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 ent of the requirements fo r the Degree of Master of Science In Entomology a t Montana State College Approved; a c . J t i ^ 4 . In Charge o f Major Soxfe nlng Committee Graduate Committee Bozeman, Montana June, 1933• t - v a r ? TJBLS Of COKTUITS Page ISTH0W8T1OK .................................. ............................................................ ... 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 BIBLIOGRAPHY ............................................................................................... ... . 66 4454L -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 de ta ils of the freezing and hardening o f Insects, with one notable exception. Beamur (1736)* published an ac(fount of h is experiments on the freezing of Insects, using h is newly Invented thermometer. The translation o f th is account is included for reference in th is paper in order to dhow the remarkable clearness, accuracy and value of Bsamur1 s 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 l i te ra tu re on the subject i s not voluminous, and only a email part of i t i s the resu lt of fundamental research. The p a ra lle l subject of the freezing and cold-hardening o f plants is much older 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 sects . This subject appears to be founded on so many unstable assumptions that 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 insects. The following work Is the resu lt o f th is attempt to learn anything a t a l l about th is extremely complicated subject. The w riter wishes to acknowledge h is gratitude to Dr. A. L. S trmd ♦-Reference i s made hy author and year to L iterature 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 Prof. W. D. Tallaan. fo r helpful criticism s end assistance. acrra# o? tlTBRATOEK %e subject of cold-hardiness and the freezing o f Insects has developed from a theoretical and p ractical study o f Insect hibernation. The h isto ry of 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 of bees; Vaudoner1 s discovery (1827) that some Insects displayed period ic ity end could hibernate In the presence o f h i # temperature and abundant food; lo b l l l and Mellonl’e (1831) use o f the thermocouple to determine the temperature of insects; and Seudder1S discussion (1887) of the subject in h ie "Butterflies o f Eastern Dnited States and Canada*. Sesmur1S contributions are eo Important that a translation Is 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 nests, they remain quite exposed to the rigors of winter. 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 ircu lates freely on a l l sides, o u # t not to be long in acquiring In i t s In te r io r the same degree of coldness of the a i r surrounding I t . These extremely small larvae, then, which thereby see® to be very - delicate, must therefore be strong enou# to re s is t the cold. I -U- have been curious to find out what degree they could re s is t , . and above a l l , what degree of cold was capable o f k il l in 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 that i t saves us from insects which are multiplying too fa s t, and which would have defoliated our trees in the spring and to the end of the summer. But the experiences that I have had have taught me that we have nothing to hope in th is country fo r the destruction of th is kind of c a te rp illa r by the cold of our worst winters, since they are in a s ta te of resis ting a greater cold than tha t of 1709. *fe know how £o make ice in any season, Tby surrounding with lee mixed with s a l t the th in vessel in which i s the water one wltiies to freeze. Physicians know also tha t the degree of cold that one can produce by suitable mixtures o f ice and certa in sa 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 fa r as the greatest cold of 1709, about lU l / 4 degrees below the point where the freezing of water begins (-17.9*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 ice and sea-salt; the liqu id o f the thermometer dropped to 15 degrees (-15.8*0), i . e . , about 3M of a degree below the point where the greatest cold of 170$ would have made i t drop. At the same time that I sank my thermometer into th is mixture of sa lt and ice , 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 ice , was open; I l e f t i t there nearly h a lf an hour. Ihen I took tb& small larvae out of 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 of an hour they were in such a s ta te that I could see that they were alive: they s tir re 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 liqu id In the thermometer drop to more than 17 degrees below freezing (-21.25% .). In th is second t r i a l , the larvae had then to re s is t a degree of cold nearly three degrees greater than tha t o f 1709; i t k illed none o f them. The sudden passage o f a i r su ffic ien tly tempered ( fo r when I carried on these experiments the liqu id of the thermometer was about e i # t o r nine degrees above freezing (10-11*0.), the passage, I eay, o f air,tempered by a i r o f such excessive coldness, should be fo r them a much more rigorous te s t than tha t o f the same coldness o f longer duration, which would become such only by B aceees lv e eceoaralatlone made during a great number o f days, aa happen# In winter. Also I have made these larrm sustain a cold o f 19 degrees without having them perish. "L ister has already remarked that ca 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 ice , and eo rig id tha t in dropping them in a glass they made a noise lik e that which would be made by a small stone, o r a small stick which i s dropped; that la th is s ta te , however, they were a live , and that 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 liqu ids had been fro sen, easts back to l i f e , th is was a true resurrection; fo r since a l l circu lation , a l l movement of the liquids are stopped, the animal Is a dead animal; a t le a s t , we have no other conception o f the s ta te of death. I believed It o u # t to be proved i f the ca te rp illa rs Whose liqu ids have actually been frozen, come back to l i f e , as I t were, Our common ca 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 other species had the ab ili ty to re s is t such a great cold. One o f those Wiose resistance against cold I wished to ts e t was the Fine ca te rp illa r , of which we shall speak soon; and o f those Which were hatched and raised on th is species of tree in the v ic in ity o f Bordeaux. I put several of them in a glass tube and'made them suffer, 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, or Ilbw harder Ice. I broke several o f them as one breaks a soft 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 of cold much less than that Which affec ts the common ones Is su ffic ien 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 -IUiO.) was su ffic ien t fo r the la t te r ones. I have taken from the tube Which had attained 8 or $ degrees of cold (-10 to -12% .), 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 of l i f e , and soon regained th e ir few er v i ta l i ty . But these larvae had not heen frozen completely. Al thou# they had a certa in amount o f r ig id ity When taken from the tube, they s t i l l had a degree of 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 s tiffn ess Wileh they had, only came from vapor Wileh was frozen around them; a vapor similar to tha t Wiieh freezes on the outside surface of the vessel Wilch contains the mixture o f su it and Ice. • th a t 1® certa in , 1® that I have never seen larvae Which were rea lly frozen, those liquid® had turned to ice , Shich were, not k illed . S tarting when a l l movement o f th e ir liquids ceased, they were perfectly dead c a te rp illa rs , ju s t as any other animal la a sim ilar case would be a dead animal. But there remain always these peculiar fac ts , that in sp ite o f the small amount o f heat in the body o f certa in species o f ca 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 of cold considerably more than tha t o f our hardest winters. That there are specie# of ca te rp illa rs much la rg e r, end in appearance much stronger. Whose liqu ids can be frozen by a degree o f cold­ ness much le ss than that which does not a ffec t the liqu ids of others. The Mnd of blood, the liquids Whitih circu late In the vessels of different species of ca 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 la t te r w ill be hardened, reduced to ice , by a degree o f cold much le ss than mother degree of cold, under which a very strong brandy w ill a l l remain as a liqu id . " I t i s known that movement of water Is an obstacle to freez­ ing; quiet water, tha t of a ditch o r pond, freezes. While the water of a riv e r remains a liquid ; the more rapid the Current, the less chance of so lid ify ing . I f the c ircu lation of the liquids of our small consaon larvae were more rapid than the circulation of the pine ca te rp illa rs , from that 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 the irs ; but th is consideration has l i t t l e o r no part in the effect we are considering. I cut o ff the head o f three of 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 live and healthy; I lo n re d the tube into a Mature of ice and sa lt which made the liqu id o f the thermometer drop to 15 degrees below freezing (-1818^0.), When I took the ca te rp illa rs out of the tube, those Which had had th e ir heads cut o ff were p liab le and soft lik e the others; th e ir liqu ids had not been frozen. PreaAshtie I t follows that these liqu ids do not need to be in the movement o f a rapid circu lation in order to conserve th e ir f lu id ity against a degree of cold o f 15 degrees belew freezing (-18.8*0.). Ie are not surprised that o f the inflammable o r spirituous liqu ids, and of the liquid# charged w ith ,sa lts A ich re 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 tha t a liqu id Which i s not a t a l l inflammable, A itih seems to us very Insip id and quite watery, that such a l iq 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 spite of great cold* That liqu id la not, then, so simple tha t we judge i t by the same standards we usually use to discover the nature of liqu ids. "The blood of large animals, b irds, quadrupeds, and ourselves, easily coagulates; besides, they are more easily frozen than the blood of insects. The blood of a pigeon, Which was made to flow warn Into a glass tube, was reduced to hard ice by a degree of cold of 7 o r 8 degrees below freezing (-9 to *10% .), and could have been frozen by a le ss cold. The blood of a lamb sustained three degrees of cold (-3.75*3.) without freezing, but a cold of . 5 degrees (-6.2*0.) converted i t Into ice. Large animals have in th e ir bodies a heat and a principle of beat which i s not found in those of Insects. Mg animals, then, have no need o f having a blood which freezes as d if f ic u lt ly as that o f Insects. 't "Whoever made Insects seems also to have constituted th e ir blood d ifferen tly according as they are exposed to endure greater o r less cold. We have seen, besides, that numbers o f species of insects, a f te r having lived in the form o f c a te rp illa rs , pass the Whole winter In the fora of ch iy sa lld if , and that there are chrysalids' which during th is harsh season are attached to walls, 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 of s i lk o r some other m aterial. Such i s the chrysalis of the most handsome of the cabbage c a te rp illa rs , and such are nenbere o f other chrysalids of the kind Which have the Industry to suspend them­ selves by means of a band o f s ilk threads. I have subjected several o f these chrysalids to very great degree# of cold, cold o f more than 15 to 16 degrees below freezing (-18 to -20% .), without th e ir freezing. We know that other chrysalids pass the winter down In the ground; there, they are not exposed to as great a cold as they are In any part of the a ir . I have subjected to a cold of 7 o r 8 degrees below freezing (-9 to -10% .) several o f those Wtilch stay underground; i t was su ffic ien t to make then perish. Thus the Insects Which remain exposed to great cold are in & position to withstand i t . Hiose which are more sensitive 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 re s is t . I say that they act as thou# they foresaw, because i t i s not the approach o f winter o r the actual cold s&ich causes than to enter the ground; we have seen that there are ca te rp illa rs which burrow in July 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 into chrysalids; and I t Is not u n til the following year that the bu tte rfly leaves I t s chrysalis." ; TJvarov (1931) gives an excellent review o f the subject, In Which Ie Included a table l is t in 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 of cold resistance In Insects, Uvarov s ta r ts with Beanmr1S explanation In 1736 of the resistance of an Insect to apparently complete freezing. Bemir explained that While an Insect may appear to be completely frozen, there remain some flu ids in the tody Which freeze a t a much lower temperature, and that death occurs only When a l l o f the flu ids are frozen. This viewpoint, considerably older than that o f EachnaetJew, i s nevertheless much more accurate. Although BachmetJew (1901) made a few technical errors In h is work which led 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 thermoelectric method. Hle observations showed that an Insect can be cooled gradually to a low "c ritic a l point", about -10eG. At th is point, Which Is called the under-coolIng point by present- day workers, ice begins to fora in the tissues and the temperature rises due to the libera tion o f heat o f fusion. Hie point to Which the temperature r ise s is not designated by any particu la r name by Bachmetjew, but I t I s , o f course, below 0*0. This point w ill be referred to In th is paper as the "rebound point*. 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. Further gradual cooling causes the Insect to become frozen quite hard. In th is la ten t o r anablotlc s ta te no metabolic processes are possible I but the Insect can be reanimated by warning. I t Is only When fhe Insect Is cooled to a certa in " fa ta l point* tha t I t Is k illed , and Bachmetjew places th le point a t the same temperature level as the " c r it ic a l point". The theory Is based on the purely physical conception of the supercooling of flu id s, hut i t has no hearing on the free sing o f insects, a fac t which has been demonstrated not only by la te r workers, but by much of Baehraetjew*s own data. A graphical representation of BachmetJew1 s conception i s given In Fig. I . He Considered that the " c r it ic a l point" depended on several con- • dltlons! ( I ) , the velocity of cooling; (2 ), the development and sex of the specimen; (3 ), the nu tritional s ta te of the insect; (U) the repetition of cooling; (5 ), the time of exposure, and (6), the "sap coefficient". Bachmetjew1 s theory was severely c r itic iz ed soon a f te r i t s appearance by Kodls (1902), according to whom the super-cooling of body flu ids has nothing to do with the freezing of water in the protoplasm, with which the fa ta l effect i t connected. Unfortunately th is critic ism escaped notice and was ignored by BachmetJew in la te r works in which he repeated and developed h is views. Maximov (1913) offered the following serious critic ism o f Bachmetjew1S theory. By determining the quantity o f ice formed in the pupae o f Celerto euphorbia# L .. BachraetJew found tha t a l l the flu ids in them froze completely a t -U. 5°3. while the c r i t ic a l point was -IO0C. These figures seen to support the theory. However, the specific heat o f the frozen pupae within the lim its -6 .7 to -16.3 was found by BachraetJew to be 0.$17, very l i t t l e lower than tha t o f the body flu id s, (1 .01). Since the specific heat of ice i s only h a lf that o f water, Maximov concluded that freezing was got complete a t -U .5°C. The same conclusion Is reached by considering the fact that of the U$ sa lt content -9 - - 10- B r DcakK 7$E.T-mancnt Iitutt jh u f fo f Ternj^orahy aHu /V = Uccjinnincj oj- Keak sh j^o r «5u ^ >rd - Oj>K m a I ZLone k.2 * O j> hm v rn Su b -oj>hmal zone = 3 e c |in n i 'n ( j c o l d jKjjx**- ~Fej-riporary co ld 3Kj^or- -su^ g lcd - n » "Temporary cold 3hujx>r 1— - ; ■ Zones of v i ta l i ty and death of an in sect. (Re-drawn after Bachmetjew1 IQOJ) 1 Fig. I . - I l - of Insect blood, about 80$ Is sodium chloride, which has a eu tectic point of about -22%. Thus, even I f no other sa lts were present, the body flu ids could not possibly freeze completely a t -U.5*C. Eecent experiments by Sacharov (1928, 1930) show that I t Is Impossible to freeze completely the body fluids o f certain Insects even a t -21.2% . Payne (192?) found that com­ p lete freezing occurs only In the neighborhood of -bO* or lower. TJvarov points out that Bachmetjew's principal mistake lay In M s re­ garding the "body flu ids of an Insect as a homogeneous liqu id which follows the re la tive ly simple physical laws o f supercooling and freezing. This viewpoint Is obviously Incorrect since apart from dissolved e lec tro ly tic substances, there are Insoluble fa te , pro teins, end other colloids which are bound to » - ' affec t the physical properties o f the flu ids. 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 lines has c la r if ie d the subject In many de ta ils , althou^i the actual physiological phenomena, Shlch a f te r a l l form the true basis of the problem, are as yet very vague. P eriod icity la cold resistance has been one of the main points of attack , and In th is connect­ ion more work has been done with plants than with !a sse ts . Chandler (1913) and Bosa (1921), showed that ce rta in p lants exhibit period ic ity gad tha 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 variation In the cold resistance of Insects. They observed that larvae of Cossas Coewus I . survived repeated freezing a t -20* In winter, but larvae of the same species taken la spring succumbed a t -I?* . Knight (1Q22) supercooled P erlllue Meculatus in winter -12. to -17% and la one case even to -26* without freezing, yet In MastSh and la te r a temperature o f -10* caused freezing and death. Bodine (192L, 1923) found that the to ta l water content of certain 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 of her f i r s t works (1926a) she found that In the ease of oak borer larvae, which are normally subjected to extremes of temperatures, freezing points vary with individuals and seasons. She found an excellent correlation o f freezing and undercooling points with average monthly temperatures. In the f a l l , the undercooling point f a l ls ahead o f the outside temperature, ac ting as a factor o f safety. In the spring, the hardiness i s lo s t with rising temperatures and i t i s a t th is time that a sudden cold snap Is 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 ic ia l ly by holding non-hardy insects 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-water 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 resistance, and vice versa, ("low* and "high* are used In th is paper in a geometrical sense, not arithmetical; l . e . the sign is considered.) The points were determined by means of a thermocouple and a pyrovolter, but the author does not s ta te the method of freezing. J t may be pointed out here that Payne1S use of the term "freezing point* i s not exactly correct, the true freezing point being s ligh tly higher than the -13- "retioxmd point11, as w ill be pointed ont la te r . Payne (1926b) soon afterward* selected for comparison thrw ecological groi^si ( l ) the oak borers, normally exposed to extremes o f temper, atnre; (2) the aquatic insects, never exposed to temperatures below G*C,, and (3) stored product Insects, representing, supposedly, a trop ical o r a sub. tropical group. In these experiments the oak-borer larvae showed marked period icity , as already stated. !Rie aquatic insects showed no period icity , nor was there any significant difference among Individuals, species, orders, o r stages of development. The mean undercooling of 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 pests, Payne found no period icity , but found more variation 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. tr ied to harden them by a moderate lowering of the temperature over a long period o f time, but the resu lt was death. !Rie natural conclusion o f these workers was that those insects 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 ic ia l ly exposed, even when th is exposure Is made gradual. TW hardening of certain lneeete in the f a l l , o r when placed a r t i f ic ia l ly a t moderately low temperatures, as evidenced by a drop in th e ir undercooling and freezing points, led Boblnson to apply to Insects the * bound- water" theory already developed hy Hewton and Oortner fo r p lan ts. According to th is theory, mder certain stim uli, ( la th is case low temperatures), the hydrophyllc colloids present In the Insect body are capable o f adsorbing or •binding* water. %e water, on being "bound*, loees most of the typical physical properties of water. Ib r example, the freezing point of bound water Is greatly depressed, and indeed i t is on the assumption that a t -20%. none o f the bound water but f i l l o f the free water Is frozen, tha t Ibblneon1S (1931a) method of determining the bound water content of a system i s based. Bie method as applied to an Insect, Is b rie fly as follows: The Insect, o f known wel#it. Is frozen a t a constant temperature o f -20%. fo r several hours and then transferred quickly to a calorimeter, where & determination Ie made of the number o f calories required to melt the ice formed within the tissues, th is determination Ie based on the fac t that to melt on# gram of lee without raising i t s temperature requires 80 ca lo ries o f heat. By calculation, the amount o f free water per gram of so lid i s determined. The fin a l step is to dry the material 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 of the bound water in the specimen. Bie theory of water binding i s o f great Importance in winter harden­ ing. The adsorption of the water occurs on the surface o f the collo idal 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 large surface. Under a fa llin g temperature, the p a rtic le s a t tra 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 greater than the diameter o f the p a rtic le . Bte water on the Inner layers i s held by Inconceivably blg& -15- pressures 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 tr ic ity ; I t w ill not dissolve such substances as sugars; I t can be considerably compressed; and i t s freezing po in t le greatly lowered. With fa llin g temperatures of autumn and adsorption o f water by the colloids In the insect tissue . I t i s obvious tha t the remaining aqueous solution w ill be more concentrated and the freezing point w ill drop. A certa in degree of protection against cold weather Is thereby established. Hewton and Gortoer (1922), working with hardy varie ties of winter ^aeat, established the fact tha t fo r plants there is a d irect correlation between winter hardiness and percent of bound water. Boblnson (1927), was the f i r s t to show that the same held fo r certain Insects. He tested the hardy & the moderately hardy * uramethea. end the non-hardy granary weevil, SltooMlus granarlus. In arriv ing a t the same conclusions as Hewton and Gortoer. He hardened the f i r s t two species both naturally , out­ doors, and a r t i f ic ia l ly in a constant temperature cabinet held a t -13% ., Just 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# of the water was bound. This Increased during the experiment 6e* *12-52#. I t Ie In teresting to note tha t the to ta l water content remained the same. - BoMneon stresses the Importance of the per cent of water botmd before equilibrium Ie reached. I f am Insect In a non-hardy rummer condition Ie placed In a refrigera ting cabinet representing winter conditions, i t is exposed to an unnaturally abrupt -change and may be k illed 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 of water adsorbed ahd how quickly; (2) water- holding capacity, to Whow the a b i l i ty to re ta in bound water under conditions o f rapid rises in temperature, which Ie especially Important in spring mortality. I t has ju s t been sta ted tha t Eoblneon (1927) in M s experimental hardening of Telea nolybbenme and Callcsamla oromethea found that the to ta l water content remained the same. Payne (1926b) s ta te s tha t the most pronounced feature of hardening was the low mole ture 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. Periodicity was thus exhibited In moisture content. The to ta l water content o f Smchroa nm etata varied from 31*1$ In Pebruary to In August. That o f Dandreidss canadensis varied from 57.1# to 73*53. The larvae were haked fo r four hours a t $0%. The adequacy o f th is method of desiccation w ill he questioned. In the same paper Payne describes a multiple freezing experiment. Repeated freezing of the same insect o r tissue exhibited no hyste resis , and the rebound and undercooling points remained the same. Samples o f blood from the aortas showed definite c ry s ta ls , while transparent larvae were also seen to have crysta ls within at the time the freezing point was recorded.. The process o f freezing In th is group was Interpreted as c ry sta llo ld a l, the f i r s t o r primary freezing point being that of the blood. Payne found farther tha t the hardened oak-borer larvae survived freezing, and that on lowering the temperature s t i l l mere, second undercooling and freezing points were recorded. The secondary freezing point occurred n«ar fo r the oak-horer gro-up and was always fa ta l. The tissue ft©#*. !»€ a t th is temperature was not defin itely Iso lated , W t the nervous tissue and fa t were suspected, Payne went so fa r as to sta te that Insects are k illed 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 that Insects with h i # freezing points were never able to withstand long exposures to low temperatures. However, insects with low freezing points could be k illed 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. The undercooling point recorded was-hS•; the rebound point - h $ \ , Apart from the seasonal varia tion In cold hardiness, there ex ist variations during the individual development o f an Insect. Ludwig (192S) found considerable difference in the a b i l i ty to withstand low temperatures among the various in sta rs of Japanese beetle larvae. 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 winter Is usually passed. Payne (1927a) c a lls a tten tion to the fac t that two factors o f heat eaorgy are involved in the study o f cold hardiness: ( I) the Quantity factors (2) the In tensity Factor. Cold hardiness may thus be e ith e r the a b il i ty to withstand long periods of moderately low temperature, (the quantity fac to r). -IS - o r the ab ility to w lthatm i short periods of Intensely low temperatures, (the Intensity fac to r). In her experimente, aquatic Insects, considered highly specialised along the quantity factor, endured long periods a t 0*, hut none survived freezing, even though the freezing point was only about I e lower. Another group that may be specialized along the quantity fac to r i s that group o f so il lneecte normally liv ing below the fro s t lin e . Most o f the stored product Insects cannot withstand doraacy. TM oak-borers develop a b ili ty to survive doraacy In September and October, but a t tha t time are • t i l l non-hardy to the in ten sity factor and are k illed by freezing. Upon fu rther low temperature exposure, o r else dehydration, they become hardy to the In tensity factor. In considering cold hardiness to the in tensity fac to r only, Payne * (1927c) brings out the Importance o f the water content. She fnr m d 4 i ■ «ad g tacrls la y lrg ln lca to be self-dehydrating In the f a l l . Whereas Pon lllla japonlca did not exhibit th is phenomemn. She considers tha t the f i r s t two species le s t a ll, o f th e ir free water. Boblnson (1927) found that In hardening Telea Polyphemus and Galleseala nromethea. the to ta l water content remained the same. I t therefore appears that certain 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 Ponlllla laoonlaa. 1]# A r Dsmdraldea Bk* for Synchroa wunctata. and 20# for mfulnm. Tha oak borars are ee lf - dehydrating, she sta tes , hut never Ioee a l l o f th e ir free water. Payne In the same paper p lo tted blood conductivity readings against survival tmqysTeture# !Toir Popillla japonloa. Pl#*Tl#ia vlrelnlca aaad Demdrais^ GanatSmwl*.. and found an excellent correlation. I t Ie extremely unfortunate tha t she does not s ta te her method of determining survival beyond the f@@% that the in tensity fac to r only was considered. The method o f free sing end thawing and of handling m e t ce rta in ly affect the resu lts . In a la te r paper, Payne (1928) discusses the various factors affec t­ ing the hardiness of the Japanese beetle to the In tensity facto r. They are ( I ) dehydration; (2) disease; (3) nu tritiona 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 selective. Hte survivors were cold- hardy. larvae kept a t 20%. and 100$ rela tive 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 tive humidity for one month, they also lo s t h a lf th e ir weight, but 25$ survived freezing. The freezing points were high. Some very in te resting points are brought out In the earn# paper in regard to starvation and cold hardiness. "In general", s ta te s Payne, "early stages of starvation are accompanied hy an Increase in cold hardiness, l a te r stages ty a decrease. The point o f decrease of hardiness comes when the digestive tra c t c lears, fresh ly molted larvae cannot withstand freezing w t l l they have eaten. Pre-pupae with c lear digestive tra c ts are not cold hardy*. This refers to both the quantity and In tensity facto rs. Larvae o f the Japanese beetle frequently exuded a f lu id upon thaw­ ing. When th is gave a te s t fo r amino acids and pro teins, 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 oo# t to be due to oxidative enzymes libera ted by a change In c e ll permeability. -19- -20- No change in pH o r reBplratory quotient mas associated with hardiness to e ith e r factor, hat the respiratory rate in hardened larvae was much lamer than in non-hardy ones, Also, as the length of time the larvae mere kept a t 10%. increased, the percent survival a f te r f reeling decreased. Fayse con­ cluded that the two types of hardiness are inversely rela ted a f te r a certa in point has been reached. I t cannot he in terpreted as a lose of v ita l i ty , since larvae kept under such conditions can complete th e ir development with a normal death rate i f removed to room temperature. In fac t, the development i s accelerated ty th is doraacy. Sacharov (1930) studied changes in the f reeling point o f ca te rp illa rs of the Brown-tail moth, Hramia sbaeorzhaea Don., taken stra igh 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 fa t decreased. The cold hardiness mat greatly reduced hy the three to four days o f feeding. Comparing hardy wood-holing larvae o f Plaaionotue arcuatne L. and the non-hardy Mney bee, t e l l a e lllfe ra . he found tha t the hardy insect contained only 5 ^ of water, hat 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 that these data prove convincingly that 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 of recording temperatures was le f t un­ changed. The general set-up is shown in Hg. 2. Temperatures were read hy means o f thermocouples and a sensitive galvanometer. A description of the galvanometer Is 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 ritica 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 fee t long, Txmt into the form o f an arc of a c irc le whose center was represented by the galvanometer mirror. The radius ~ was 3 fee t. A 3-volt galvanometer lig h t bulb was mounted In front o f and s ligh tly below the galvanometer mirror, so that i t s reflec tion was thrown by the m irror onto to the scale. The K ale was calibrated from+ 1*0. to -13% ., represent- ing an external resistance o f ho ohms, smd from +2%. to •29% ., representing a resistance of 100 ohms. The degrees were marked o ff by means o f black adhesive tape. The calib ration 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. of d is t i l le d ra te r in a v ia l. The v ia l was lowered into a s a l t and lee solution and cooled very slowly. In order to eliminate the lag of the thermometer mercury behind the thermocouple. The calib rations were checked several times - 22- CONSTANTAN COPPED Fig. 2. Diagram of apparatus used. A, Galvanometer; B, Galvanometer ligh t bulb; C, Scale, calibrated as shown in Degrees Centigrade; D, Resistances o f to and 100 Ohms; E, Dewar flask known junctions, containing ice and water; F, Thermocouple used to record insect temperature; J, Thermocouple used to record environmental temperature; K ,Calcium chloride bath; L, Refrigeration c o ils . v durlng 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 flask , the 0*6. mark wws the same on both scales. I t w ill be seen tha t with such a large scale as that presented by a 16 foot board, and with the sensitive but well-damped galvanometer used, great 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 per minute and the observer Is recording the time, an accuracy of 6.1* Is a l l that 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 refrigeration cabinet used was la I t s e l f en 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 refrigera tion c e lls running around the Inner sides. The Internal dimensions rare 23 Inches x 11 inches x 20 inches in depth. The coil# rare cooled by.the expansion of ammonia, regulated by an expansion valve situated ju s t above the cabinet. The refrigeration plant consisted of an ammonia compressor end a 2 H.P, motor Which rare used to cool an adjoining store room. Dy closing the valve to the -23- coil* in th is room, the valve above the refrigera tion box eonld be opened and the la t te r cooled. Many d lf f lcn ltie e were met with here, - elm## the w i t va* not automatic and the expansion valve me Inadeqwte. 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 ircu lation and a nlchrome wire heating w i t operating through an e lec tric relay from a mercury toluene thermo regulator. Ih is type o f thermo regulator hae come into common use so i t w ill net be da- eerlbed here. With th is apparatus, the temperature as read by a thermometer inserted th rou# 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 insects, Which were frozen In a sim ilar r i a l and sim ilar position. Due to the un­ re l ia b il i ty of the ammonia refrigeration apparatus, constant atten tion was needed to keep the cabinet a t a constant temperature, so tha t 10 hours was about the maximum time of running. Later in the work a constant temperature hath was substitu ted, and found to be much more convenient and re liab le . A bucket o f eWlelw chloride solution wittx a f reeling point o f •$$• was placed in the refrigera tion cabinet, and in i t were arranged a motor s t i r r e r , a knife heater, and a Micmry toluene thermo regulator. A quart s i lk bo 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 of the milk bo ttle was sufficient to allow the passage of the insect to he fro sen. Bie temperature o f the a i r in the s i lk bo ttle wee recorded by means of e thermocouple situated about h a lf an inch away from the suspended insect. By throwing a switch, e ith e r the temperature o f the-insect = -25- e r that of the a i r near I t could be read from the scale. Since the bath liqu 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 heater. Twm then on, the bath cooled a t a ra te o f about 0.1* per hour, so that 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 of a thermostat. Occasional use of the knife heater would keep the temperature constant to within 0,1* or 0.2*. The thermocouple used In recording the temperature of the Insect was designed to f i t under the e ly tra o f the adult potato beetles, on Which p rac tica 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 ce rta in copper and Constantsa wires were used. Only two se ts o f thermocouples were used In th is work, and they were iden tica 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 i t . The bead was then f iled u n til i t was f la t , and the contact of 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 of the thermocouple and cemented Into such a position that the thermo junction extended about h a lf an Inch beyond the edge of the glass tubing. The connected wires are o f course Insulated, and fo r frees- Ing adult potato beetles they are heat Into an arc sim ilar to tha t presented by the e ly tra o f the beetles. The thermocouple then f i t s snugly between the e ly tra and the so ft body and forme an excellent semi-internal contact. —26— LOW T5MPEBATOB3 SUBTTTiT. SXPBmTWBaT: in experiment was undertaken to determine the time-temperature mortality relationships of Leotinotarsa deceallneata M olts. This experiment was performed during the la s t part of AogAst and the f i r s t part o f September, on Insects taken d irectly from the fie ld . Tea series of five beetles each, In ten eosfced v ia ls , were placed in the refrigeration cabinet which was held a t a constant temperature fo r ten hours, At the end of each hour one v ial was removed from the cabinet and allowed to warm a t room temperature fo r 2U hours. At the end of th is time, the mortality was determined. Due to the habit of these beetles of feigning death, they were subjected to a heat of 30* to 35*0. fo r about ha 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 lec tric lig h t bulb held close by. Any movement except a very slow muscular contraction was considered to indicate l i f e . The constant temperatures used were -5 ° to -12* In one degree in te r ­ vals, and most of these were duplicated. The experiment required one day fo r each temperature, since only one cold cabinet wae available. H g. 3 shows graphically the resu lts o f these experiments. A temperature of -5* produced no mortality in ten hours, while temperatures o f - ll* and-12* produced 100$ mortality during the f i r s t hour. The curves a t temperatures o f -8 , -9 and -10* are intergrades, as shown. 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 uniformity o f resu lts could be obtained. The explanation - 27- -ig - ?• Time-mortali t y carve of non-hardy adults of Leptlnotarsa decemlineata. - 25- probably lie* la the fact that th is temperature range I* tha 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 certa in o f the Insects are frozen by exposure to -6® o r -7®, eh lle others do not reach th e ir undercooling points a t such temperatures, the amount o f mortality w ill be affected. I t l s assumed that an Insect which Is held unfrozen at a temperature "7* for 10 hours has a much t e t t e r chance o f survival than one which Is held, frozen, a t -7* fo r the same time. This factor w ill be seen to affect the -8® curve to some extent, making I t Irregular. 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 that In such a case the mortality 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 Insect. 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 beetles were treated In the same way a t -6®, without the rebound taking place, althou#i th is temperature I* below the normal undercooling points of many o f these Insects. 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 beetles o f sim ilar condition showed that only U beetles out of 120 had a freezing point of lower than -5®. ,The beetle# were, then. In an undercooled condition when they were subjected to ag ita tion , yet th is was not su ffic ien t to s ta r t the c ry s ta llisa tio n o f Ice. -29* FB2EZIRG CUMFS The apparatus used la much that shea a sample Is being frozen, the complete time-temperature reactions from Oe down Is presented on the scale in front of the observer1 e eye. In order to preserve th is visual data, the idea was early conceived of recording time as well as temperature, and p lo tting the two as a time-temperature curve. This idea has proven extremely useful,' since I t enables one to In terpret what happened to the Insect as the temperature dropped. A sample curve of d is t i l le d water is shown in Fig. U. Water, being a homogeneous system o f inown properties, shows very well the various stages of which the curves are composed. F irs 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 the rebound point, usually s ta r ts fa s t and ends slowly. Third, from the rebound point, the temperature begins to drop again, but more slowly, since the heat of fusion of the freezing ice must be dissipated in to the environment. As more and more ice is formed, the specific heat i s lowered, since tha t o f ice i s only about ha lf that o f water. Thus the curve gradually becomes steeper, as may be seen in the figure. As the temperature approaches that o f the environment, however, the temperature d iffe ren tia l becomes le ss and less and the curve approaches the environmental temperature assyoptotically. In the figure shown, 0*3253 grams of water were frozen in a small v ia l , with the cabinet a t a temperature of exactly -IO0C. For comparison, a time-temperature curve is given fo r a potato beetle TE M PE RA TU RE I N - 30- TIME I N MINUTES Fig. U. Freezing curve o f O.3253 grams o f d is t i l le d water. The lower curve represents the temperature o f the refrigeration cabinet. adult wiggling 0.1U60 grams. This Is shoim In Hg. $. cabinet temperature was constant a t -12, the ca lc lm chloride hath being used. The final stage Is not represented In th is curve since the beetle was needed alive for fu rther freezing. I t w ill be noticed that the temperature Srsps from the rebound point almost In a straigh t lin e , whereas in the ease of water, ■ , the temperature remains at the rebound point fo r tome time, and then f a l ls In a gradually accelerating decline. The difference i s probably due to the lew water content of the beetle. A Tijralld larva weighing 1.1S20 grams, and the same welgit of d is t i l le d water, were therefore frozen under Identical 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 larva. The outside of the larva was carefully dried befbre freezing. Both freezing curves are given In H g. 6. I t w ill be seen that th e Tlpulld larva remained almost a t the rebound temperature even longer than the water. A h lg i water content is therefore assumed, In contrast to the potato beetle o f Hg. 5* - 32- ' 500 +00 TIME IN SECONDS F ig .5. Freezing curve of 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 i l le d water under almost identical conditions. The lower curves represent the temperature of the refrigerating cabinet. MtiiTiPLi ramtim Payne (1926b) describes a moltlple freeslne e ^ e rle en t. Bepested freezing of the same In s is t o r tissue e AIM ted no hysteresis, she found, end the Tmdercooling and rebound jo in ts remained the came. Two th ird -lne 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 that 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. -3L_ TJLBLS I . T rial I 2 5 — 5— 5 b 7 g 9 10 Corrected freezing uoint -6 .7 -7 .4 -6 .6 -5 .5 Bone -6 .7 - # . lf, Sone None Son# Temp, a t A lch removed I i ~9.0 -9 .0 -9 .0 -11.5 —20.0 —10.0 -19.0 -17.0 -17-0 -17.0 % The other Insect was also frozen 10 times, but each time was removed when I t had reached a temperature of -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. Hte resu lts are l is te d in Table I I . The cabinet temperature varied from -12.5* to -24.2*0. TJLBLS I I . x Trial I 2 1S 4 9 6 7 I Q 10 Corrected freezing -3 .5 -4 .4 ue Int i ' -3 .9 -4 .5 -4 .7 -4 .2 -4 .0 . # 5 .4 .3 -4 .7 Temp, a t A ich -S0.0 -10.0 removed -10 .0 «10.0 -10 .0 -10 .0 -10 .0 -10 .0 -1 0 .0 -10 .0 Time to cool to -10*5. 12 11 ihSb» f I l IU 19 13 10 9 g g iao ther experiment was carried on to determine the effects 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 of -IOeOt O. A thermometer Inserted th rong a cork into a sim ilar v ia l and in a sim ilar position, was used to record the cabinet temperature. A strong a i r circulation was provided by a fan, and heat ms# obtained from co ils of nlchrome wire connected th rou# a relay to a mercury- toluene thermo regulator. The temperature as recorded by the thermometer varied by about 1.0% . In freezing the Insects, 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 defin ite ly reached, the insect was removed from the cabinet and allowed to : - , ' ' warm a t room temperature. The ra te of cooling varied from about 1 .5* to 2.5* per minute, calculated from 0* to the undercooling point. Table J l I gives the corrected freezing points of 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 tha t there is no defin ite trend in the changes of the freezing po in ts . The insects used were taken from the f ie ld just before use, and were kept in v ia ls a t room temperature and fed potato leaves during the experiment. They were, of course, in a non-hardy condition, four out of 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. -35- TABLS I I I , freezing Pointe and the Average Magnitude of the Rebound, in Degrees Centigrade Beetle S b , . , .. I 8/26 -2.3 -1 .4 S / a 8/19 9/1___9/1 1 /6 q/7 O O t O ° ° O % O 0 «0° f o t O ° O ° O1 ° 0O ' ° uO 0O0O O O O °n 00 00 O ’ Ov O O O ° % 0 ° ° R A T E O F C O O L I N G I N D E G R E E S P E R M I N U T E . Correlation table showing the relation between freezing points and rate o f cooling of Len t in o t a r s a adults. Fig. 7 -Io- polnts so that they could be kept alive fo r re-free zing. For th is reason, any statements made in th is paper regarding the occurrence of only one rebound must be lim ited to the temperature range used. Below th is temper- ature, farther rebounds ml^ it occur, bat th is has not been determined as yet fo r Ieotlnptarsa decemllneata. -U l- ITOLTOPIg BSBOTma Dartng the course o f various experiments on the freezing of Leptlnotarsa ^ecemllneata adults, there were frequent cases In which more than one rebound occurred. The same phenomenon has been observed In the f re e s - Ing of grasshopper nymphs of a hibernating species. In such cases, however, the maximum number o f rebounds recorded has been two. Potato beetle larvae have been given as many as five d is tin c t rebounds In a time in terval of le ss than three minutes and a temperature range of -4 .9 to -6.3*5. Pig. 8 shows the particu lar case referred to , In which four rebounds are defin ite , and the hesita tion a t -4 .9* i s considered as a rebound. The free sing apparatus used was such that the minor rebounds were recorded accurately, since the galvanometer was quite sensitive 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 un it, since the c ircu lation of a i r in the cabinet was adequate and the beetle 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 po ss 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 sec t's body, exactly as In the case of the major rebound. Before each of these rebounds, necessarily, there was under- cooling, and since an aqueous solution cannot possibly undercool In the presence o f ice, each rebound must represent the independent freezing of separated systems. While multiple rebounds have have been o f frequent occurrence during - 42- ZOO TIME I N S E C O N D S F ig .8. Freezing curve of a non-hardy Lentlnotarsa adult, showing multiple rebounds. th is freezing work with potato beetle adults, no regularity has been observed, even with‘the same individuals. One factor seeming to affec t the occurrence o f more than one rebound Is the rate o f cooling. In running the experiment l is te d under "Multiple Freezing", the cabinet temperature used was -10e. In th is experiment i t was noticed that 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 that he can te 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 Is the only rebound, I t Is of f a i r magnitude; i t begins with a fast rise in temperature, gradually slowing as i t approaches the rebound point, Where i t h es ita tes 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 r ise in temperature to the rebound point is very hesitan t and Irregular. Moreover, in such cases the rebound point Ie no sooner reached than the temperature begins to drop again quite fa s t. When the la s t rebound point i s reached, the temperature drop i s very slow, These phenomena are I llu s tra ted in Figs. 10 to lU. The w riter therefore feels confident that 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 insects had been allowed to cool fu rther. This seemed to be the case only when cabinet temperatures of -IOe 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$ of the insects had more than one rebound. For comparison, the occurrences of Multiple rebounds under varying cooling conditions is given in Table IV. TABLB IV . Cabinet temp, used Ho, cases Multiple Rebounds Total Ho. frozen Percentage of -1010.5*0. 0 117 0 - 7.5 to -13.5*0. 0 Ug 0 -1210.2*0. 32 50 6U.0 -13.5 to -25*0. 11 92 12.0 —20 to —30 eC. IS 107 lU.O I t Is suggested tha t In the case of the loimr ra te of cooling there Is lees 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 Irregu lar cooling ml#it lead to the freezing of the outer tissues sooner than the Inner ones. However, a change of 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 greater difference In the rate o f cooling than would th is two degree difference in cabinet temperature, so tha t the above suggestion seems to be un justified . Further analysis Is needed, but w ill not be under­ taken here. . HARDENING 0? I3PTIB0TABS1 ADOLTS Ia order to determine the effect o f moderately low temperatures ©a Laotlnotarsa o r the quantity facto 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 beetle removed a s soon as the rebound point had been defin itely reached. The beetles were divided Into four groups of five , 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 of about 20 ce. capacity, the ones a t 8* and 11* being fed potato tuber. L itt le or bo feeding was done a t these temperatures. Each series was removed and frozen as above a t weekly Intervals fo r s ix weeks. The refrigera ting 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 fif teen cases out of one hundred and seven, o r lW , more than one rebound occurred. Table 7 gives the corrected freezing points 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 multiple points are comparable to the single ones. Troa observatlpn of the table, I t w ill be seen tha t no hardening occurred a t 8* o r a t 11*, and that hardening was greater a t 5® than a t 2*. In th is experiment, as In others that the w riter carried 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- M RZ IfG POINTS 07 SERIES 0 ? LBPTIWTAMA TKCawr.Tmg^ CORBBCTBD ACGOHDIM TO fOBfOLA. . TABLE V. (Cab, T«mm. "Bmm m -20» to -1SOtO.) Bo. 0 iteeki I week 2 3 weeks 4 week# 5 weak# 6 week#I -3.99 -5.it2 -u.55 -7.36 , Dead 2 -U.81 -U.72 -K 58 -7.54 -9.31 -7.97 Dead 2"C. - -U.05 -6.00 -7.37 -6.33 DeadU -3.60 4M» -4.36 -7.07 -4.08 Dead 5 -5.22 - -U.U7 -6/59 -5.76 Dead Awr. -U.5U -U.38 -U.Ul -4.37 -7.38 —6.13 " 6 -3.02 -6.96 - U.35 -7.83 -7.79 -9.36 Dead 7 -5.92 -3.86 -6.50 -8.77 -9.19 Dead 5"Q. g -6.35 -5.39 -6.91 -7.28 -7.64 Dead 9 -3.U3 -5.95 —6.81 -6.21 Dead 10 - -3.78 -3.9U —6.82 -7.75 -10.31 Dead Ayer. -U.27 -5*60 -5.1? -4.93 -7.99 -9.62 11 -3.65 -5.03 -2.92 52.18 Dead 12 -6.35 _ -U.27 Dead s«e. 13 -U.U6 -5.2U -U.13 -4.62 -3.70 -4.18 IU - -3.83 -5.05 Dead 15 -3.89 -5.95 -U.07 -5.27 -2.20 -2.19 -2.97 Aver. ^ .9 9 -5. Ul -3.8U -U.98 -2.69 -2.19 -3.58 16 -5.59 -U.07 -1.15 -3.15 —1.06 -1.35 -3.14 17 -3.97 -U.18 -3.94 Dead • I l eC. 18 -3.57 -3.18 -2.U2 -3.36 -3.15 -2.24 -3.43 19 . -5.1U -3.62 -3.75 -3.21 Dead 20 -3.21 — -1.64 —1.86 Dead ; Aver,, —^.30 -3.76 -2.58 -2.90 -2.10 -1.80 -3.28 above result*, therefore, are valid only from a temperature standpoint. The feet that the v ia ls were corked and that potato tuber was placed with the series a t 8e and 11* would produce a M^hi humidity In the series which mt^it ac t against hardening. Another experimental hardening of Ieu tlno tarsa was carried on during the winter. The Insects used were removed from an outdoor so 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 refrigera ting cabinet a t -12*. Time m s recorded, correct to I second, so that time-temperature curves might be con­ structed 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 of the curve beyond th is point. The Insects 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 In i t ia l freezing, the eerie# of 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 Identical conditions, the Insects being weighed each time before freezing. The variations In weight are shown In Table VI. TjfflLS TI Bamomur#. In ##«*# W fIA t . le » i 0 0.1277 grams T * I 0.1090 " I It. 7 2 0.1006 21.3 3 0.1092 1U.5 U O.O976 23.6 The irregu la rity In the flgaree fbr three seeks1 exposure Is probably due to the fact that between the second and th ird week seven o f the se ries 'd ied and were substituted from the "other IO beetles A lch 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 sign ifican t. Here again, however, humidity probably has it# effect and the resu lts must be considered from a temperature standpoint only. The object o f th is experiment when I t was sta rted warn two-fold. Tl re t, i t was thought that by making a Correctioh fo r the variation in weights o f the insects used and bringing them a l l to a common wel^xt basis, the time- temperature curves would be enouA alike to s tr ik e an average of the 10 curves each week. Second, I t was thought that I f the insects lo s t o r bound water in the hardening process, thereby decreasing the percentage o f free water In the * body, the slope of the curve,particularly tha 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 theoretical only, o r a t leas t to apply ohly to a homogeneous system lik e water. Obviously, the freezing curves of d ifferent weights of 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 readily seen by examination of figures $ to lU. The individual insects correspond as indicated hy th e ir nxsnbere. figure 9 shows the I n i t ia l free sing curves of the 10 |.ia tl«n ta rsa adults, before any hardening had been Induced, idien b rou# t to a common welgit basis o f 0.1 gran. This correction waa made by multiplying the time by O .l/x , Where x Ie the welgit of the Insect In grams, figure 10 chowe the sane curves, uncorrected fo r wei^it. SSlnce the weight correction seems to complicate matters rather than to simplify 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 orig inal ten beetles are Included In the three weeks' exposure curves, and only two In the h weeks' exposure curves. In comparing these charts, the main variation seems to come during the f i r s t week of exposure. The undercooling and rebound points dropped about one or two degrees. 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 of the f i r s t week. I t Ie noticeable, however, that the ten Insects vary considerably among themselves and that i t is u se less 'to attempt an average curve. Single cases w ill be noticed In figures 12, 13 and lU where the rebound points were abme really hid&. Bie w riter recognizes that a f te r examination o f these curves, a certa in c ritic ism may be ju s tif ied . I t w ill be noticed tha t the rebound po in t In p rac tica 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 . IN S E C O N D S F ig .9- Freezing curves o f 10 non-hardy Leutinotarsa adults, with time corrected to a weight basis of 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 sects as shown in Fig. 9» without weight correction. Freezing curves one week. a fter an exposure to + 5*0. forF ig .11. - 53- 300 450 T IM E : IN SELCOND5 Fig. 12. Freezing curves a fter an exposure to 4 5°5. for two weeks. - 5% - t i 300 450 T I M E IN S EC O N D S . . Freezing curves a fter an exposure to +50C. three weeks. Fig.13 for - 55- 300 450 T IM E . I N S ECOND S F ig .lU. Freezing curves a fte r an exposure to -AR0G. for four weeks. i» merely the point ehere the temperature eeaeee to rise and s ta r ts to fa ll I t might he charged that the rate of cooling i s so fa s t that the attainment o f equillhrlxsa i s not secured, so tha t the rebound points l is te d are meaningless. Xt i s admitted that the rate o f cooling used i s much g reater than that Which would ever be obtained in nature. However, the correlation 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 tif ie d in concluding that since a variation in the rate o f cooling between '0#7 and 8.6 degrees per minute does not affect the freezing point, a variation between 0.0 and 0.7 degree• per minute would not a ffec t i t e ith e r. I f such," I* the case, the rebound points I llu s tra ted 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, free and to ta l water content, and specific heat# o f the various tissues of the in sect. In sp ite of the fact that th is series m s selected fo r having high rebound points and only one rebound, a l l the charts show a fa ir ly large proportion o f multiple 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 adults in U series o f 10 each were placed under the same harden- lag conditions as the insects in the experiment ju s t described. Eaeh series was welded in i t ia l ly , and each week one series was removed from the 5* hardening cabinet, weighed, desiccated a t 105* fo r six hours, and re-weighed. The resu lts are given in Table TXX, -56- i m a m -57- 1.745 * 1.3W0 I . USlg 1.552 1.2675 1.352» 0.»79 « 0.537 0.3975 0.U595 Trm these data Ijr Is seem that the percentage to ta l water content remained practically constant. Tkt the f reccing points dropped during each treatment, especially during the f i r s t week. To secure this result, the body solutions had to become more concentrated. Two explanation# teem plausible. Wre t, the conclusion to Which IoMneen came, tha t water was bound by the hydrophilic co llo ids, being removed thereby from the role of solvent, with a resulting depression o f the f rooming point. 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 rac tica 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 I f# weight decrease. But I t i s these e lectro ly tes which a ffec t the freezing point depression to the greatest extent. Therefore, with the same amount of electro ly tes and a decreased absolute water content, a mere concentrated solution resu lts and the f rooming point Ie depressed. These two theories are not contradictory, or even mutually exclusive. They are given as possible explanations and i t may be that 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? vm x.T,*pn„Tp* a m o r T g , mmmcT %* ( A In rerlewliig the li te ra tu re ©ae cannot help being lapreeeed with a certain weakneee. P rac tica lly a l l of the knowledge ©f the subject td date I s the resu lt of experimental work, but very few workers have described th e ir methods and apparatus such that others could get comparable resu 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 Is the use o f new terms without adequate defin ition. One thing i s certa in ; i f previous - 1 - ' authors had described th e ir experimental method bo that la te r ones could c r i t ic is e and make Improvements, the subject would be much more advanced today. The tern 1 f 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 ice formed In the bo^r has llh e ra ted 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 that when the f i r s t ice cry sta ls are formed the remaining solution is more concentrated. Thus the observed depression of the freezing v point Is the freezing point of th is more saturated solution. Gortner (1929) gives a very good explanation of th is physical phenomenon In Ms discussion o f freezing point depression. I f a gram of pure water is uniereooled to - I eC. before Ice c ry sta lliza tion begins, one-elghtlsth o f the water w ill separate In the form of lee. Since the la ten t heat of fusion of water Ie SO ca lo ries, the o n e -e l^ tle th of a gram of ice formed lib e ra tes one calorie o f heat. Also since the specific heat o f water i s 1.0, the temperature of th# g tm o f water Is raised one degree, o r from - I* to 0^8. Had the water ropercooled to «»3*0. before c iy e td lltsa tle a began, three»el^htlethe of the gram e f water would have f rosea, lib e ra ting three ca lo ries of heat which would raise the temperature o f the water three degrees, o r from «»3e to 0*. Using these values, one can easily correct the observed depression o f the f reeling point %y means of the fommUa, ^ ( V - '% j ) V or A = A ' - O. o i z s a * ^ ' shere T ■ wel^it of the water, (solvent). A = corrected depression of the f reeling point. A = observed depression o f the freeslng point. M s degrees o f undercooling before Ice-separatlon begins. The true freeslng po in t, therefore, cannot be recorded by a ther- mometer, e le c tr ic a l o r otherwise, M t must be calculated from other data. The "observed depression o f the freeslng 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, of course, to the uee of 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 tissue flu id s were agitated so that super­ cooling could not take place. This criticism i s probably un justified because i t i s based on unnatural conditions. Another objection Is based on the fact tha t insect tissue Ie heterogeneous, and i t s various components freese a t Atffereat temperatures. This viewpoint I s sabstantlated ty Payne«s (IQZGb) Alscoveiy o f e eeconA w Aeicoollng and fieeslag o f oek bower Imrvee mt eboet end by the w rite r 's observation of multiple 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 ln o t* ^ Aecemllneat^ adult Anrlng m time In terval of almost three minutes steady cooling. Another aspect o f the same question Is tha t of the completeness o f freezing. Insect tissue does not remain a t the rebound point temperature u n til co^jletely frozen, but s ta r ts to cool again almost Instan tly . Evidence I s given by various authors, as stated above, that freezing In not complete u n til extremely low temperatures are reached. An In teresting problem la presented by the magnitude o f the rebound. Assuming, as seems necessary, th a t the insect body Ie a * water-system*, l .e . tha t water Is the solvent, the magnitude of the rebound represents the number •, ' ■ o f calories libera ted Iy the f i r s t Ice formation, pey gran o f water. I f some method were developed for measuring the quantity of heat represented by the rebound, the amount of water (solvent) representing the f i r s t part o f the insect to freeze could easily be calculated. I t Iz suggested that th is might be the "free.water* content of the system. The undercooling point has been used by many authors. Carter (1925) p la tted undercooling points egelnst rebound points fo r Brochue obteetam. md found correlations o f 0.779*0.053 fo r the adu lts , 0 .GlW±0.0A fo r the pupae, and 0.690* 0.028 fo r the larvae. This Is to be expected. Inasmuch a# under­ cooling Is a physical process with certain lim its , and i s mainly dependent on - 60- th# of ag itation of the cooling liquid . Carter also p lo ts unden- ooolla* points against rates o f cooling la degrees per minute. Payne (1926a) meed undercooling points in much of her wo Ac, giving Undercooling and rebound points equal consideration. I t w ill he seen from the discussion of the Mention given above that the degree of supercooling Ie a fac to r Is the correction of the observed freezing point, so tha t i f the corrected freezing point Ie used, the supercooling point may be disregarded. Another tem which has often been used without adequate defin ition i s "survival temperature*. The survival of an insect a f te r subjection to low temperature i s dependent on many factors. Even i f such things as development, nu tritional s ta te , water content, e tc . are constant, o r even assumed to be constant throu^iout a se ries , time le so Important a factor that i t must be given i f the data are to W sign ifican t. When cold resistance to the quantity factor Ie being studied, time Is o f course always given. But in cold resistance to the in tensity facto r, most authors do not mention anything but the lowest temperature to which the insect was cooled. The time involved in acquiring th is temperature, l . e . , the ra te o f cooling, must surely be considered. This zute of cooling depends on the apparatus used fo r freezing, fo r Instance, the insect may he placed In a warn environment which is then cooled a t a certa 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 certain ra te . The la t te r case Involves such things as the specific heats o f the various parts o f the insect, the surface exposed, and a i r currents. Carter (1925) plotted usdsresellng points of Bruchum obtsetus egslnet ra te of cooling In degrees per minute and found no relationship. Sines he did so t cover the range of Me correlation chart, however, but determined 6 # o f -<2- Me TOdeicoollng poin t• a t a cooling ra te of 0*5* per mtmite and 11$ a t about 2* per minute, h ie data are o f l i t t l e value. I t has beam ehoen, howver, that • lth in certain lim it# at le ae t, the relatlenChlp beteeem freeslng point# of Lamtlnotaraa decemllneata and rate o f eooling la very alldht. I t might be veil to point out here a alight ambiguity In Payom#a (l$27a) use o f the tern "Qomtlty fee tor*. She s ta te s , "In the metric system fo r the quantity fac to r the ca lo rie Is the u n it . . .* "%@amtity* here Ie not the same as she use* i t , since obviously the exposure of an Insect to a f a ir ly lew temperature fo r a long time has nothing to do with ca lo ries, nor does time ea te r into the defin ition o r measurement.of the ca lo rie . The erro r i s t r iv ia l , however, since Payne makes no mention o r use o f a u n it o f h e r "quantity 'factor* In her subsequent work. The w riter has made a study o f Bohlnson1S method o f determining bound water. The method Is open to criticism . (L) Becent evidence a l l goes to show that freeslng i s nowhere near complete even a t .20^0.; (2) The formula used must be regarded as empirical, since the denominator has not been sa tis fac to rily derived o r explained; (3) the formula involves specific heat, the determination of which Involves the specific heats o f a concentrated solution, ice , and so lid matter, each o f which should be considered separately. S&yre (1932) compared the cryoecopie, calorimetric and dilatemeter methods o f determining bound water, using plant tissue , and decided tha t the calorimetric method ( th a t of Buhner, improved by BoMneon) i s the most rapid, easy, accurate and re liab le . He recommends i t fo r the measurement o f bound TOter in p rac tica lly a l l material#, in spite o f the objections lis te d . Bobinson made an enormous number of determinations of the bound water content e f Insect!, and him data seem to indicate accuracy of the method, A discrepancy has been noticed between the resu lts o f Bobinson and those of Payne regarding the to ta l water content o f insects the hardening process, Bobinson found that the percentage o f to ta l water did not I* IESlJSiL Callossala nromethea. Be dSsiocated the insects a t 100*0, to constant weight. Payne, on the other hand, found a decrease in percentage o f water content during the hardening process, but determined th is to ta l w te r ty desiccating a t go* fo r k hours. Such a low tsspsratus# would W t desiccate the insects to an appreciable extent in U hours; in fac t, i t is w*y l i t t l e above the upper le th a l lim it o f the insects Payne used. The admit# o f Lentlnotare^ deoemllneata shleh were hardened by the eetbor TSgr storing 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 el# t. These reeulte ew flra BoMneon1S obeerrations. The l ite ra tu re on the subject Is reviewed and discussed, sad certa 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 de ta il and i t s advantages pointed out. Time-mortality curves are given for the mortality o f Lentlnetarea decemllneata adults under exposure to low temperatures. I f the Insect Is frozen for a few hours a t a temperature su ffic ien tly low fo r the undercooling point to be reached, death resu lts . Time-temperature freezing curves of water, Leptlnotarsa decsmllmsata adults and Tlpulldae larvae are given for comparison and analysis. The resu lts o f repeated freezing of Melamonlum d iffe ren tia lle nymphs and Lentinotarsa deccmllneata adults show no defin ite hysteresis. The freezing points o f Lentlwtarwa deceWLlmeata adults are correlated with ra te o f cooling. Ho mlgnlflcant relationship was found. The problem of multiple rebounds as observed in Leotlnotare^ d## W llneata adult a I s discussed, suggesting opportunities o f fu rther worts: on th is phenomenon. The hardening of 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 insects a t weekly in te rva ls during hardening a t 5* are given and compared. Complementary data on to ta l water content, le ss o f weight, and the concentration of the body flu ids during th is hardening, are given. The effec 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 ea te r content o f lneects remains the same during the hardening process Is substantiated. UBKRArome cisco BachmetJewl P. 1901. Ezperlmentelle entomologlche Studlen ron phyelcall«;h- chemlechen Standpunkt ami. Leipzig. Iodine, J.H. 1921. Ractore lnelumcing the water content and the ra te of metabolism in certain Orthoptera. Jour. Exp.Zool. 137-16U. 19^3. Hlbematloa in Orthoptera; I . Physiological changes during hibernation in certain Orthoptera. Jour.Ixp .tool. 2£tH57-b76. Carter, Walter 1925. The effectoof low temperature# on Bruehue obteetue Say, an lneeet affecting seed. Jour.Agr.Bee. Chmdler, W.H. 1913. The k ill in g o f p lan ts by low temperature. Mo.Agr1BxpeSta. Res. Bel. S. Cortner, R.A. 1929« Outlines of Biochemistry . John Wiley & Sons, Hew To*. Oueylard and P o rtle r 1916. 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P a a ll l ia lmmmnlaa Hsrnm. Biol.Bui. 25flQkl79. %"*#**** ------ 1929. Abrolute humidity as a facto r In insect cold hardiness, with a note on the effect o f nu trition on cold hardiness. Ann.Bnt.Soc.Aner. 22:601-6%). I 1736. wMemoln pour S e rrlr & VHletolre dee Iaeeetew1 7 o l.I I , pp, I*)-!*?. BoMnson, Hm1 1926, Low temperature and moisture me factors la the ecology of the Rlee weevil, CTrw L. a&d the Orenary weevi l , Sltouhllue * Ualv1 o f Miea1 Agr1 Bxp1 Sta. Tech, Bal1 %. 1926a. Aa e lec tric method of determining the moisture content of liv ing ItlsssM,. BeoTlcgy &3(&"370. 1927. Hater Mndlng capacity o f co llo ids a defin ite factor In winter Ihardlneee e f laeeete. "Jcur.Bcon.Ba*. IgQfSa.**. 1928. Bewpenee and adaptation of Insects to external S tlaM l1 Jknzt1 Bnt. Ste*,. Amer1 ab*07-*&7. 1928a. A study of the effect o f surgical shock on Insects. Jour. Agr.Bes, 7*0-7**. 1928k. Determination o f the natural undercooling end f roesing points In Insects. 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