Effect of Carbon dioxide (CO₂) on mortality and reproduction of Anagasta kuehniella (Zeller 1879), in mass rearing, aiming at the production of Trichogramma spp.

Abstracts

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 4^th and 5^th 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 influenced by carbon dioxide.

mass rearing; factitious host; abiotic factor; carbon dioxide

Ovos de Anagasta kuehniella (Zeller, 1879) são muito utilizados para a criação massal de Trichogramma spp. e de outros parasitóides e predadores, sendo comercializados em muitos países. O objetivo deste trabalho foi avaliar o efeito do dióxido de carbono (CO₂), proveniente do metabolismo larval, em parâmetros biológicos de A. kuehniella, principalmente na postura. Para que este objetivo fosse atingido, foram avaliados a produção de dióxido de carbono (CO₂) por bandeja de criação de A. kuehniella e o efeito do CO₂ na viabilidade do período ovo-adulto e na postura de A. kuehniella. Por meio dos resultados obtidos pôde-se estimar que uma bandeja de criação, com lagartas entre o 4° e 5° ínstares, inoculada com 10.000 lagartas produz, em média, 30,67 ml de CO₂ por hora. A maior produção de ovos de A. kuehniella foi obtida quando as lagartas foram mantidas em salas com concentração de CO₂ inferior a 1.200 ppm, produzindo 23% mais ovos nesta condição em relação a de insetos provenientes de salas com concentrações maiores. Em salas com alta densidade de bandejas (70 bandejas/sala), a concentração de CO₂ ultrapassou 4400 ppm. A viabilidade do período ovo-adulto não foi influenciada pelo dióxido de carbono.

criação massal; hospedeiro alternativo; fator abiótico; dióxido de carbono

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 difficult to obtain in eggs of natural hosts. The flour-moth, Anagasta kuehniella (Zeller 1879), is one of the 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). High temperatures also favor the emergence of the parasitoid larvae Habrobracon hebetor (Say 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 (CO₂), 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/m²) inside of the rearing rooms is very high, which can lead to excessive accumulation of CO₂, and can ultimately cause losses in the rearing.

Given the characteristics of mass rearing of A. kuehniella, this paper aims to evaluate the CO₂ production per rearing tray of A. kuehniella and the effect of CO₂ on the viability of adult-egg period and oviposition of this moth.

MATERIALS AND METHODS

Carbon Dioxide (CO₂) Production Per Rearing Tray of A. kuehniella

The test to evaluate the production of CO₂ per rearing tray of A. kuehniella was developed at the Laboratory of Forest Ecology and Entomology, ESALQ/USP. To measure the CO₂ production per rearing tray of A. kuehniella, the trays remained in rooms of 3.60 m² or 9.79 m³ with the temperature adjusted to 25 ± 3°C and a photophase of 14h. Ten, twenty, thirty, forty, sixty and seventy rearing trays (40 × 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/m³, respectively.

Each tray contained 1.4 kg of diet (97% whole wheat flour 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 CO₂ production occurs at this instar. After 24 hours, we measured the CO₂ concentration inside the rearing rooms, using a CO₂ environment meter Testo^®, model 535/CO₂, and the CO₂ 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 CO₂ concentration with the number of trays per room in order to estimate the amount of CO₂ produced by a single tray of A. kuehniella.

To express the amount of CO₂ produced by trays in milliliters (mL), we made a conversion, where the amount of CO₂ produced in the room was subtracted by the amount of CO₂ in the control room. The remaining amount of CO₂ was converted into percentage using a rule of proportionality, considering that 1,000,000 ppm corresponds to 100% CO₂. Afterwards, the percentage of CO₂ in the room was transformed into mL, considering the volume in liters in the room and the percentage of CO₂ inside each room, to obtain the amount of CO₂ produced per hour. Once again, a rule of proportionality was used to quantify the amount CO₂ produced in 24 h and the quantity produced in 1h.

Effect of CO₂, on the Egg-to-Adult Viability and Oviposition of A. kuehniella

The experiment to evaluate the effect of CO₂ on A. kuehniella was also carried out at the Laboratory of Forest Ecology and Entomology, ESALQ/USP. In this study, rearing trays of A. kuehniella (40 × 25 cm of base and eight inches tall), containing 1.4 kg of a diet (97% whole wheat flour and 3% yeast) and “inoculated” with approximately 10,000 eggs (Parra 1997) were kept in three rooms (3.60 m² or 9.79 m³) 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/m³, respectively.

We performed daily measurements of CO₂ 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 first adults. After emergence, five 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 first day of emergence of each treatment. For the daily egg counts, these couples of adult insects were kept in glass tubes (8 × 2 cm) in a room with temperature controlled at 25 ± 1°C, RH 60 ± 10%, photophase of 14h and atmospheric CO₂ 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 filter paper inside. The dishes were kept at a temperature of 25 ± 1°C, RH 60 ± 10%, photophase of 14h and atmospheric CO₂ 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 first 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).

RESULTS

Production of Carbon Dioxide (CO₂) Per Rearing Tray of A. kuehniella

The production of CO₂ 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 number of trays, the higher the concentration of CO₂. 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 CO₂ in the rearing room to 9.79 m³ at 75 ppm (Figure 1), i.e., a tray produces approximately 730 mL of CO₂ inside the room in a period of 24h. In the room with higher density of trays (7 trays/m³), the amount of CO₂ produced was 39.40 L, thus increasing the concentration of CO₂ inside the room to 4,400 ppm (Table I).

Figure 1
Linear regression of CO₂ production at different densities of rearing trays of A. kuehniella (with approximately 10,000 larvae in the 5^th instar) per m³, 9.79 m³ rooms, 25 ± 3°C, photophase: 14h.

TABLE I
Concentration of CO₂ (ppm) produced by trays of A. kuehniella, inside the rooms of 9.79 m³, the amount of CO₂ into the rearing rooms (mL) and the amount of CO₂ produced per tray per hour (mL): Temperature: 25 ± 3°C, photophase: 14h.

Number of trays/room	Proportion of trays/m3	CO2 (ppm)	CO2 (mL)	mL CO2/tray/h
1	0.1*	75**	736**	30.67**
10	1	909	5,130	21.39
20	2	1,533	11,260	23.45
30	3	2,143	17,240	23.95
40	4	2,888	24,560	25.58
60	6	3,688	32,410	22.51
70	7	4,400	39,400	23.45

• ^*
  proportion of one tray kept in a room of 9.79 m³,
• ^**
  Estimated values.

Effect of CO₂ on the Viability of the Egg-to-Adult Period and Oviposition of A. kuehniella

The concentration of CO₂ 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 CO₂ 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 to lower pupal metabolism (Chapman 1998) and sharply reduced the CO₂ concentration inside the rearing rooms (Figure 2). In the room containing 10 trays at maximum, the CO₂ concentration was 1,195 ppm, while in the room with 35 trays, the CO₂ concentration reached 1,763 ppm and in the room with 70 trays, the maximum CO₂ concentration was 4,425 ppm, a concentration 13 times higher than the atmospheric CO₂ concentration (approximately 300 ppm) (Figure 2).

Figure 2
Concentration of CO₂ (ppm) during the egg-to-adult period of A. kuehniella, inside the rearing rooms with different quantities of trays per m³. Temp.: 25 ±3°C, photophase: 14h. The arrows indicate the maximum production of CO₂ in the three conditions studied, corresponding to the 5^th instar of the larval stage.

Females of immature stages kept in rooms with lower CO₂ concentrations (1,195 ppm) produced, on average, 22% more eggs than females from rooms where the CO₂ concentration was higher than 1,500 ppm (F=7.37; p<0.001; n=20). Females in rooms where the CO₂ concentration reached 1,763 ppm and 4,424, laid the same number of eggs (Table II).

TABLE II
Number of trays per m³, 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 CO₂ concentration (metabolic) and maximum concentration of CO₂ (ppm) inside the rooms. Temperature: 25 ±1°C, RH: 60 ±10%, photophase: 14h, atmospheric CO₂ concentration.

Number of trays/room	Density of trays/m3	Weight of females (mg)1,5±EP4	N° of eggs/female2±EP4	Life span (days)		Maximum concentration of CO2 (ppm)
				♂3±EP4	♀1±EP4
10	1.02	21.08 ± 0.64	427.55 ± 23.67 a	12.25 ± 0.58 a	7.47 ± 0.24	1,195
35	3.57	22.04 ± 0.49	328.65 ± 14.07 b	13.50 ± 0.61 a	8.00 ± 0.44	1,763
70	7.14	21.89 ± 0.63	343.15 ± 20.09 b	7.70 ± 0.48 b	8.05 ± 0.41	4,425

• ¹
  Data showed no statistical differences in the Tukey test, at 5% of probability.
• ²
  Means followed by the same letter did not differ in the Tukey test at 5% of probability.
• ³
  Means followed by the same letter did not differ in the Log-rank test.
• ⁴
  Standard error of the mean.
• ⁵
  Females on the first day of data collection.

The weight of females, on the first day of collection, in rooms with different CO₂ concentrations were similar (F=0.77; p=0.47; n=25), likewise so were their life span (T=2.2; p=0.338; n=20) (Table II). Males that emerged in trays kept in rooms where the CO₂ concentration reached 4,425 ppm had a shorter life span than males emerged in trays kept in rooms where the CO₂ 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 CO₂ 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).

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 CO₂ concentrations. Temperature 25 ±3°C, RH: 60 ±10%, photophase: 14h, atmospheric CO₂ concentration.

Number of trays/room	Total weight of insects/tray1(g)±EP3	Estimated number of insects/tray1±EP3	Estimated viability/tray1±EP3	Number of estimated eggs/tray2±EP3
10	64.78 ± 3.1	4,513 ± 218	45.1 ± 2.2	964.9 ± 46.71 a
35	63.8 ± 1.1	4,728 ± 95	47.3 ± 0.9	777.1 ± 15.72 b
70	67.38 ± 1.7	4,468 ± 107	44.7 ± 1.1	766.7 ± 18.52 b

• ¹
  Data showed no statistical differences in the Tukey test, at 5% of probability.
• ²
  Means followed by the same letter did not differ in the Tukey test at 5% of probability.
• ³
  Standard error of the mean.

The viability of eggs laid by females maintained during the immature stages at different CO₂ 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 CO₂ concentrations to which the immature eggs were exposed (F=0.07; p=0.93; n=10) (Table IV).

TABLE IV
Viability and weight of eggs from females of A. kuehniella emerged in rearing trays kept in rooms with different concentrations of CO₂. Temperature: 25 ± 3°C, RH 60 ± 10%, 14h photophase, atmospheric CO₂ concentration.

Number of trays/room	Viability of eggs (%)1± EP2	Egg weight (µg)1 ± EP2
10	97 ± 1.5	24.0 ± 0.9
35	98 ± 1.3	24.4 ± 0.8
70	97 ± 1.5	24.4 ± 0.8

• ¹
  Data showed no statistical differences in the Tukey test, at 5% of probability.
• ²
  Standard error of the mean.

DISCUSSION

Production of Carbon Dioxide (CO₂) Per Rearing Tray of A. kuehniella

Analyzing the value of CO₂ 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 m³, we can estimate a production of 7,300 liters of CO₂ per day, considering the 5^th instar of larvae (Table I). However, we must consider that the 7,300 L of CO₂ provide a higher concentration inside the room of 625 m³, because the density of trays in the room is 16 trays/m³, resulting in a CO₂ concentration above 10,000 ppm, 30 times higher than the atmospheric air (which is approximately 300 ppm).

The CO₂ production showed an average rate of 23 mLCO₂/tray/h (Table I), much smaller than the CO₂ production of a human being at rest, which produces 12,000 mLCO₂/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 mLCO₂/larave/h or 2.3 µLCO₂ /larvae/h.

According to Williams (2009) exposure to high concentrations of CO₂ 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 CO₂ 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.

Effect of CO₂ on the Viability of Egg-to-Adult Period and Oviposition of A. kuehniella

The effect of CO₂ on the fertility of A. kuehniella was reported in the literature by Janish (1924 apud Lum and Flaherty 1972) who reported that anesthesia with CO₂ reduces the frequency of copulation and oviposition of A. kuehniella. The smaller number of eggs laid by females in rearing rooms with higher CO₂ 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 CO₂ concentrations of 96% (960,000 ppm), suppresses the development of the ovaries and the number of eggs laid reduces significantly, even when the insects are transferred to atmospheric concentrations of CO₂. Lum and Phillips (1972) reported that when Plodia interpunctella (Hübner 1813) is anesthetized with 96% of CO₂ (960,000 ppm), there is a 63% reduction in egg laying of the insect, when compared with those not treated with CO₂. The authors argued that the CO₂ can immobilize the sperms inside the female, which eliminates the stimulus for oviposition. CO₂ 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 CO₂.

According to Schroeder et al. (2006), the increase of CO₂ in the soil to 550 ppm has a beneficial effect on egg production of Diabrotica virgifera virgifera (LeConte 1868) raising fertility by 50%. However, the authors concluded that CO₂ increases egg production because insects recognize the presence of their host by means of the CO₂ emitted by roots and, thus, the high concentration of CO₂ in the soil induces the insect to lay more eggs.

Under the conditions adopted in the present study, the different CO₂ concentrations during the immature stages did not affect the weight of females on the first day of collection, nor their life span (Table II). The shorter life span of males, in rooms where the CO₂ concentration reached 4,425 ppm, indicates that males have greater sensitivity to the conditions of CO₂ 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 CO₂ concentrations above 600,000 ppm, even when the O₂ 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 CO₂ concentration.

According to Perron et al. (1972) the anesthesia of CO₂ for 15 minutes affected the mortality, life span and fecundity of Drosophila melanogaster Meigen, 1830, with the most pronounced effect occurring in newly-emerged insects between 0 and 3 h. According to Hooper (1970) the use of CO₂ to anesthetize Ceratitis capitata Wiedemann, 1824, for a period of 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 CO₂ to which the insects were subjected in the above mentioned studies are much higher than the concentrations of CO₂ 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 CO₂ (960,000 ppm), the viability of eggs of this insect was reduced by 73%, compared with eggs laid by females not treated with CO₂. AliNiazee and Lindgren (1970) reported that eggs of Tribolium confusum Du Val, 1868 and Tribolium castaneum (Herbst 1763) from females kept in atmospheres with CO₂ above 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 CO₂ 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 CO₂, 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 CO₂ can reduce egg production and cause problems for the staff engaged in the daily handling of these insects in rooms with high concentrations of CO₂.

The CO₂ 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 CO₂ concentration inside the rooms be kept below 1,200 ppm in order to increase egg production.

However, in order to maintain the CO₂ 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 CO₂ 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.

We thank Janet Reid, JWR Associates, for the English revison. We also thank the National Counsil of Technological and Scientific Development-CNPq and the National Institute of Semiochemicals in Agriculture-INCT for financial support.

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Publication Dates

• Publication in this collection
  June 2013

History

• Received
  16 Mar 2012
• Accepted
  17 Oct 2012