*er the "re­
bound point".
All "freeslng points* lis te d have been corrected according to
th is formula. Values o f-ti up to 6*0. have been observed by the w riter, making
necessary a correction of 0,U*C.
There are objections, o f course, to the uee o f the term "freeslng
point* In any case, since th is point would represent the temperature a t which
the Insect would freese only i f the tissu e flu id s were ag itated so th at super­
cooling could not take place.
This criticism i s probably u n ju stifie d because
i t i s based on unnatural conditions.
Another objection I s based on the fact
th a t insect tissu e Ie heterogeneous, and i t s various components freese a t
- 60 -
Atffereat temperatures.
This viewpoint I s sab stan tlated ty Payne«s (IQZGb)
Alscoveiy o f e eeconA w Aeicoollng and fiee slag o f oek bower Imrvee mt eboet
end by the w rite r's observation o f m ultiple reborn As In various Insects.
As many as four rebounds have been observed between -U.Q and -6.3*0. in an
WhmrAmeA l e o t l n o t * ^ Aecemllneat^ adult Anrlng m time In terv al o f almost
three minutes steady cooling.
Another aspect o f the same question I s th a t o f the completeness o f
freezing.
Insect tissu e does not remain a t the rebound point temperature
u n til co ^ jletely frozen, but s ta r ts to cool again almost In sta n tly .
Evidence
I s given by various authors, as stated above, th at freezing In not complete
u n til extremely low temperatures are reached.
An In te re stin g problem la presented by the magnitude o f the rebound.
Assuming, as seems necessary, th a t the in sect body Ie a *water-system*, l . e .
th a t water Is the solvent, the magnitude o f the rebound represents the number
•,
'
■
o f calo ries lib e ra te d Iy the f i r s t Ice formation, pey gran o f water.
I f some
method were developed fo r measuring the quantity o f heat represented by the
rebound, the amount o f water (solvent) representing the f i r s t p a rt o f the
in sect to freeze could e a sily be calculated.
I t Iz suggested that th is might
be the "free.water* content of the system.
The undercooling p oint has been used by many authors.
C arter (1925)
p la tte d undercooling points egelnst rebound p oints fo r Brochue obteetam. m d
found co rrelatio n s o f 0.779*0.053 fo r the a d u lts, 0 .GlW± 0 .0 A fo r the pupae,
and 0.690* 0.028 fo r the larvae.
This I s to be expected. Inasmuch a# under­
cooling Is a physical process with ce rtain lim its , and i s mainly dependent on
th#
of ag itatio n of the cooling liq u id .
C arter also p lo ts unden-
ooolla* points against rates o f cooling la degrees per minute. Payne (1926a)
meed undercooling points in much o f her woAc, giving Undercooling and rebound
points equal consideration.
I t w ill he seen from the discussion o f the
M ention given above th at the degree of supercooling Ie a fa c to r I s the
correction o f the observed freezing point, so th a t i f the corrected freezing
point Ie used, the supercooling point may be disregarded.
Another te m which has often been used without adequate d efin itio n
i s "survival temperature*.
The survival of an in sect a f te r subjection to low
temperature i s dependent on many facto rs.
Even i f such things as development,
n u tritio n a l s ta te , water content, etc. are constant, o r even assumed to be
constant throu^iout a se rie s, time le so Important a facto r th at i t must be
given i f the data are to W sig n ific a n t.
When cold resistance to the quantity
facto r Ie being studied, time Is o f course always given.
But in cold resistance
to the in te n sity fa c to r, most authors do not mention anything but the lowest
temperature to which the in sect was cooled.
The time involved in acquiring
th is temperature, l . e . , the ra te o f cooling, must surely be considered.
zute of cooling depends on the apparatus used fo r freezing,
This
f o r Instance, the
insect may he placed In a warn environment which is then cooled a t a c e rta in
ra te , o r i t may be placed in a cold environment to which I t gives o ff I t s heat
a t a ce rtain ra te .
The l a t t e r case Involves such things as the sp ecific heats
o f the various p a rts o f the in se c t, the surface exposed, and a i r currents.
Carter (1925) plotted usdsresellng points o f Bruchum obtsetus egslnet
ra te of cooling In degrees p e r minute and found no relationship.
Sines he did
so t cover the range of Me co rrelatio n ch art, however, but determined 6 # o f
-<2M e TOdeicoollng p o in t• a t a cooling ra te of 0*5* per mtmite and 11$ a t about
2* p er minute, h ie data are o f l i t t l e value.
I t has beam ehoen, how ver, that
• lth in certain lim it# at le a e t, the relatlenChlp beteeem freeslng point# of
Lamtlnotaraa decemllneata and rate o f eooling la very alldht.
I t might be v e il to point out here a alight ambiguity In Payom#a
(l$27a) use o f the tern "Q om tlty fee tor*.
She s ta te s , "In the metric system
fo r the quantity fa c to r the c a lo rie I s the u n it . . . *
"%@amtity* here Ie not the
same as she use* i t , since obviously the exposure o f an In sect to a f a ir ly lew
temperature fo r a long time has nothing to do with c a lo rie s, nor does time
e a te r into the d efin itio n o r measurement.of the c a lo rie .
The e rro r i s t r i v i a l ,
however, since Payne makes no mention o r use o f a u n it o f h e r "q u an tity 'factor*
In her subsequent work.
The w rite r has made a study o f Bohlnson1S method o f determining
bound water.
The method I s open to criticism .
(L) Becent evidence a l l goes
to show th at freesln g i s nowhere near complete even a t .2 0 ^0 .; (2) The formula
used must be regarded as em pirical, since the denominator has not been
s a tis fa c to rily derived o r explained; (3) the formula involves specific h ea t,
the determination o f which Involves the specific heats o f a concentrated
solution, ic e , and so lid m atter, each o f which should be considered separately.
S&yre (1932) compared the cryoecopie, calorim etric and dilatem eter
methods o f determining bound water, using p lan t tissu e , and decided th a t the
calorim etric method (th a t of Buhner, improved by BoMneon) i s the most rapid,
easy, accurate and re lia b le .
He recommends i t fo r the measurement o f bound
TOter in p ra c tic a lly a l l m aterial#, in sp ite o f the objections lis te d .
Bobinson made an enormous number of determinations o f the bound w ater content
e f In se c t!, and him data seem to in dicate accuracy o f the method,
A discrepancy has been noticed between the re su lts o f Bobinson
and those of Payne regarding the to ta l water content o f in sects
hardening process,
the
Bobinson found th at the percentage o f to ta l water did not
I* IESlJSiL
a t 100*0, to constant weight.
Callossala nromethea. Be dSsiocated the insects
Payne, on the o th er hand, found a decrease in
percentage o f water content during the hardening process, but determined th is
to ta l w t e r ty desiccating a t go* fo r
k
hours.
Such a low tsspsratus# would
W t desiccate the in sects to an appreciable extent in U hours; in fa c t, i t is
w *y l i t t l e above the upper le th a l lim it o f the in sects Payne used.
The
admit# o f Lentlnotare^ deoemllneata shleh were hardened by the eetbor TSgr
sto rin g a t 5*0. retained the same percentage
to ta l water content fo r three
eeek8S although they lo s t approximately 1 # o f their w e l# t.
e w f lr a BoMneon1S obeerrations.
These reeulte
The lite r a tu r e on the subject Is reviewed and discussed, sad c e rta in
criticism s and suggestions are added.
Aa Improved apparatus fo r the study o f Insect freeslng, whereby the
temperature o f the cooling Insect can be followed closely. I s described la
d e ta il and i t s advantages pointed out.
Time-mortality curves are given fo r the m ortality o f Lentlnetarea
decemllneata ad u lts under exposure to low temperatures.
I f the Insect Is
frozen fo r a few hours a t a temperature su ffic ie n tly low fo r the undercooling
point to be reached, death re s u lts .
Time-temperature freezing curves o f water, L eptlnotarsa decsmllmsata
adults and Tlpulldae larvae are given fo r comparison and analysis.
The re su lts o f repeated freezing o f Melamonlum d iffe re n tia lle
nymphs and Lentinotarsa deccmllneata adults show no d efin ite h y ste re sis.
The freezing points o f Lentlwtarwa deceWLlmeata adults are co rrelated
with ra te o f cooling.
Ho mlgnlflcant relationship was found.
The problem o f m ultiple rebounds as observed in Leotlnotare^
d##W llneata adult a I s discussed, suggesting opportunities o f fu rth e r worts: on
th is phenomenon.
The hardening o f Leotlnotarma deceaillneata adults Ie more rapid a t
5° than a t 2*, while temperatures o f 8* and I l e produce no hardening.
Ths
curves of in sects a t weekly in te rv a ls during hardening a t 5* are given and
compared.
Complementary data on to ta l water content, le s s o f weight, and the
concentration o f the body flu id s during th is hardening, are given.
The e ffe c t
o f humidity on th is subject Ie recognised, but i t Is not Included in th is work.
. . .
-Sg- -
BeMnson1S conclusion that the percentage to ta l e a te r content o f lneects
remains the same during the hardening process Is substantiated.
UBKRArome cisco
BachmetJewl P.
1901.
Iodine, J.H .
1921.
19^3.
C arter, Walter
1925.
Chmdler, W.H.
1913.
Ezperlmentelle entomologlche Studlen ron phyelcall«;hchemlechen Standpunkt ami. Leipzig.
Ractore lnelum cing the water content and the ra te of
metabolism in certain Orthoptera. Jour. Exp.Zool.
137-16U.
H lbem atloa in Orthoptera; I . Physiological changes during
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The effectoof low temperature# on Bruehue obteetue Say, an
lneeet affectin g seed. Jour.Agr.Bee.
The k illin g o f p la n ts by low temperature.
Res. Bel. S.
Cortner, R.A.
1929« Outlines o f Biochemistry .
Mo.Agr1BxpeSta.
John Wiley & Sons, Hew T o * .
Oueylard and P o rtle r
1916. Recherehee ear l a resistance au Rrold dee Chenilles de
ComsUW #t Camocsmsa. Compt. Rend.Soc.B iol. 7Q«77*1.777.
Harvey, R.B.
1918.
Hardening process In p lan ts and developments from fro st
in ju ry . Jour.Agr.Bee. 15? 83-112.
Kirby, Urn. and Spence, Wme
1818. An Introduction to Shtomology; or; Element# o f the Haturml
H istory o f In sec ts. 2%IQO-W).
Knight, H.H.
1922.
Kodle, E.K.
1902.
Studies on the L ife Bietory o f P e rillu s M oculatue Rabr..
Including observations Sn the nature o f the color p attern .
Rep.St. Bbt. Wim.
(Supercooling o f Animal organisms). (In Russian).
Bui, Acad. S cl. St. Petersb. 1&130-1W.
Im W g, D.
1928*
Baaimev, B.jLc
1912.
1
*
■
Develop^Bt o f cold herdlaeee la the la rm e o f the Jsem ese
B##tle. Boelogr 9;303-306.
(CM the freeslag m d cold reeletm ce o f p le a ts. Bsperlmmtal
aad c r itic a l observations). (In Roeelaa). M itt. I b r s tln s t.
S t, petersb.
.
•
Iewton and Gortner
1922, A method fo r estim ating the hydrophyllc co llo id content of
expressed p le a t Jnlee flu id s. Bot. Gas.
I e b ill e t Mellon!
1831. Becherehes ea r pleusieurs phenomenes calorifigue# entreprises
en raoyen da thsrmswmltip llo a te n r. Ann. Chlm.et Phys.gi 198-217.
Pays*, l.M.
192S.
' '
Ieadmasks In the h isto ry of the study o f in sect hibernation.
Bnt. I*"* ID 99-101/
*
1926a. The effect o f environmental temperatures on in sect freezing
p o in ts. Ecology D 99-106.
4________ '
.
1926b. Pressing sad survival o f Insects a t low temperatures.
Qpart.Bwv.Biol. D 270-282.
—
—
.i n -
•
1927'
Pressing and survival o f la sects a t low temperatures.
Jour, Mbrphu
1927a. Two factors o f heat energy involved In Insect cold hardiness.
Ecology *pl9*-196.
1927b. Changes in perm eability associated with the absolute
v ita l temperature. Aaer.Jour.Phyeiol.
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Biol.M L.
1928,
Cold hardiness la the ,IqpmswM, %*#$!#. P a a lllia lmmmnlaa Hsrnm.
Biol.Bui. 25flQ kl79.
%"*#****
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M 7B
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