Using Low Oxygen Atmospheres to Control Two Insects pests Attacking Historic objects in Egypt

The efficacy of controlled atmospheres of high nitrogen concentration (99.9%) and various carbon dioxide concentrations (25, 50,75and 99.9%) was investigated against adults and larvae of two insects attacking historic objects in Egypt, the black carpet beetle and the cigarette beetle. Also, the effect of temperature and exposures time on the mortality of insects was examined. Results revealed that the efficiency of CO2 and N2 gases was increased with increasing gas concentration, temperature and exposure time. Carbon dioxide gas was more toxic to both insects than nitrogen gas at the same conditions. Larvae of both species were more susceptible than adults to both gases.


INTRODUCTION
The black carpet beetle Attagenus fasciatus is considered to be a pest in the museums where it attacks organic articles, such as furs, hides, insect specimens, wool articles and oil seeds (Back and cotton, 1936).Carpet beetle larvae cause damage by feeding on a wide variety of materials including fur, feathers, wool and silk cloth, wool felt, hair and skins.There are many species of carpet beetles, but one of the most commonly found in museums is the black carpet beetle A. unicolor which is the most abundant and destructive of the carpet beetles (National Park Service, 1998).Cigarette beetle Lasioderma serricorne is a pest of stored tobacco, but is also a serious pest of flax, spices, crude drugs, seeds, and most importantly for museums books and dried plants (Rust et al. 1996).
In the past, chemical applications were widely used in museum to eradicate pests.Such application may be harmful to museum articles (Dawson, 1988).Studies have shown that fumigation with traditional chemicals can cause harm to museum operators, destroy the environment, and may damage rare antiquities and artifacts (Florian, 1988).Recent changes in public attitude and government regulations have increased the pressure to minimize the use of pesticides and have encouraged the use of preventative measures and less toxic materials and methods.
Use of modified atmospheres for pest control in museums has received an increasing amount of interest during the last decade (Kigawa et al. 2001).

1-Rearing Technique of Stock Cultures:
The black carpet beetles were reared according to the technique described by Ali (2010).Larvae were reared in child milk contains the following nutritive 100g: 11.9 g protein, 27.7 g fat, 55.4 g carbohydrate plus pure wool textiles.Adults were fed on tiny drops of honey placed on a sheet of paper.The cigarette beetles were reared on wheat flour containing 10% dry brewer's yeast.Insect cultures were kept under controlled conditions of 27-30 ºC and 55 ± 5 % R.H in the incubator of the pest control laboratory, Center of Research and Conservation of Antiquities, Supreme Council of Antiquities.Adults of each insect species were introduced into the jars to laying eggs under controlled conditions.After hatching, all adults were separated from the jars and introduced to new jars and kept again in the incubator.This procedure was repeated several times in order to obtain large numbers of the larvae and adults needed to carry out the experiments.

Exposure Procedure:
A circulatory multi flask apparatus was established to provide an exposure room suitable for gas concentrations applied (Fig. 3).The dreshel flasks with a volume of 0.55 litres were connected to each other with Polyvinyl chloride (PVC) tubing and joints were greased.

Gases Used:
Carbon dioxide (CO 2 ) and Nitrogen (N 2 ) were provided as pure gases of about 99.9% in pressure steel cylinders.Each cylinder was connected to a pressure regulator.The dilution method was used to achieve the required CO 2 concentrations in the flasks of the apparatus by using a gas tight pump.For the atmospheres of nearly pure N 2 , the valve of the N 2 cylinder was opened for two minutes in order to fill the dreshel exposure flasks with the gas after filling , the flasks were directly closed tightly by using two metal clips and glass rods.Concentrations of 25, 50, 75 and 99.9% CO 2 and mixture from various concentrations of oxygen, nitrogen and carbon dioxide were prepared.

Determination of Gases Concentrations:
CO 2 was monitored using gas analyzer model 200 -600 (Gow-Mac-Instrument CO, USA) Nitrogen concentration was determined inside the dreshel flasks using Oxygen Analyzer 572, Servomex, England .

Preparation of Insects for Gas Treatment:
Batches of 25 of each 3 rd and 5 th instars larvae and 25 of newly emerged adults of A. fasciatus and L. serricorne were put in 4 replicates and placed in wire gauze cages (Fig. 5) (14 mm diam.and 45 mm long), filled with about 10 gm of child milk powder for A. fasciatus and 10gm wheat flour with dry brewer's yeast for L. serricorne and the cages were closed with rubber stoppers.The cages were then introduced into the 0.55-L gas tight dreshel exposure flasks.Insects in the flasks were treated for different exposure periods (24, 48,72,96 and120 hours) for C.A of high N 2 content and (48 &120 hours ) for C.A of various concentrations of CO 2 at 30±1 º C and 20± 1 º C and 65 ± 5 % R.H.After the desired exposure periods, the flasks were aerated and the insects were transferred into Petri dishes and kept at the above mentioned conditions prior to mortality assessment.

Bioassay Tests of Gases:
After the desired exposure period mortality assessment was performed.Both larva and adult mortalities of A. fasciatus and L. serricorne were determined after 24, Using low Oxygen Atmospheres to control two insects pests attacking historic objects in Egypt 63 48, 72, 96 and 120 hours for C.A of 99.9% N 2 and (48 &120 hours) exposure periods for C.A of various concentrations of CO 2 .

Statistical Analysis of the Data:
Lethal time values were determined by probit analysis using a computer program of Noack and Reichmuth (1978).

RESULTS
Efficiency of controlled atmosphere of carbon dioxide and nitrogen against larvae and adults of Attagenus fasciatus and Lasioderma serricorne was tested.

1-Susceptibility of A. fasciatus to Controlled Atmosphere (CA) of Carbon dioxide (CO 2 ).
The efficacy of different concentrations of CO 2 against 3 rd and 5 th instars larvae and adults of A. fasciatus was tested after 48 and 120 hours at two different temperatures (20±1ºC and 30±1 ºC) and 65 ± 5% RH.The results are shown in tables (Tables 1 to 6) and illustrated graphically in figures (Figs 1 to 12).
Data in Tables 1 &2 and Figs 1 & 2 shown that, larvae of A. fasciatus were more susceptible to all tested gas concentrations than adults.At 20±1ºC and 65 ± 5% RH, the LC 50 values after 48 and 120 hours were 53.893 and 37.623 % for 3 rd larval instars, respectively.The LC 50 values of CO 2 for 5 th larval instars after 48 and 120 hours were 62.144 and 45.458 %, respectively.Also, the results indicated that, LC 50 values of CO 2 for adults after 48 and 120 hours were 64.656 and 48.382 %, respectively.
The efficacy of CA of 100%, 75%, 50% and 25% CO 2 against larvae and adults of A. fasciatus at 30±1ºC and 65 ± 5% RH is presented in Tables 1& 2 and shown graphically in Figs 1 to 4. The results illustrated that, the LC 50 values of CO 2 for 3 rd and 5 th larval instars and adults after 48 were 52.739, 60.004 and 61.909 %, respectively.
After 120 hours the LC 50 values were 35.310, 43.957 and 47.295 for 3 rd and 5 th larval instars and adults, respectively.
As shown in tables (Tables 1& 2) and figures (Figs. 1 & 4) the mortality of A. fasciatus increased as the gas concentration increased.According to LC 50 values for A. fasciatus, the concentrations of CO 2 were more effective at higher temperatures than lower temperatures and the adults were more tolerant to CA contain CO2 than larval stages.Also the mortality of A. fasciatus increased as the exposure time increased.Presented data revealed that, at 20±1ºC, larvae of L. serricorne were more susceptible than adults to all tested gas concentrations.The LC 50 values of CO 2 for 3 rd and 5 th larval instars and adults after 48 were 55.440, 63.769 and 65.692 %, respectively (Table 3).
After 120 hours, the LC 50 values were 39.059, 45.926 and 49.445 for 3 rd and 5 th larval instars and adults, respectively (Table 4).
The efficacy of CA of 100%, 75%, 50% and 25% CO 2 against adults and larvae of L. serricorne at 30±1ºC and 65 ± 5% RH is presented in Tables 5& 7 and shown in and Figs 5& 7. The results illustrated that, the LC 50 values of CO 2 for 3 rd and 5 th larval instars and adults after 48 were 53.999, 60.964 and 62.962 %, respectively (Table 3).
After 120 hours, the LC 50 values were 35.847, 44.810 and 47.379 for 3 rd and 5 th larval instars and adults, respectively (Table 4).
According to LC 50 values for L. serricorne, the concentrations of CO 2 were more effective at higher temperatures than lower temperatures and the adults were more tolerant to CO 2 than larval stages.Also the mortality of L. serricorne increased as the exposure time increased.

3-Susceptibility of A. fasciatus to Controlled Atmosphere (C.A) Of High Nitrogen (N 2 ) Content.
The efficacy of CA of 99.9% N 2 against A. fasciatus at 20±1ºC and 65 ± 5% RH is shown in Table 5 and Fig. 9.The results indicated that, the time needed to obtain 50% mortality of A. fasciatus was 3.155, 3.510 and 3.894 hours for 3 rd , 5 th larval instars and adults, respectively.Also, the results indicated that, the time required to obtain 95% mortality of A. fasciatus was 40.529, 47.320 and 79.168 hours for 3 rd , 5 th larval instars and adults, respectively.
The efficacy of CA of 99.9% N 2 against A. fasciatus at 30±1ºC and 65 ± 5% RH is presented in Table 5 and Fig. 10.The results illustrated that, LT 50 and LT 95 of 3 rd larval instars were 0.681 and 12.731 hours, respectively.
Data in Table 5 showed that LT 50 and LT 95 values of 5 th larval instars of A. fasciatus were 1.970 and 18.871 hours, respectively.
As illustrated in Table 5 and Fig. 9 the time required to obtain 50% mortality of A. fasciatus adults was 4.184 hours and the time required to obtain 95% mortality was 55.900 hours.Using low Oxygen Atmospheres to control two insects pests attacking historic objects in Egypt 67 As shown in Table 5 the mortality of A. fasciatus increased as the exposure time increased.Also the mortality increased as the temperature increased.
According to LT 50 and LT 95 values for A. fasciatus the concentration of 99.9% N 2 was more effective at higher temperatures than lower temperatures and the adults were more tolerant to CA than larvae stages.

4-Susceptibility of L. serricorne to Controlled Atmosphere (CA) Of Nitrogen (N 2 ).
The efficacy of CA of 99.9% N 2 against L. serricorne at 20±1ºC and 65 ± 5% RH is given in Table 6 and shown graphically in the Fig 10 .The results indicated that, LT 50 of L. serricorne were 3.360, 3.510 and 19.280 hours for 3 rd , 5 th larval instars and adults, respectively.Also LT 95 values were 47.320, 55.005 and 114.118 hours for 3 rd , 5 th larval instars and adults, respectively.
The efficacy of CA of 99.9% N 2 against L. serricorne at 30±1ºC and 65 ± 5% RH is shown in Table 6 and Fig 11.The results illustrated that, LT 50 and LT 95 values of 3 rd larval instars were 2.416 and 16.000 hours, respectively.LT 50 and LT 95 values of 5 th larval instars were 3.067 and 27.860 hours, respectively.
The results indicated that, LT 50 and LT 95 values of adults were 3.394 and 70.444 hours, respectively.As shown in Table 6 and Figs 11&12 the mortality of L. serricorne increased as the exposure time increased, also the mortality increased as the temperature increased.
According to LT 50 and LT 95 values for L. serricorne the concentration of 99.9% N 2 was more effective at higher temperatures than lower temperatures and the adults were more tolerant to CA than larvae stages.Data in tables (Tables 5&6) illustrated that larvae and adults of A. fasciatus were more sensitive to N 2 gas than Larvae and adults of L. serricorne.Also, the presented results show that N 2 gas was generally more effective against larvae and adults of A. fasciatus and L. serricorne than CO 2 gas.

DISCUSSION
The efficacy of CA of 100%, 75%, 50% and 25% CO 2 and (99.9%)N 2 against A. fasciatus and L. serricorne were tested at 20 and 30±1ºC and 65 ± 5% RH.According to LC 50 values of CO 2 and LT 50 values of N 2 for A. fasciatus and L. serricorne, results showed that, adult stage was more tolerant to CO 2 and N 2 than larval stages.Also the mortality of A. fasciatus and L. serricorne increased as the exposure time and gas concentration increased.The presented results show that N 2 gas was generally more effective against larvae and adults of A. fasciatus and L. serricorne than CO 2 gas.These findings agree with El-Lakwah et al. (1998) who found that controlled atmosphere of around 99.5%N 2 was found to be effective than controlled atmosphere of 96% CO 2 .
The exact physiological mechanism of nitrogen anoxia is not completely understood.The sensitivity to low oxygen concentrations varied with insect species, developmental stage and the environmental conditions to which the insects were exposed before as well as during the anoxia treatment.
Most insects are capable of restoring all body function after an exposure to lowoxygen atmospheres for several hours, sometimes even for days, but die if the exposure time is extended.Researchers agree that desiccation is an important mechanism influencing insect mortality during exposure to low-oxygen atmosphere.Jay and Cuff (1981) demonstrated that weight loss of different stages of Tribolium castaneum (H.) was 1.6 to 4.8 times higher after exposure to low-oxygen atmosphere (99% nitrogen) than after exposure to air under identical humidity and temperature conditions.They therefore suggested that water loss was the major cause of death.The process of water loss is closely linked to respiration, the exchange of oxygen and carbon dioxide.An insect's respiratory system (tracheal system) has openings to the atmosphere (spiracles) that can be partially or completely closed to prevent water loss (Blum, 1985) this feature allows insect to maintain a sufficient water reserve in environments with low relative humidity.However, air containing low oxygen levels about 2%, or sufficient concentrations of carbon dioxide above 10% cause the spiracles of a well hydrated insect to open fully, resulting in a higher rate of water expiration (Bursell, 1970).After critically low moisture content is reached, the insect tends to reduce its oxygen consumption to minimize further water loss.Of course, reduce oxygen consumption could enable the insect to survive anoxic conditions for an extended period (Gilberg, 1989).Also, (Ali, 1972) referred the death of insects to insect's consequent accumulation of abnormal quantities of various metabolic end products.More recent study by (Adler, 1994) confirm that the accumulation of lactate in insects during exposure to a nitrogen atmosphere lead to acid imbalance in an insects body.This seriously inhibits the anaerobic metabolic pathway.
In the present study the two gases were more effective at higher temperatures than lower temperatures.These results may be illustrated as insects generally show increased metabolic rates and consume more oxygen when the temperature rises.When a certain critical temperature is reached, the insect might use evaporative cooling by opening its spiracles to control body temperature promoting increased water loss the same mechanism was illustrated by (Bursell, 1970), these two phenomena may explain the greatly increased mortality rate of insects when anoxia treatments are carried out at higher temperatures.
Also, several studies by (Ali, 1972); Lindgren and Vincent (1970); Soderstrom et al. (1986) and Rust et al. (1996), showed that raising the temperature from 15ºC to 35ºC can reduce the required exposure time for death by 25% to 65% for each 5ºC increase.

Table 2 :
Susceptibility of A. fasciatus to (C.A) of CO 2 after 120 hours at 20 and 30±1ºC and 65 ± 5% RH 2-Susceptibility of L. serricorne to Controlled Atmosphere (CA) of Carbon dioxide (CO 2 ).The efficacy of different concentrations of CO 2 against 3 rd and 5 th instars larvae and adults of L. serricorne was tested after 48 and 120 hours at two different