CHANGES I N SORBITOL CONTENT AND SUPERCOOLING POINTS IN OVERWINTERING EGGS OF T H E EUROPEAN

RED MITE (PANONYCHUS ULMI (KOCH))'

LAURITZ S ~ M M E Division of Entomology, The Norwegian Plant Protection Institute, Vollebekk, Norway

Received June 1 7 , 1965

Abstract Overwintering ems o l the European red mite ~Pa'amns~~rhns aimi (Kmh)),

stored at h 'C i n the laboratory, were found to accurnulnte surbitol during the fall, while a decrease in concenttations took place from January to March. -4 linear ccrrrelation suEgests that increased sorhitor content causes a deprewion of the supercoaling points. Significant rhangrs in supercooling puints <luring storage were found in ~ g g s Iron1 m o Iwa~iorlu in eastern Korrr-ay, while only srilall changes took plate in ems from rl location in Noru-a>.. Chan~rs in sorbitol content \\,ere signihcant in from all lacations. 34aximum con- centratinns uf sorhital wpre obwrvetl at ihe time diapause terminated. J u d ~ n g fro111 lhe supercoolirlg poirlts, eggs rollwtcrl irl eastern S o m y were sliglliI>- more rold-hardy than were ems [ram tnilrler climates in weqtern Somay .

In t roduct ion During their overwintering' on branches and twigs of fruit trees, eggs of

the European red mite (Panonychus ulmi (Koch)) are exposed to the tem- peratures of the surrounding air. MacPhee (1961) has shown that these eggs are killed by freezing, but supercool a large amount. Eggs from Nova Scotia were killed by higher temperatures than were eggs from colder areas in New Brunswick and Quebec.

In insects, a depression of the supercooling point is found in direct relation to the accumulation of glycerol in the haemolyinph (for example, Salt 1961; S@mme 1964). The present investigation was started to see if a similar mecha- nism was present in mites, and if differences in cold-hardiness could be shown in eggs from different climatic areas of Norway.

Methods Eggs of P. ulmi were collected in October 1964 from apple trees (Malus

domestics) from one location in western Norway (Ullensvang, Hordaland) and from two locations in eastern Norway (Frogn, Akershus, and Nes, Hedmark). The average January temperatures are -0.6 "C in Ullensvang, and -4.0' and -6.5 OC in Frogn and Nes respectively. All eggs were stored on their twigs a t 6 "C in the laboratory, and samples were taken for exam- ination a t different times during the winter.

Supercooling points were measured with a copper-constantan thermo- couple and a recording potentiometer a t a cooling rate of 1" to 3 "C per minute. Each determination of average supercooling points was based on 1 2 to 20 eggs. Quantitative data on content of polyhydric alcohols were obtained by paper chromatography of egg extracts in the same way as described earlier

'This work was carried out with financial support from the Norwegian Research Council of Agriculture. Canadian Journal of Zoology. Volume 43 (1965)

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882 CANADIAN JOURNAL OF ZOOLOGY. VOL. 43. 1965

for insects (Sqimme 1964). Two or three separate samples of 200 to 300 eggs were prepared from each group to be analyzed. The average weight of one egg is approximately 2 pg.

Results No traces of glycerol could be detected in any of the eggs a t any time of

the winter. Large spots of another substance did, however, show up on the chromatograms. This substance was identified as sorbitol on descending chromatograms in four different solvent systems. The solvents were run off the papers, and glucose was used as a reference (Smith 1958). The resulting R, values for some sugars and polyhydric alcohols are given in Table I , to- gether with values for the substance present in the eggs. I t appears that this substance travelled as far as sorbitol in all solvents, and was clearly separated from mannitol in three of them. Separation from dulcitol was best in IsoPr and EtAcPy.

The content of sorbitol varied in eggs from all locations during the winter. An accumulation took place during the first part of storage, followed by a decrease in concentrations from January till the early part of March (Table 11). When average supercooling points from Table I1 are plotted against their corresponding data for sorbitol content, a linear correlation is found between these factors. The regression coefficient (r) for this line is 0.653, which is significant a t the 5% level.

While eggs transferred from 6 O C to room temperature in October and November were all in diapause, this was broken in more than half of them in January. Thus the termination of diapause corresponds to the time of maxi- mum sorbitol content.

Analysis of variance based on the individual observations showed signifi- cant changes during storage in sorbitol content in eggs from all locations, and in supercooling points in eggs from the two eastern ones (Table 11). The ability to supercool also changed in eggs from western Norway, but the differences were not significant. For supercooling points, there were signifi- cant differences between locations (F = 3.33; significant for P equal to 0.05),

TABLE I R, values from chromatograms of extracts of eggs of P. ulmi and of various sugars and poly-

hydric alcohols

Solvent*

IsoPr BuPy EtAcPy IsoPrBu

Eggs of P. Glucose Galactose Fructose Mannose Sucrose Dulcitol Mannitol Sorbitol Glycerol

ulmi

*IsoPr: iso-propanol water (4: 1). BuPy: n-butanol. p~ridine. water (2:2: 1); EtAcPy: ethyl acetate, pyridine. water (14: 5 : I ) ; ~so~rbu: iso-propahol. n-butanol. water (7: 1 : 2).

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SBMME: SORBITOL A N D SUPERCOOLING POINTS 883

TABLE I1 Changes in supercooling points and sorbitol content in eggs of P. ulmi from three locations,

stored at 6 "C

Location Days at 6' Month Supercooling point kg sorbitol per egg

Nes

Ullensvang

Frogn 0 Oct. 44 Nov. 89 Jan.

147 Mar. F valueb

0 Oct. 45 Nov. 82 Jan.

137 Mar. F valueb

0 Oct. 42 Nov. 83 Jan.

138 Mar. F valueb

-31.3 kO.2la -31 .1+0 .19 -33.2 k0 .70 -30 .5k 0.27

9.26** -31 .4k0 .48 -32.1 k 0 . 2 9 - 3 2 . 2 k 0 . 4 0 -30 .9k0 .33

3.22* -30 .7k0 .20 -31 .Ort0.23 -31.4rt0.21 - 3 0 . 8 k 0 . 3 1

1.71 (not sign.)

standard error. *Differences between storage times. 'Significant for P equal to 0.05. **Significant for P equal to 0.01.

and a significant interaction between locations and times (F = 3.71; signifi- cant for P equal to 0.01). In sorbitol content, there was no significant differ- ence between locations (F = 1.12; not significant), but a tendency for inter- action between locations and times (F = 2.73; significant for P equal to 0.1).

Discussion As mentioned in the introduction, the supercooling points in insects may

be lowered by increased glycerol content. The present data indicate that a similar mechanism is present in eggs of P. ulmi, but that the depression of supercooling points is caused by sorbitol. A relation between supercooling and accumulation of substances other than glycerol has also been suggested by Tanno (1964), who found high levels of fructose, glucose, and trehalose in overwintering adults of solitary bees. Sorbitol also accumulates in some insects (Chino 1957; Salt 1961), but its relation to cold-hardiness is not known.

Analysis of variance showed differences in ability to supercool between eggs from different locations, but no differences in sorbitol content. This disagreement may be a result of less accuracy in the determination of sorbitol than of supercooling points, partly because sorbitol determinations were based on only two or three replications. Since the average supercooling points of the eggs from the two eastern Norway locations were quite similar, the results suggest that the eggs from western Norway were slightly less cold- hardy. The significant interaction between locations and times for super- cooling points is also a result of smaller variations during the winter in the western Norway eggs. The tendency for interaction between locations and times for sorbitol content can not be explained in the same way, but the experi- mental errors are probably too large for any particular tendencies to appear.

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884 CANADIAN JOURNAL OF ZOOLOGY. VOL. 43. 1965

Acknowledgments I thank Dr. R. W. Salt, of the Canada Agriculture Research Station,

Lethbridge, Alberta, for valuable criticism of the manuscript, and Mr. Chr. Stenseth, of the Norwegian Plant Protection Institute, for advice about the statistical analysis.

References CHIN& H. 1957. Conwrsion of ~ l y m ~ e n to sorhitol and glycerol in the diapat~se m of the

Bomhyx silkworm. Nntore, 180, hOl~-O07. MACPHEE, A. W. lObl. Mortality of minter cgp~. al the European red mite, Punonychscs ult?rl:

(ICoch), at low tetnperatures, ant1 i t s erological significance. Can. J. Znol. 39, 229-243. SALT. R. LT. 1961. I'rincioles nf insect cold-hardiness. Ann . Rev. En tomol. 6. 5 S 7 4 . SMITH, 1. 1858. Su~ars. 1;r Chrtmratnwaphic terhniclues, clinical and biochemical applications.

CY. Heinemann Lld., London. Interscirl~re Ptil,lishrrs Inc., Kew York. pp. 164-177. S~MME, L. 1964. E1k t s of ~lyccral on ccdcl-hardiness in insects. Can. J. Zml. 42, 87-101. TAKNO, K. 1964, High sugar levels in ilre solitary bee Cerutirm. ( I n japar~ese, English sum-

mary). Low 'I'crnp. Si. Scr. R, 22, 51-57.

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