Microbial biopesticides affected age-stage life table of the tomato leaf miner, Tuta absoluta (Lepidoptera – Gelechiidae)
© The Author(s) 2018
Received: 18 September 2017
Accepted: 6 December 2017
Published: 8 February 2018
The tomato leaf miner, Tuta absoluta (Meyric), second instar larvae were exposed for 1 day to LC50 of Bacillus thuringiensis (Berliner) subsp. kurstaki or Beauveria bassiana (Balsamo) on treated tomato foliages. Treated larvae had the longest development period while non-treated larvae had the shortest development period. The highest survivorship (l x ) of adults was obtained by the non-treated larvae while the lowest survivorship was obtained by B. thuringiensis-treated larvae. The lowest age-specific fecundity (m x ) of females was obtained by individuals treated as second instar larvae with B. bassiana. The intrinsic rate of increase (r m ) reached its maximum with non-treated individuals while this value decreased to the minimum values with biopesticide-treated individuals. Therefore, development, survival, and reproduction of treated individuals were lower than those of non-treated individuals. The reproduction period and adult longevity were the shortest considering biopesticide-treated individuals. The highest and lowest net reproductive rates (R0) were recorded for non-treated and treated individuals, respectively. The mean generation time was increased with biopesticide-treated individuals.
The tomato leaf miner, Tuta absoluta (Meyric) (Lepidoptera – Gelechiidae), is a pest of great economic importance in a number of countries. Its primary host is tomato although potato, eggplant, common weed, and various wild solanecous plants are also suitable hosts (Siqueira et al., 2000 and Lietti et al., 2005). The tomato leaf miner, T. absoluta larvae, can significantly reduce both yield and fruit quality by direct feeding and the secondary pathogens which may enter through the wounds caused (Silva et al., 2011). The infestation of tomato plants occurs throughout the entire crop cycle. Feeding damage is caused by all larval instars and throughout the whole plant. On leaves, the larvae feed on the mesophyll tissue, forming irregular leaf mines which may later become necrotic. Larvae also can form extensive galleries in the stems and attack fruits.
Since T. absoluta was detected in the Mediterranean Basin, the most common control practice has been based on the use of chemical insecticides (Bielza, 2010). Pesticides may have effects on the natural enemies and also may lead to insect resistance (Lietti et al., 2005). Microbial control include bacteria and fungi; the comparison of entomopathogens with convention chemical pesticides is usually solely from the perspective of their efficacy and cost (Lacey et al., 2001). As an alternative to chemicals, commercial formulations based on the entomopathogenic bacteria, B. thuringiensis subsp. kurstaki and entomopathogenic fungi, B. bassiana have been tested against T. absoluta (Giustolin et al., 2001; Marta et al., 2006 and Gonzalez-Cabrera et al., 2011).
In order to continue to evaluate potential of the entomopathogens, its effects on some development attributes and population parameters are needed. The population dynamics of any insect pest, the age-stage, and two-sex life table is the most important techniques which help in qualitative and quantitative female and male populations, the stage differentiation, and variable developmental rate among individuals (Chi and Liu, 1985 and Chi, 1988). Comparison of life table parameters of T. absoluta individuals treated with the microbial biopesticides with non-treated individuals may refer to important aspects that may be useful for future control programs.
The objective of this study is to elucidate novel aspects considering development, survival, and fecundity of the tomato leaf miner, T. absoluta, after treated the second instar larvae with the two microbial biopesticides, Bacillus thuringiensis (Berliner) subsp. kurstaki and Beauveria bassiana.
Materials and methods
The insect colony
The tomato leaf miner, Tuta absoluta, colony was established with larvae collected from tomato fields at EL-Natron Valley area (30° 30′ 26″ N, 13° 30′ 03″ E). The moth adults emerged from larvae reared on leaves of different tomato cultivars were collected to be used in the development of the colony. In order to obtain the same age eggs, 15 pairs of both sexes of the moth were kept inside a plastic oviposition container (30 cm diameter, 20 cm height) sealed at the top with a fine mesh net. After 24 h, the laid eggs were collected from the container. Each egg was transferred into a 10-cm Petri dish. Fresh tomatoes’ leaves were provided for larval feeding in dishes and were replaced every other day. The fourth instar larvae were transferred into small plastic tubes (3 cm diameter, 6 cm depth) till pupation. Experiments began following the rearing of three generations of T. absoluta under laboratory conditions. The insect colony and the experiments were conducted in a (3 × 4 m) greenhouse equipped with a drip irrigation system located at Biological Control Laboratory, Agricultural Research Center, Giza. The average temperature during the experiments was 25 ± 3 °C with relative humidity ranging from 70 to 85% and a photoperiod of 14:10 h (L:D).
The microbial biopesticides
The first microbial biopesticide, Bacillus thuringiensis subsp. kurstaki, is a member of the genus Bacillus, adverse group of spore-forming bacteria Dipel-2x. The commercial formulation (wettable powder) of B. thuringiensis kurstaki (32,000 IU/mg) was used in this bioassay. The second biopesticide used in this study was Bio-Bower, the commercial formulation of entomopathogenic fungus, Beauveria bassiana provided by T. Stanes and Company Limited, India.
Bioassay activity of the microbial biopesticides.
This study was designed to determine the efficacy of B. thuringiensis and B. bassiana when T. absoluta larvae fed on treated tomatoes leaves. Five concentrations of 0.05, 0.1, 0.2, 0.4, and 0.5 mg B. thuringiensis powder/ml H2O and also five concentrations of 1 × 107, 0.5 × 107, 0.25 × 107, 0.125 × 107, and 0.063 × 107 B. bassiana Bio-Bower/ml H2O were used. Five replicates (each replicate involved 10 larvae) per concentration were tested. Tomato seedlings’ pots of approximately 45 days old were embedded in each concentration then transferred to a clean filter paper for allowing water to evaporate. The second instar larvae (2 days old) were used in this bioassay. The larvae were starved for 24 h before the begging of feeding. After 24 h of treatment, treated larvae were transferred to new clean Petri dishes (10 cm × 3 cm) with new clean fresh tomato seedling pots. The numbers of larval mortality were recorded daily and also the pots of tomatoes were changed daily until pupation. The average cumulative mortality percentage of larvae was calculated for each concentration. Mortality was corrected with Abbott’s formula (Abbott, 1925), and all B. thuringiensis or B. bassiana data were subjected to probit analysis (Finney, 1971) also the LC50, LC90, and 95% confidence limits.
Host plant source
Young foliage (45 days old) of tomato plants was used in this study. All foliage tested in this laboratory experiments was collected from field grown plants free of pesticide and chemical fertilizers.
Development and survival of the immature stages
Ten female and ten male moths (newly emerged) were collected from larvae reared on normal host plants. The moths were provided with 10% sucrose solution and allowed to mate for 1–2 days in containers (25 × 15 × 10 cm). The mated moths were transferred to new cages (15 × 10 × 5 cm), one female/cage. The top of the cage was cut-off and replaced with a covering of fine mesh gauze. Host plant leaves were replaced with a fresh one on a daily basis. Thirty 2-day-old larvae (second instar) were treated with LC50 of B. thuringiensis kurstaki while other 30 larvae of the same age were treated with the LC50 of B. bassiana and left for 24 h. After treatment, the larvae were transferred to new clean Petri dishes provided by normal tomatoes’ foliage. Non-treated 30 larvae of the same age used as control. Development of larvae and pupae were observed in the growth chamber under similar conditions. Survival rate and developmental time were recorded daily for all immature stages; also, the sex of emerged adults was determined.
Reproduction and population growth parameters
Adult longevity and reproduction
Moths emerged from larvae treated as second instar by the LC50 of B. thuringiensis or B. bassiana and also those emerged from non-treated larvae were allowed to mate for 1–2 days. Mated moths were transferred to a new container (25 × 15 × 10 cm, one female per container) and supplied with 10% sucrose solution. The females were provided with fresh foliage every day for oviposition. The foliage was collected every day to determine the number of eggs deposited until the death of each adult female. The pre-oviposition period, oviposition period, post-oviposition period, adult longevity, and age-specific fecundity were determined.
The intrinsic rate and life table parameters
where x is the age (days), l x is the age-specific survival, and m x is the average number of female offspring of a female at age x. The r m is the intrinsic rate of increase for the population. In addition to r m , the other life table parameters, including net reproductive rate (R0 = Σ l x m x ), generation time (T = Σ xl x m x /R0), finite rate of increase (λ = e rm ), and population doubling time (DT = ln 2/r m ) were calculated.
Firstly, variables were tested for normality before analysis. Data were analyzed by one-way analysis of variance (ANOVA) followed by comparison of the means with Tukey test at α = 0.05 using software GraphPad Instat (2009).
Results and discussion
Toxicity of biopesticides
Lethal concentrations of B. thuringiensis and B. bassiana against second instar larvae of T. absoluta
95% confidence limits
95% confidence limits
B. thuringiensis (mg/ml)
1.33 ± 0.34
B. bassiana (spore/ml)
1.8 × 107
1.2 × 107–9.5 × 107
48.9 × 107
10.9 × 107–100.4 × 107
0.48 ± 0.24
Development of treated second instar T. absoluta larvae
Development of T. absoluta treated as second instar larvae by LC50 values of B. thuringiensis or B. bassiana
Developmental period (mean ± SE) days
Total developmental time
5.93 ± 0.13a
14.75 ± 0.03a
10.94 ± 0.26a
31.57 ± 0.42a
6.3 ± 0.12a
15 ± 0.04a
10.77 ± 0.24a
32.07 ± 0.51a
5.96 ± 0.14a
10.96 ± 0.15b
8.29 ± 0.14b
25.21 ± 0.38b
In this study, the mean larval periods varied from 10.96 to 15 days considering the non-treated and B. bassiana-treated larvae. Pereyra and Sanchez (2006) found out that at 25 °C the larval periods of the tomato leaf miner were 12.14 and 14 days on tomato and potato plants, respectively. Erdogan and Babaroglu (2014) reported that total larval period of T. absoluta was 10.97 days at 25 °C when fed on non-treated tomato cultivars; the same result was obtained also by Nouri-Ganbalani et al. (2016). The pupal period of the tomato leaf miner varied from 8.29 to 10.94 days taken into consideration the non-treated and B. thuringiensis-treated larvae, respectively. Torres et al. (2001) found the pupal period of the tomato leaf miner ranged from 7 to 9 days when the larvae reared on normal (non-treated) tomato cultivar.
In our study, the total developmental time of T. absoluta varied from 25.21 days on non-treated cultivar to 31.57 and 32.07 days on B. thuringiensis- or B. bassiana-treated cultivar, respectively. According to Cuthbertson (2011), the mean developmental time of T. absoluta was 35 days under 25 °C while Erdogan and Babaroglu (2014) showed that the mean total of developmental time of T. absoluta was 31.18 days on non-treated tomato leaves at 25 °C. The treated T. absoluta in our study developed slowly compared to the non-treated individuals. The relatively longer developmental time of T. absoluta on microbial biopesticide-treated cultivar may be attributed to the mode of action of the microbial biopesticides. The developmental time of the herbivore insects are strongly affected by the nutritional qualities of the host plant, which in turn influences its population growth (Du et al., 2004).
Age-specific mortality of T. absoluta treated with the two tested microbial pesticides
Age stages (x)
Number alive at begging of x(l x )
Number dying during (x)
% mortality (100qx)
% cumulative surviving (100rx)
Comparison of T. absoluta life span treated as second instar larvae by LC50 values of B. thuringiensis or B. bassiana
Mean ± SE (range)
Mean ± SE (range)
9.48 ± 0.44a (7–11)
10.75 ± 0.41a (9–12)
9.63 ± 0.48a (7–12
10.8 ± 0.33a (9–12)
11.17 ± 0.32b (9–12)
13.2 ± 0.26b (12–15)
Obtained data indicated that the microbial biopesticides can also affect survival, growth, and reproduction of herbivore insects. Insect individuals receiving lethal concentration (LC50) of the microbial biopesticides may survive and complete their development but with differences in growth and development as a result of their varying nutritional requirements (Lacey et al., 2001). Differences in life span and reproduction period of T. absoluta in this study indicated that the microbial biopesticides had deleterious effects on the treated individuals. The longest female life span was obtained by the non-treated individuals, whereas the shortest life span was obtained by the treated individuals. The same result was obtained considering male life span.
Oviposition periods and age-specific fecundity
Oviposition periods and fecundity (mean ± SE) of T. absoluta treated as second instar larvae by LC50 values of B. thuringiensis or B. bassiana
(Mean ± SE)
Pre-oviposition period (day)
2.4 ± 0.13 a
2.13 ± 0.13 a
2 ± 0.1 a
Oviposition period (day)
8.93 ± 0.23 a
7.85 ± 0.24 b
7.5 ± 0.31 b
Post-oviposition period (day)
1.67 ± 0.21 a
0.5 ± 0.02 b
0.3 ± 0.02 b
202.4 ± 3.95 a
146.87 ± 4.4 b
134.4 ± 7.34 b
Daily ovipositional rate
22.78 ± 0.55 a
18.46 ± 1.1 b
18.07 ± 1.03 b
The fecundity was affected significantly by the microbial biopesticide treatments as shown in Table 5. The fecundity of female from non-treated larvae was 202.4 ± 3.95 eggs with daily oviposition rate of 22.78 ± 0.55 eggs. Fecundity of those females from B. thuringiensis- or B. bassiana-treated larvae were 146.87 ± 4.4 or 134.4 ± 7.3 eggs with daily oviposition rate of 18.46 ± 1.1 and 18.07 ± 1.03 eggs, respectively. Statistically, a significant difference of fecundity was found between the non-treated larvae and treated larvae (F(2,32) = 53.18; P = 0.018). Similarly, a significant difference was obtained considering the daily ovipositional rate of females from non-treated and treated larvae (F(2,32) = 11.16; P = 0.033). No significant differences were obtained in comparison with the fecundity and daily ovipositional rate of females from B. thuringiensis- or B. bassiana-treated larvae (Table 5).
The age-specific fecundity (m x ) of T. absoluta females from non-treated larvae and those from B. thuringiensis- or B. bassiana-treated larvae is shown in Fig. 2. The first oviposition occurred at age of 3 days of female lifetime considering non-treated and treated females. The highest daily fecundity (m x ) of T. absoluta females were 42.8, 30.4, and 27.8 female offsprings per female per day considering females from non-treated, B. thuringiensis-treated, and B. bassiana-treated larvae, respectively. The highest daily fecundity was obtained at the age of 6 days. Treatment of T. absoluta larvae by the tested microbial biopesticide affected the age-specific fecundity of females. The obtained oviposition period of females from B. thuringiensis-treated larvae was 7.85 ± 0.24 days while this period for treated larvae with B. bassiana was 7.5 ± 0.31 in comparison to oviposition period of 8.93 ± 0.23 days for females from non-treated larvae.
Differences in life span and reproduction period of T. absoluta in this study indicated that the microbial biopesticides had deleterious effects on the treated individuals. The longest female life span was obtained by the non-treated individuals, whereas the shortest life span was obtained by the treated individuals. The same result was obtained considering male life span. The total female fecundity in this study ranged from 202.4 to 134.4 eggs considering non-treated and B. bassiana-treated individuals, respectively. The low number of eggs laid by B. thuringiensis- or B. bassiana-treated individuals could have been affected by the more indirect route of biopesticide treatment. It was observed that young tomato leaf miner individuals treated with the biopesticides fed less and developed more slowly than the non-treated individuals. Different values have been reported for pre-oviposition period, female fecundity, oviposition period, and longevity of T. absoluta.
Population growth parameters
Life table parameters (mean ± SE) of T. absoluta treated as second instar larvae by LC50 values of B. thuringiensis or B. bassiana
(Mean ± SE)
The net reproductive rate (R0)
101.3 ± 18.9 a
38.73 ± 11.98 b
44.57 ± 11.92 b
The intrinsic rate of increase (r m )
0.142 ± 0.01a
0.097 ± 0.01 b
0.099 ± 0.01 b
The finite rate of increase (λ)
1.15 ± 0.01 a
1.10 ± 0.01 b
1.11 ± 0.01b
The mean generation time (T0)
32.69 ± 0.4 a
38.09 ± 0.83 b
38.57 ± 0.58 b
Doubling time (DT)
4.9 ± 0.3 a
7.1 ± 0.4 b
7.0 ± 0.3 b
The population growth parameters of T. absoluta significantly varied in comparison with microbial biopesticide-treated and non-treated tomato cultivars in this study. This indicated a deleterious effect of the biopesticides on the treated individuals. The net reproduction rate (R0) is a key statistic in population dynamics (Richard, 1961) that summarizes the physiological traits of an insect related to its reproduction capacity. The net reproduction value of T. absoluta treated as second instar larvae with B. thuringiensis was the lowest. Moreover, R0 values observed from B. thuringiensis- or B. bassiana-treated individuals were significantly lower than R0 of non-treated individuals. The intrinsic rate of increase (r m ) reached its maximum for non-treated individuals while decrease to the minimum values for biopesticide-treated individuals. Intrinsic rate of increase is important because it reflects the overall effect of the biopesticides on development, reproduction, and survival of the insect species (Southwood and Henderson, 2000). Therefore, the reproduction, development, and survival of T. absoluta treated with B. thuringiensis or B. bassiana were low. The mean generation times (T0) of biopesticide-treated individuals were longer than those of non-treated individuals. The daily finite rate of increase (λ) was significantly lower for biopesticide-treated individuals in comparison with that for non-treated individuals. Also, higher doubling times (DT) of T. absoluta were obtained with the treated individuals. In conclusion, the microbial biopesticides (B. thuringiensis subsp. kurstaki and B. bassiana) affected the development, survival, reproduction, longevity, and population parameters of T. absoluta. In this study, individuals of T. absoluta receiving lethal concentrations (LC50) of B. thuringiensis or B. bassiana and directly not dead, have long developmental time and lower m x as well as the lowest r m value of immature and low survival rate (l x ). Such biopesticide effects could cause reductions in survival fitness of the pest insect. Also, prolonged developmental time could increase the exposure of the insect to its natural enemies.
T. absoluta is an important pest of tomato and many other plants of Solanaceae family. Because the primary management tactic for T. absoluta control is often the chemical methods although it is not a sustainable option in the long run due to insecticide resistance. An attractive alternative tool to control this pest is the use of microbial biopesticides due to their eco-friendly and target selective characteristic. The microbial biopesticides (B. thuringiensis and B. bassiana) were affected the population parameters of T. absoluta in addition to their acute effects. Our study provides useful data for developing management tactics depending on the changes in the population density of the pest.
AAY and NZMZ conceived and designed the research. HAA and RF conducted the experiments. AAY and RF analyzed the data and wrote the manuscript. AAY, HAA and NZMZ made critical reviewed and approved the final version. All authors reviewed and approved the final manuscript.
The authors declare that they have no competing interests.
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