The Thermal Dependence of the Rates of Glycogen and Triglyceride Synthesis in the Mosquito

Eggs obtained from Aedes sollicitans brought from the field were stored and hatched when needed. Larvae were reared on a liver-yeast dog biscuit diet (Lea, 1963). A light cycle of 12 hr. was maintained throughout. Unmated females were collected within 6−12 hr. after emergence, and maintained in groups of twenty in plastic cups of 400 ml. with access to water only. The mosquitoes, when not disturbed, sit rather still and make only occasional flights across the cup. The experiments were carried out in incubators with temperatures held constant to ± 0·5°C.

Females were starved for 7 days and then fed on 1 mg. of sugar by easing the proboscis into a calibrated micropipette filled with 2μl. of a 50 % solution of glucose/fructose 1:1. Within minutes after feeding (zero values) and at appropriate time intervals thereafter, depending on the temperature of incubation, three pools of three mosquitoes each were analysed for glycogen, triglycerides and sugar (Van Handel, 1965 b). Zero values for glycogen and triglycerides did not exceed 0·01 mg. for either product. The experiments were repeated 4 to 8 times, until the standard errors of the means of the pooled data were smaller than 5 %.

Medial neurosecretory cells were surgically removed as described previously (Van Handel & Lea, 1965).

The rate of glycogen synthesis in A. sollicitans increases with temperature from 10 to 22·5° C. Above 22·5° C. the rate of glycogen synthesis is independent of temperature (Table 1). The relationship between the reciprocal of the absolute temperature and the logarithm of the rate constant (time in which the mosquito synthesized 0·15 mg. glycogen) can be plotted as two intersecting lines, one with Q₁₀ = o or μ = o and one with Q₁₀ = 5 or μ = 30 Cal./mole (Fig. 1 a).

The maximum amount of glycogen produced from 1 mg. of sugar was 0·2 mg. for each of the seven experimental temperatures between 10 and 35° C.

The rate of triglyceride synthesis in A. sollicitans increases with temperature from 10 to 30° C. (Table 1). At 35° C. maximum synthesis remained below 0·1 mg. and the rate of synthesis was the same or slightly less than at 30°C. It is therefore not certain whether 30° C. is the transition point beyond which the rate of triglyceride synthesis does not increase or whether the result is due to heat damage. The relationship between the reciprocal of the absolute temperature and the logarithm of the rate constant (time in which the mosquito synthesized 0·10 mg. of triglycerides) can be plotted as a straight line with an estimated Q₁₀ = 3 or μ = 20 Cal./mole (Fig. 16). The points can also be plotted as two straight lines (Q₁₀ = 2·5 between 30 and 20°C.; Q₁₀ = 3·5 between 20 and 10°C.), but the experimental error is so great that the existence of different slopes cannot be regarded as established.

The maximum amount of triglycerides produced from 1 mg. of sugar was 0·13−0·14 mg. for each of the six experimental temperatures between 10 and 30°C.

The sugar fed to mosquitoes is stored in the diverticulum (crop) and absorbed from the midgut. Even if emptying the diverticulum and absorption are independent of temperature, these processes may be rate-limiting. Furthermore, the rates at which the crop is emptied or the sugar absorbed could in themselves be dependent on temperature. Injection of the precursor directly into the haemolymph bypasses these potential rate-limiting factors.

One μ. of 50 % glucose was injected into the abdomen with the aid of a drawn-out glass capillary, the controls being fed on the same amount with a micropipette. Before feeding, the starved mosquitoes contained less than 0·01 mg. of glycogen and less than 0·01 mg. of sugar. The insects were analysed individually after 4 and 8 hr. at 30°C, and after 8 and 16 hr. at 15° C.

In injected insects the rate of glycogen synthesis was not increased at 30° C, where synthesis was independent of temperature fluctuations of at least 5° C, or at 15°C, at which glycogen synthesis is strongly temperature-dependent (Table 2). In both injected and fed insects the sum of the synthesized glycogen and the remaining sugar was the same, so that injection did not affect the metabolic rate (Table 2).

These results justify the conclusion that emptying of the diverticulum and absorption of sugar are not rate-limiting factors in glycogen synthesis and, implicitly, in the much slower triglyceride synthesis.

In the female mosquito medial neurosecretory cells (MNC) restrict synthesis of glycogen from sugar. Removal of these cells greatly increases the storage capacity for glycogen at the expense of triglyceride storage (Van Handel & Lea, 1965). It would therefore be possible that MNC affect the rate of synthesis as well as the pool size. In order to investigate this possibility rates of glycogen synthesis were determined in mosquitoes from which the MNC were removed. A. taeniorhynchus was used, because in this species the maximum amount of glycogen produced by the unoperated control from a single dose of sugar is only 0·05 mg., as compared to 0·2 mg. for A. sollicitans. Almost all glycogen produced from sugar in operated A. taeniorhynchus is therefore due to the removal of the MNC (Van Handel & Lea, 1965).

Removal of the MNC increased the storage capacity for glycogen at all temperatures, but the rate of synthesis at 35° C. did not exceed that at 20° C. (Table 3). Since the failure of the rate of glycogen synthesis to increase above a critical temperature persists after removal of MNC, this failure in unoperated controls is not due to the MNC. At 10° C. the rate of glycogen synthesis is greatly depressed, in controls as well as in operated mosquitoes.

The process most frequently studied at different temperatures in intact poikilotherms is oxygen consumption. Some Q₁₀ data for processes which plot approximately as straight lines on Arrhenius-type graphs are listed in Table 4. When a range is given for the Q₁₀, the higher value applies to the lower value of the given temperature range. This may indicate a gradual transition to diminished activity, chill coma, diapause or irreversible changes leading to death. Heart-beat frequency (from 12 to 40° C.) and wing-beat frequency (from 17 to 30° C.) in roaches vary proportionally rather than logarithmically with the temperature (Richards, 1963 a, b). The same author has studied the temperature dependence of enzyme systems that might be pacemakers for some physiological functions (Richards, 1958).

The present investigation is concerned with the rate of accumulation of caloric reserves from ingested food. The maximum amount of glycogen (0·2 mg.) and triglycerides (0·13−0·14 mg.) synthesized from 1 mg. of sugar by the female A. sollicitons appeared to be the same at each temperature. This is 50% of the caloric value of the meal. Since the maximum pool size did not depend on the temperature, the observed changes in the rates of synthesis were directly due to changes in temperature, and not indirectly to changes in pool size.

The temperature-dependence of the rates of synthesis can be presented as an Arrhenius plot. From 10 to 30°C. the rate of triglyceride synthesis increases with an approximate Q₁₀ = 3 (μ= 20 Cal./mole). The rate of glycogen synthesis increases with an approximate Q₁₀ = 5 (μ = 30 Cal./mole) from 10 to 22·5° C., while further increase in temperature does not result in further increase in the rate of synthesis. These two widely different Q₁₀ values, observed in glycogen synthesis, do not occur during glycogen utilization; during starvation, the rate of disappearance of glycogen increases over the entire range from 10 to 35° C. (Van Handel, in preparation). It is not known why, above 22·5° C., the rate of glycogen synthesis is independent of temperature, but neither crop emptying and sugar absorption (Table 2) nor the medial neurosecretory cells (Table 3) are rate-limiting factors.

To my knowledge, no other measurements have been reported for the thermal dependence of the rate of synthesis of specific products in an intact animal.

1. Net synthesis of glycogen and triglycerides produced from 1 mg. of sugar by the female mosquito A. sollicitans was determined at different temperatures.

2. At each temperature 0·2 mg. of glycogen and 0·13−0·14 mg. of triglycerides was synthesized from 1 mg. of sugar.

3. The logarithm of the rate constant (the time required to synthesize 75 % of the maximum) was proportional with the reciprocal of the absolute temperature.

4. For triglyceride synthesis the Q₁₀ = 3 from 10 to 30°C. ; for glycogen synthesis the °₁₀ = 5 from 10 to 22·5°C., but from 22·5 to 35°C. the rate of glycogen synthesis was independent of the temperature (Q₁₀ = o).

5. Crop emptying and sugar absorption are not rate-limiting factors.

6. Medial neurosecretory cells, which limit the maximum amount of glycogen that can be synthesized from sugar, do not affect the rate of synthesis.

The author thanks Rosetta Warren, Jose E. Güira and Marius H. Van Handel for technical assistance and Dr Roger W. Meóla for performance of the brain surgery. This work was supported by grants (Al 05054 and Al 06587) from the National Institutes of Health.