Effect of Carbon dioxide ( CO 2 ) on mortality and reproduction of Anagasta kuehniella ( Zeller 1879 ) , in mass rearing , aiming at the production of Trichogramma spp

Eggs of Anagasta kuehniella (Zeller 1879) are widely used for mass rearing of Trichogramma spp. and other parasitoids and predators, largely commercialized in many countries. The aim of this study is to evaluate the effect of carbon dioxide (CO2) originated from larval metabolism on the biological parameters of A. kuehniella. For that purpose, we assess the production of carbon dioxide (CO2) per rearing tray of A. kuehniella and the effect of CO2 on the viability of egg-to-adult period and oviposition of A. kuehniella. Results allow to estimate that a rearing tray, containing 10,000 larvae between the 4th and 5th instars, produces an average of 30.67 mL of CO2 per hour. The highest egg production of A. kuehniella was obtained when the larvae were kept in rooms with lower concentration of CO2 (1,200 parts per million ppm), producing 23% more eggs than in rooms with higher CO2 concentrations. In rooms with high density of trays (70 trays/room), CO2 concentration exceeded 4,400 ppm. The viability of the egg-to-adult period was not infl uenced by carbon dioxide.


INTRODUCTION
Egg parasitoids Trichogramma are widely used in various parts of the world, with over 18 species being mass-reared for pest control in 16 countries, an area, estimated in the 90's, corresponding to around 18 million ha (Hassan 1997).In Russia alone, 3 to 10 million hectares were "treated" annually with Trichogramma spp. as a mean to control pests in different crops (van Lenteren 2008).Currently, in Brazil, an area of 500,000 ha of sugarcane has been treated with Trichogramma galloi Zucchi, 1988, for the control of Diatraea saccharalis.(Fabr 1794) (Parra 2010).
The successful use of these parasitoids in pest control is attributed to the condition of rearing them on eggs of factitious hosts, since the number of insects required to rear them is very large and diffi cult to obtain in eggs of natural hosts.The fl our-moth, Anagasta kuehniella (Zeller 1879), is one of the Anagasta kuehniella (Zeller 1879), is one of the Anagasta kuehniella alternative hosts that provides desirable nutritional quality to their parasitoids (Lewis et al. 1976) and, due to its mass rearing condition, it is used in biological control programs in Europe and in Brazil (Parra 1997).
The mass rearing of this host requires control of temperature and relative humidity (RH), since temperature regulates the development of all stages of the insect, compromising reproduction, if not adequately controlled (Daumal and Boinel 1994).
823-831 ALOISIO COELHO JUNIOR and JOSÉ R.P. PARRA High temperatures also favor the emergence of the pa rasitoid larvae Habrobracon hebetor (Say Habrobracon hebetor (Say Habrobracon hebetor 1836), which is attracted by the frass produced by the larvae (Parra et al. 1996).Fungi and mites are commonly found in insect rearing, favored by high RH (Parra 1997).
One factor, not much taken into account, in mass rearing of A. kuehniella is carbon dioxide (CO 2 ), which accumulates in rearing rooms, released from the larval metabolism.Commercial laboratories use trays of several different sizes, containing a large number of larvae, so the density of trays (trays/m 2 ) inside of the rearing rooms is very high, which can lead to excessive accumulation of CO 2 , and can ultimately cause losses in the rearing.
Given the characteristics of mass rearing of A. kuehniella, this paper aims to evaluate the CO 2 production per rearing tray of A. kuehniella and the effect of CO 2 on the viability of adult-egg period and oviposition of this moth.

A. KUEHNIELLA
The test to evaluate the production of CO 2 per rearing tray of A. kuehniella was developed at the Laboratory of Forest Ecology and Entomology, ESALQ/USP.To measure the CO 2 production per rearing tray of A. kuehniella, the trays remained in rooms of 3.60 m 2 or 9.79 m 3 with the temperature adjusted to 25 ± 3°C and a photophase of 14h.Ten, twenty, thirty, forty, sixty and seventy rearing trays (40 x 25 cm of base by eight inches tall) were kept in the rooms resulting in the following proportions: 1, 2, 3, 4, 6, 7 tray/m 3 , respectively.
Each tray contained 1.4 kg of diet (97% whole wheat fl our and 3% yeast) (Parra 1997), with the vast majority of larvae at the 5 th instar.Each tray contained approximately 10,000 eggs, equivalent to 0.27 g of eggs (1g = 36,000 eggs).
Measurements were taken in these larvae in the 5th instar, since preliminary tests showed that peak CO 2 production occurs at this instar.After 24 hours, we measured the CO 2 concentration inside the rearing rooms, using a CO 2 environment meter Testo ® , model 535/CO 2 , and the CO 2 concentration was expressed in parts per million -ppm.An empty room without rearing trays was used as the control.We used the results to build a scatter plot, in which we related the CO 2 concentration with the number of trays per room in order to estimate the amount of CO 2 produced by a single tray of A. kuehniella.
To express the amount of CO 2 produced by trays in milliliters (mL), we made a conversion, where the amount of CO 2 produced in the room was subtracted by the amount of CO 2 in the control room.The remaining amount of CO 2 was converted into percentage using a rule of proportionality, considering that 1,000,000 ppm corresponds to 100% CO 2 .Afterwards, the percentage of CO 2 in the room was transformed into mL, considering the volume in liters in the room and the percentage of CO 2 inside each room, to obtain the amount of CO 2 produced per hour.Once again, a rule of proportionality was used to quantify the amount CO 2 produced in 24 h and the quantity produced in 1h.
EFFECT OF CO 2 , ON THE EGG-TO-ADULT VIABILITY AND OVIPOSITION OF A. KUEHNIELLA The experiment to evaluate the effect of CO 2 on A. kuehniella was also carried out at the Laboratory A. kuehniella was also carried out at the Laboratory A. kuehniella of Forest Ecology and Entomology, ESALQ/USP.In this study, rearing trays of A. kuehniella (40 x 25 cm of base and eight inches tall), containing 1.4 kg of a diet (97% whole wheat fl our and 3% yeast) and "inoculated" with approximately 10,000 eggs (Parra 1997) were kept in three rooms (3.60 m 2 or 9.79 m 3 ) permanently closed with temperatures adjusted to 25 ± 3°C and a photophase of 14h.In these rooms, 10, 35 and 70 rearing trays were placed, corresponding to 1.02, 3.57 and 7.15 trays/ m 3 , respectively.EFFECT OF CO 2 ON Anagasta kuehniella (ZELLER 1879) We performed daily measurements of CO 2 in the rooms using a meter and the concentration was expressed in ppm.Measurements were made from the moment of placing the eggs into the trays until the emergence of the fi rst adults.After emergence, fi ve trays (randomly chosen) of each room were selected to determine the viability estimated for each treatment.
We collected adults emerged from each tray until the full emergence, at intervals of five days, using an adapted vacuum cleaner, and weighed the total of insect/tray on a scale in order to estimate the feasibility of each tray.We also estimated the number of eggs produced per tray, multiplying the estimated number of emerged females by the average number of eggs per female in each condition.
We evaluated the individual average weight for males and females of each collection day and we considered a gender ratio of 0.5 (Stein and Parra 1987), thus estimating the number of insects emerged in relation to the number of "inoculated" eggs.
We performed the assessment of the number of eggs laid and life span of adults by separating 20 couples on the fi rst day of emergence of each treatment.For the daily egg counts, these couples of adult insects were kept in glass tubes (8 x 2 cm) in a room with temperature controlled at 25 ± 1°C, RH 60 ± 10%, photophase of 14h and atmospheric CO 2 concentration.Life span was assessed up until the death of all adults.
The viability of eggs laid was assessed by separating the eggs, from the second laying day onwards, into batches of 10 and placing them in Petri dishes (9.5 cm diameter) containing fi lter paper inside.The dishes were kept at a temperature of 25 ± 1°C, RH 60 ± 10%, photophase of 14h and atmospheric CO 2 concentration.The remaining eggs were stored for 10 days for subsequent weighing, separated into in batches of 50 and weighed on an analytical balance.

STATISTICAL ANALYSIS
Data on the number of eggs per female, individual weight of females on the fi rst day of collection, viability of the trays, the total weight of insects per tray, estimated production of eggs, their weight and egg viability were analyzed in terms of normality, homoscedasticity and presence of outliers by the optimal transformation of Box-Cox (with the aid of statistical software SAS ® ).Subsequently, data were subjected to analysis of variance and means compared by Tukey test at the level of 5% probability.
The data on life span were analyzed using the survival curve, where the means and standard error were computed with the Kaplan-Meier of the survival function and corresponding duration, and averages compared by Log-rank (p<0.05).

PRODUCTION OF CARBON DIOXIDE (CO 2 ) PER REARING TRAY OF A. KUEHNIELLA
The production of CO 2 per rearing tray of A. kuehniella showed a linear and proportional relation at an average temperature of 25 ± 3°C and a photophase of 14h, being that the greater the Figure 1 -Linear regression of CO 2 production at different densities of rearing trays of A. kuehniella (with approximately 10,000 larvae in the 5 th instar) per m 3 , 9.79 m 3 rooms, 25 ± 3°C, photophase: 14h.ALOISIO COELHO JUNIOR and JOSÉ R.P. PARRA number of trays, the higher the concentration of CO 2 .Through the linear equation obtained with the collected data, we were able to estimate that a single rearing tray of A. kuehniella, with approximately 10,000 larvae in the 5 th instar, increases the concentration of CO 2 in the rearing room to 9.79 m 3 at 75 ppm (Figure 1), i.e., a tray produces approximately 730 mL of CO 2 inside the room in a period of 24h.In the room with higher density of trays (7 trays/m 3 ), the amount of CO 2 produced was 39.40 L, thus increasing the concentration of CO 2 inside the room to 4,400 ppm (Table I).
to lower pupal metabolism (Chapman 1998) and sharply reduced the CO 2 concentration inside the rearing rooms (Figure 2).In the room containing 10 trays at maximum, the CO 2 concentration was 1,195 ppm, while in the room with 35 trays, the CO 2 concentration reached 1,763 ppm and in the room with 70 trays, the maximum CO 2 concentration was 4,425 ppm, a concentration 13 times higher than the atmospheric CO 2 concentration (approximately 300 ppm) (Figure 2).
Females of immature stages kept in rooms with lower CO 2 concentrations (1,195 ppm) produced, on average, 22% more eggs than females from rooms where the CO 2 concentration was higher than 1,500 ppm (F=7.37;p<0.001; n=20).Females in rooms where the CO 2 concentration reached 1,763 ppm and 4,424, laid the same number of eggs (Table II).
The weight of females, on the fi rst day of collection, in rooms with different CO 2 concentrations were similar (F=0.77;p=0.47; n=25), likewise so were their life span (T=2.2;T=2.2; T p=0.338; n=20) (Table II).Males that emerged in trays kept in rooms where the CO 2 concentration reached 4,425 ppm had a shorter life span than males emerged in trays kept in rooms where the CO 2 concentration reached 1,763 and 1,195 ppm (T=13.3;p=0.001; n=20) (Table II).
The total number of adults emerged, as well as their weight per tray of different CO 2 concentrations were similar in all treatments (F=0.85;p=0.45; n=5/ F=3.37; p=0.071; n=5), likewise so was the estimated viability of the egg-to-adult period for each treatment (F=0.85;p=0.45; n=5) (Table III).However, when estimating the number of eggs produced per tray, relating the number of eggs laid per female and number of emerged females, we could observe a higher production in trays maintained at concentrations below 1,200 ppm (F=14.98;p=0.0005; n=5).It can be inferred that these trays produce about 180,000 eggs, i.e., approximately 5 g more than those kept at concentrations above 1,200 ppm (Table III).The concentration of CO 2 inside the rearing rooms was equal until the 8 th day of larval development (450 to 550 ppm).From the 9 th day onwards, the difference in the CO 2 concentration inside the rooms was more pronounced, rising gradually in each room as each instar increased, until the 25 th day, considering that this time the larvae had reached the 5 th instar, and were about to pupate.After the 25 th day, the metabolism of insects decreased due EFFECT OF CO 2 ON Anagasta kuehniella (ZELLER 1879)

Density of trays/m 3
Weight of females (mg)   The viability of eggs laid by females maintained during the immature stages at different CO 2 concentrations were not affected, remaining equal for all concentrations and above 90% (F=0.16;p=0.86; n=10); nor was egg weight was affected by different CO 2 concentrations to which the immature eggs were exposed (F=0.07;p=0.93; n=10) (Table IV).
instar of larvae (Table I).However, we must consider that the 7,300 L of CO 2 provide a higher concentration inside the room of 625 m 3 , because the density of trays in the room is 16 trays/m 3 , resulting in a CO 2 concentration above 10,000 ppm, 30 times higher than the atmospheric air (which is approximately 300 ppm).
The CO 2 production showed an average rate of 23 mLCO 2 /tray/h (Table I), much smaller than the CO 2 production of a human being at rest, which produces 12,000 mLCO 2 /h, according to the National Institute for Occupational Safety and Health-NIOSH, USA (Williams 2009).Considering that each tray had 10,000 larvae, we can estimate a production of 0.0023 mLCO 2 /larave/h or 2.3 μLCO 2 /larvae/h.
According to Williams (2009) exposure to high concentrations of CO 2 can cause to humans, visual disturbances, headaches, idleness, the sensation of breathlessness and dyspnea and induce narcosis, similar to nitrous oxide.Williams (2009) noted that human beings at rest can withstand, without restriction, concentrations of up to 15,000 ppm.Analogically, concentrations of 30,000 ppm are tolerated for up to 15h by humans at rest, while people running heavy duty can tolerate this concentration for just 30 minutes.When the CO 2 concentration is too high, about 70,000 ppm, the tolerance, even at rest, is 30 minutes, while people performing heavy work collapse and become unconscious for an undetermined period.Analyzing the value of CO 2 production in a single tray found in this study, 0.73 L, for mass-rearing, in which about 10,000 trays are kept in the rearing room, 625 m 3 , we can estimate a production of 7,300 liters of CO 2 per day, considering the 5 th EFFECT OF CO 2 ON Anagasta kuehniella (ZELLER 1879) EFFECT OF CO 2 ON THE VIABILITY OF EGG-TO-ADULT PERIOD AND OVIPOSITION OF A. KUEHNIELLA The effect of CO 2 on the fertility of A. kuehniella was reported in the literature by Janish (1924 apud Lum andFlaherty 1972) who reported that anesthesia with CO 2 reduces the frequency of copulation and oviposition of A. kuehniella.The smaller number of eggs laid by females in rearing rooms with higher CO 2 concentrations (up to 1,200 ppm) may be related to lower ovarian development.According to Press et al. (1973) exposure of females of Tribolium castaneum (Herbst 1797), newly emerged, to CO 2 concentrations of 96% (960,000 ppm), suppresses the development of the ovaries and the number of eggs laid reduces signifi cantly, even when the insects are transferred to atmospheric concentrations of CO 2 .Lum and Phillips (1972) reported that when Plodia interpunctella (Hübner 1813) is anesthetized Plodia interpunctella (Hübner 1813) is anesthetized Plodia interpunctella with 96% of CO 2 (960,000 ppm), there is a 63% reduction in egg laying of the insect, when compared with those not treated with CO 2 .The authors argued that the CO 2 can immobilize the sperms inside the female, which eliminates the stimulus for oviposition.CO 2 can also inhibit the development of eggs or block stimulus for oviposition when the oocytes are already mature.Kumar and Saxena (1978) reported mating delay of Empoasca devastans Distant, 1918 anesthetized with CO 2 .
According to Schroeder et al. (2006), the increase of CO 2 in the soil to 550 ppm has a benefi cial effect on egg production of Diabrotica virgifera virgifera (LeConte 1868) raising fertility virgifera virgifera (LeConte 1868) raising fertility virgifera virgifera by 50%.However, the authors concluded that CO 2 increases egg production because insects recognize the presence of their host by means of the CO 2 emitted by roots and, thus, the high concentration of CO 2 in the soil induces the insect to lay more eggs.
Under the conditions adopted in the present study, the different CO 2 concentrations during the immature stages did not affect the weight of females on the fi rst day of collection, nor their life span (Table II).The shorter life span of males, in rooms where the CO 2 concentration reached 4,425 ppm, indicates that males have greater sensitivity to the conditions of CO 2 during the immature stages, and that is not possible to make such assessment in females, because they die soon after they oviposit.The results described are different from those reported by Edwards and Patton (1965) who observed that anesthesia with CO 2 concentrations above 600,000 ppm, even when the O 2 concentration is maintained similar to atmospheric conditions (210,000 ppm), has deleterious effects on size, weight and growth of nymphs of Acheta domesticus (Linnaeus, 1758).The authors also reported that the heartbeat of insects decreased with the increase of CO 2 concentration.
According to Perron et al. (1972) the anesthesia of CO 2 for 15 minutes affected the mortality, life span and fecundity of Drosophila melanogaster Meigen, Drosophila melanogaster Meigen, Drosophila melanogaster 1830, with the most pronounced effect occurring in newly-emerged insects between 0 and 3 h.According to Hooper (1970) the use of CO 2 to anesthetize Ceratitis capitata Wiedemann, 1824, for a period of Ceratitis capitata Wiedemann, 1824, for a period of Ceratitis capitata 30 minutes, causes unwanted adverse reactions such as increased mortality, decreased number of eggs per female and decreased viability of these eggs, when compared to the use cold and nitrogen which did not affect the insect.We should take into account that the concentrations of CO 2 to which the insects were subjected in the above mentioned studies are much higher than the concentrations of CO 2 of this work, since the authors cited aimed to anesthetize insects in their studies.
The viability and weight of the eggs found in this study showed no difference, but Lum and Phillips (1972) reported that when Plodia interpunctella (Hübner 1813) was anesthetized with 96% of CO 2 (960,000 ppm), the viability of eggs of this insect was reduced by 73%, compared with eggs laid by females not treated with CO 2 .AliNiazee and Lindgren (1970) reported that eggs of Tribolium confusum Du Val, 1868 and Tribolium castaneum (Herbst 1763) from females kept in atmospheres with CO 2 above ALOISIO COELHO JUNIOR and JOSÉ R.P. PARRA 25% (250,000 ppm) were not viable.The authors concluded that the decrease of oxygen and the increase of nitrogen had no effect on the incubation period and viability, but the increase of CO 2 concentration was effectively responsible for deleterious effects on the incubation period and viability.The authors argued that the accumulation of lactic acid resulting from anaerobic metabolism may possibly interfere with physiological processes of insects.
Thus, the accumulation of CO 2 , yet little studied, should be considered for mass rearing of A. kuehniella for the production of Trichogramma spp.and other natural enemies, because the high concentration of CO 2 can reduce egg production and cause problems for the staff engaged in the daily handling of these insects in rooms with high concentrations of CO 2 .
The CO 2 produced by the metabolism of the larvae interfered with oviposition of A. kuehniella, and females reared at low concentrations (1,195 ppm) laid more eggs than those kept at concentrations exceeding 1,200 ppm.For mass rearing of A. kuehniella, it is suggested that the CO 2 concentration inside the rooms be kept below 1,200 ppm in order to increase egg production.
However, in order to maintain the CO 2 at such levels, there is need for assessment on expenses involving electricity, air exchange and cooling of the room, to evaluate the cost/benefit ratio of such operations.
It is obvious that CO 2 is not the only factor affecting egg production of A. kuehniella in mass rearing for the production of Trichogramma spp. or predators, as Parra (1997) reported that temperature, humidity, the ectoparasitoid Habrobracon hebetor (Say 1836) and even ants, mites and fungi as other factors may be decisive in rearing such moth.Therefore, carbon dioxide is an additional parameter to be evaluated, especially in mass rearing that is becoming increasingly common, for the production and commercialization of parasitoids and predators in programs for Biological Control.

Figure 2 -
Figure 2 -Concentration of CO 2 (ppm) during the egg-to-adult period of A. kuehniella, inside the rearing rooms with different quantities of trays per m 3 .Temp.: 25 ±3°C, photophase: 14h.The arrows indicate the maximum production of CO 2 in the three conditions studied, corresponding to the 5 th instar of the larval stage.
II Number of trays per m 3 , weight average of females, number of eggs per female, life span of ♂ and ♀ of A. kuehniella, emerged in rearing trays kept in rooms at different CO 2 concentration (metabolic) and maximum concentration of CO 2 (ppm) inside the rooms.Temperature: 25 ±1°C, RH: 60 ±10%, photophase: 14h, atmospheric CO 2 concentration.ALOISIO COELHO JUNIOR and JOSÉ R.P. PARRA

TABLE I Concentration of CO 2 (ppm) produced by trays of A. kuehniella, inside the rooms of 9.79 m 3 , the amount of CO 2 into the rearing rooms (mL) and the amount of CO 2 produced per tray per hour (mL): Temperature: 25 ± 3°C, photophase: 14h.
EFFECT OF CO 2 ON THE VIABILITY OF THE EGG-TO-ADULT PERIOD AND OVIPOSITION OF A. KUEHNIELLA

TABLE III Average of total weight of insects, estimated average of the number of insects, viability of the egg-to-adult period and number of eggs produced per tray, originated from A. kuehniella emerged in rearing trays kept in rooms with different CO 2 concentrations. Temperature 25 ±3°C, RH: 60 ±10%, photophase: 14h, atmospheric CO 2 concentration.
Data showed no statistical differences in the Tukey test, at 5% of probability. 2Means followed by the same letter did not differ in the Tukey test at 5% of probability. 3Standard error of the mean. 1

TABLE IV Viability and weight of eggs from females of A. kuehniella emerged in rearing trays kept in rooms with different concentrations of CO 2 . Temperature: 25 ± 3°C, RH 60 ± 10%, 14h photophase, atmospheric CO 2 concentration.
Data showed no statistical differences in the Tukey test, at 5% of probability. 2Standard error of the mean.