J. srored Prod. Res. Vol. 31, No. I, pp. 49-54, 1995 Copyright 0 1995 Elsevier Science Ltd

0022-474X(!M)OOO35-2 Printed in Great Britain. All rights reserved 0022-474X/95 $9.50 t 0.00

The Effect of Carbon Dioxide Induced Anaesthesia on Fecundity of Callosobruchus

maculatus (Coleoptera: Bruchidae)

COLIN DAWSON

Department of Pure and Applied Zoology, University of Reading, Whiteknights, Reading RG6 2AJ, U.K.

(Received 1 August 1994)

Abstract-The use of COz to anaesthetise freshly emerged adult Cdlosobruchus muculutus (F.) is shown to affect fecundity. Increasing the time of exposure to 100% CO, atmosphere from 30 s to 32 min significantly decreased 40 h fecundity compared to controls. A significant dose-response was seen when beetles were exposed for 8 min to increasing concentrations of CO, atmosphere. Lifetime fecundity was significantly reduced after exposure to 100% CO1 atmosphere for 8 min.

Key words-CO,, anaesthesia, fecundity, Callosobruchus maculatus.

INTRODUCTION

The use of carbon dioxide (CO,) to anaesthetise insects, to facilitate easy handling, is a common laboratory procedure. There is a growing body of evidence showing that even a brief exposure of insects to high concentrations of CO* can have major long term effects. Exposure to high concentrations of CO, has been shown to increase mortality and reduce fecundity in Drosophila melanogaster Meigen (Perron et al., 1972), change behaviour in D. melanogaster (Dijken et al.,

1977), increase mortality in Mediterranean fruit flies, Ceratis capitata (Wied.) (Hooper, 1970; Shermand, 1953), retard growth in the German cockroach, Blattella germanica (L.) (Brooks, 1957; Tanaka, 1982), reduce oviposition in the codling moth, Cydia pomonella (L.) (White et al., 1970) and reduce larval growth in Heliothis zea (Boddie) (Edwards, 1968). See Nicholas and Sillans (1989) for a review of the effects of CO, on insects.

Callosobruchus maculatus is a commonly cultured insect. It is extremely active at laboratory temperatures. Therefore it is desirable to have methods to anaesthetise it when handling. Exposure to 100% CO, atmosphere for 30 s will anaesthetise all C. maculatus but they recover quickly- within l-2 min (unpublished observation). If large numbers of insects are required for delicate handling it is usual, once anaesthetised, to place them on a sintered glass plate that has a continuous stream of CO, flowing through it. This keeps the insects anaesthetised until ready for handling. C. maculatus still recover within l-2 min even if they have been kept on the plate for a long time (unpublished observation). If large numbers are used it is possible for an insect to be exposed to high concentrations of CO2 for an hour or more.

In these experiments the effect of CO, induced anaesthesia on both short term and lifetime fecundity in C. maculatus is investigated.

49

50 Colin Dawson

MATERIALS AND METHODS

Insects

All insects used in these experiments were from a Brazil (also known as ‘Campinas’ by Credland et al., 1986) strain of C. maculutus that has been maintained at 30°C and 70% r.h. in a controlled temperature and humidity room (CTH room) for several years (Giga and Smith, 1983). All experiments were carried out under these conditions.

Three experiments were done to determine the effect of CO? on the fecundity of C. maculutus- see below.

Statistical analysis

Data were analysed using Minitab DOS Microcomputer Version (release 7.1).

1. Time of exposure to 100% carbon dioxide

Several hundred freshly emerged insects, 24 h old or less, were taken from a single culture jar. They were all from the same generation and had experienced similar conditions immediately before the experiment. Groups of approximately 60 insects were chosen at random and placed in 150 ml glass jars that were then sealed with a filter paper. One jar chosen at random was used as a control. The control was not injected with air but was treated the same as the experimental jars in every other respect. Each of the other jars was injected with 100% CO, gas from a cylinder for 0.5, 1, 2,4,8,16 or 32 min. The gas was introduced into the jar using a hypodermic needle pushed through the filter paper. The rate of flow of CO* was 2 l/min.

Immediately after the treatment the insects were transferred to a clean glass petri dish open to the atmosphere and allowed to recover. All treated beetles recovered from the treatment and were active after 2min. The beetles were then sexed using the elytral patterns described by Southgate et al. (1957). A male and female were then added to a small glass tube (25 x 50 mm) containing 15 cowpeas. The tube was sealed using a filter paper. Ten such pairs were taken from each treatment and from the control. It was assumed that females had mated in the first 24 h but a male was provided to ensure a mate was available if required. The pairs were allowed 40 h in the CTH room to mate (if required) and lay eggs before being removed and discarded. The seeds were returned to the CTH room for a further 10 days to allow the eggs to hatch before hatched eggs were counted.

2. Exposure to diferent concentrations of carbon dioxide

Several hundred freshly emerged insects were taken from a single culture jar in the same way as experiment 1. Groups of approximately 60 insects were placed in a 60 ml syringe sealed with a three-way tap. One end of the tap was connected to a CO* cylinder and the other end was open to the atmosphere. By turning the tap to the appropriate position it was possible to draw in air from the atmosphere or to allow CO, to flow in from the cylinder. Once the desired mix of gases was obtained the tap was closed to seal the syringe. To use the apparatus the piston was first removed from the syringe. The beetles were added and the tap was turned so that the syringe was open to the atmosphere. The piston was replaced and depressed down to the 2 ml mark. This allowed enough room for the beetles without squashing them but minimised the amount of air in the syringe. For mixes of air and C02, air was drawn into the syringe up to the desired level. The tap was then turned to allow CO* to flow steadily into the syringe up to the 60 ml mark when the tap was turned to seal the syringe. The piston was drawn back slightly, released and allowed to settle to ensure that the mixture of gases inside the syringe was at atmospheric pressure.

Five different mixtures of gases of approximately 100, 75, 50, 25% CO2 and a control (100% air) were used. The experiment was performed four times for each of the five mixtures of gases to ensure that the results obtained were consistent.

The beetles were exposed to the mixture of gases for 8 min before being removed from the syringe, placed in a glass petri dish open to the atmosphere and allowed to recover from the treatment. The rest of details were as above.

Anaesthesia and fecundity of Callosobruchus macularus 51

3. Eflects of exposure to 8min of 100% carbon dioxide on lifetime fecundity

Beans from a stock culture, containing developing larvae, were put individually into small glass tubes. Over a period of 5 days newly emerged, virgin, adults were isolated in small glass tubes. The beetles were sexed and placed into single sex groups of 10. The groups of 10 were then placed in the syringe apparatus and either air or CO2 was then drawn in to the apparatus up to the 60 ml mark. The syringe was then sealed using the tap and allowed to stand for 8 min. The beetles were then removed from the apparatus and allowed to recover. All beetles recovered from the treatment. Five groups of each sex were treated with CO2 and a further five groups of each sex were treated with air. By keeping the sexes separate it was possible to ensure that no mating took place before or during the treatment.

After exposure, a male and female beetle were added to a glass tube containing 50 beans. The tube was sealed using a filter paper and replaced in the CTH room. Three replicates of 10 pairs each were prepared from the CO,-treated insects and three replicates from the controls. The remaining pairs were each made up with one partner from the CO2 treatment and the other from the control, to investigate whether one or both sexes were affected by CO, exposure. There were thus two IO-pair replicates of treated males and untreated females, and two of treated females and untreated males.

Sixteen days after the beetles were added to the tubes they had all died and the number of eggs laid per pair were counted. If no eggs were laid in a tube the data from these pairs were eliminated from the analysis as it was not possible to determine if the treatment or other factors were the cause (wrongly identified sexes for example). As only a small number of unhatched eggs were found (58 out of 5374 eggs counted); they were also eliminated from the data set.

Fig. 1. The effect of different times of exposure to 100% CO, on the fecundity of freshly emerged Callosobruchus maculatus laying for 40 h on cowpeas at 30°C and 70% r.h. Lines represent standard errors.

Significant difference from control indicated by *(P < 0.05) or **(P < 0.01).

52 Colin Dawson

coalIo1 25% 50% 15% 100%

Perecent carbon dioxide beetles exposed to for 8 tin

Fig. 2. The effect of exposure to different concentrations of CO, for 8 min on the fecundity of freshly emerged Cullosobruchus macularus laying for 40 h on cowpeas at 30°C and 70% r.h. Lines represent

standard errors. Significant difference from control indicated by **(P < 0.01) or ***(P < 0.001).

RESULTS

Ir :reasing the time of exposure to 100% CO, caused progressive and significant decreases in 40 h fecu tdity (Fig. 1). There was a dose response of 40 h fecundity to increased concentration of CO1 (Fig. 2). Analysis of variance on data from experiment 2 showed that there were no significant differences in fecundity between each replicate experiment at any particular concentration (P = 0.303).

The control values for 40 h fecundity from experiments 1 and 2 were not significantly different (two sample t test, tn = 1.78, n.s.). However, the number of eggs laid by pairs held in 100% CO* for 8 min was higher in experiment 2 than in experiment 1 (two sample t test, t,, = 2.17, P = 0.045).

Lifetime fecundity was significantly reduced by exposure to 8 min of 100% CO2 (Fig. 3). Analysis of variance between the replicate groups within a treatment showed that there were no significant difference between them (P = 0.274). Whereas lifetime fecundity of females was significantly affected by exposure to 8 min of CO*, male fertility was not significantly affected (Fig. 3).

DISCUSSION

Exposure to 100% CO, for 0.5 min is the minimum required to anaesthetise all C. maculatus treated (unpublished observation). When C. maculatus are exposed to different concentrations of CO2 for 8 min the lowest concentration to achieve anaesthesia in all beetles was 75% (unpublished observation). Therefore to achieve anaesthesia in C. maculatus using CO* it is necessary to use high concentrations of the gas. The results show that exposure to high concentrations of CO* significantly affects both short term and lifetime fecundity. Experiment 3 showed that CO, affects only the fecundity of the females.

The mechanism of CO, induced anaesthesia has not yet been fully elucidated. However, there is empirical evidence of a general mechanism of action of all anaesthetics which involves physical phenomena leading to changes in permeability of the neurone cell membrane (Mullins, 1975) and

Anaesthesia and fecundity of Callosobruchus macularus 53

Control Males only Females only

Beetles treated with carbon dioxide

Both sexes

Fig. 3. The effect of exposure to 8 mm of 100% CO, on lifetime on fecundity of Callosobruchus maculutus laying on cowpeas at 30°C and 70% r.h. Lines represent standard errors. Significant difference from

control indicated by **(P < 0.01) or ***(P < 0.001).

a theoretical model explaining this has been proposed (Trudell, 1977). From other experiments on insects it appears that CO, exerts its anaesthetic effect directly on the nervous system via the trachea and not via the haemolymph (Woodring et al., 1978).

It has been shown that a short period of CO, induced anaesthesia effects both short term and lifetime fecundity of C. maculatus. Given the multiple effects of CO2 induced anaesthesia reported in other insects (Nicolas & Sillans, 1989) it is possible that other characters in addition to fecundity could be affected in C. maculatus. The results of experiments where CO, has been used to anaesthetise C. maculatus before fecundity has been measured (such as Colegrave, 1993) should be considered suspect. Hence the use of CO, as an anaesthetic agent should be treated with great caution.

Acknowledgements-I would like to thank C. E. Dyte for suggestions and encouragement. A. R. McCaffery for critical reading of the manuscript, J. C. Millard and V. Rimmer for technical help and my supervisors R. M. Sibly and R. H. Smith. This work was funded by a NERC research assistant grant.

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