Abstract: The effects of sublethal concentrations of B.t.i. (Vectobac 12 AS, LC₂₀ = 0.15 and LC₇₀ = 0.76 mL m^-2) on life parameters of Anopheles superpictus were assessed in the laboratory for 3 generations. According to the data, sex ratios were affected by exposure to sublethal doses of B.t.i. Developmental time was prolonged significantly in groups treated with LC₇₀, while, LC₂₀ had no effect. Pre-oviposition periods were not affected by sublethal concentrations. Oviposition period in the LC₇₀ group was prolonged, whereas, in the control and LC₂₀ groups it was nearly identical. Longevity was affected by sublethal exposure and increased from F₁-F₃ in both groups. Gross and net reproductive rates were adversely affected in both exposure groups and these effects increased with increasing B.t.i. concentration. Generation time was affected by exposure to sublethal concentrations and decreased from generation to generation. Main daily fecundity did not differ significantly between treatment groups and the control group but was slightly higher in the latter. Significant differences (p<0.05) between generations in terms of survival rate were found in the LC₂₀ group but not in the LC₇₀ group. Life parameters were affected adversely and this effect was more pronounced in lines exposed to higher concentrations. Lower concentration effects were not clear and parameters fluctuated between generations when compared to the control group.

INTRODUCTION

Thirteen Anopheles sp. have been recorded in Turkey (Ramsdale et al., 2001). Of these, Anopheles (Cellia) superpictus Grassi is one of the most important and widely distributed species, especially in malarious regions of Turkey (Parrish, 1959; Postiglione et al., 1973; Ozer et al., 2001, Simsek, 2006). The efficiency of this species as a vector of Plasmodium vivax (Kasap et al., 1987) and Plasmodium falciparum (Luty et al., 2006) has been demonstrated under laboratory conditions. It is also, considered to be an important malaria vector in the Middle East, Middle Asia and Mediterranean countries and a secondary vector in other regions (Barkai and Saliternik, 1968; Zahar, 1974; Romi et al., 1997). For these reasons, An. superpictus is always taken into consideration in malaria control programs conducted in Turkey. The microbial insecticide Bacillus thuriensis var. isralensis (B.t.i.) was integrated into mosquito control programs in the last decade of the 20th century. Recently, B.t.i. has been commonly used in mosquito larvae control programs in Turkey (Matur and Ceber, 1988; Simsek et al., 2005) and B.t.i applications have increased in Anopheles larvae control programs. Larvicidal agents, if administrated at high enough concentrations, will yield complete or almost complete mortality in exposed populations. A number of chemical larvicides and mosquito control agents have been shown to manifest delayed effects at sublethal doses in the survivors. Adugelo-Silva and Spielman (1984) have shown that in the laboratory inefficient larviciding reduces larval competition among the survivors and increases the density and average body size of the resulting adult population. Hare and Nasci (1986) noted delayed mortality in surviving larvae of Aedes aegypti exposed to a median lethal concentration of B.t.i. Mulla and Singh (1991) examined some biological parameters and morphogenetic aberrations of Culex quinquefasciatus larvae, pupae and adults after larvae were treated with sublethal concentrations (LC₂₅ and LC₈₀) of B.t.i. Flores et al. (2004) have indicated that inefficient larviciding with B.t.i. reduces the developmental time and fecundity of Ae. aegypti. They examined the effects of sublethal concentrations (LC₃₀, LC₅₀ and LC₇₀) of B.t.i. on survival, longevity, fecundity and sex ratio of adults for surviving larvae and their F₁ progeny. However, it is well known that the effects of insecticides and pesticides vary from generation to generation. Therefore, in order to have a more complete understanding of the effects of sublethal concentrations of B.t.i. on mosquito populations it is necessary to monitor populations for several generations. The present study aims to fill such a gap by assessing the effects of sublethal concentrations of B.t.i. (Vectobac^® 12 AS) on different biological parameters of An. superpictus for three generations.

MATERIALS AND METHODS

Mosquitoes: The An. superpictus specimens were from a colony established in the insectary of Hacettepe University Ecological Sciences Research Laboratory (ESRL). This colony originally was obtained from the village of Magarali, 10 km southeast of Birecik (37°, 01'N and 37°57'E) district in Sanliurfa province, in the malarious region of Turkey (Simsek et al., 2005).

B.t.i.: A commercial B.t.i. product, Vectobac^® 12 AS (1200 ITU mg^-1, Valent Biosciences), was used to determine the effects of sublethal concentrations of B.t.i. on biological parameters of An. superpictus. The sublethal concentrations examined in this study were LC₂₀ (0.15 mL m^-2) and LC₇₀ (0.76 mL m^-2), one under and one above the LC₅₀ concentration. The LC₂₀ value was chosen because this was the minimum concentration available that would not violate the 10% error rate. The LC₇₀ value corresponds to the same concentration above the LC₅₀ value. These concentrations were determined in ESRL in 2004 and repeated again in 2005.

Experimental procedure: The laboratory was maintained at 26±2°C, 65%, ±5 relative humidity with a 12:12 h light:dark cycle photoperiod. Cohorts of 3000 eggs were used in the establishment of the An. superpictus colony. After hatching, 1st stage larvae were transferred into rearing pans filled with 2 L of distilled water (25±2°C). The larvae were fed twice daily with 0.01-0.04 g of powdered larval food (TetraMin^® fish food), which was spread evenly onto the water surface (Bangs et al., 2002; Kuhn, 2002). Late 3rd instars larvae were exposed to sublethal concentrations (LC₂₀ and LC₇₀) of B.t.i. Treatments were conducted on a total of 500 larvae within plastic rearing cups containing 200 mL of deionized water and 25 larvae each (total 20 cups). The 1st cohort exposed to sublethal concentrations was named the F₁ generation. After 24 h of exposure the surviving larvae were transferred to fresh containers including 200 mL of distilled water. Every 24 h the larvae cups were checked and surviving pupae were collected and transferred to adult cages. After adult emergence 40 females and 40 males were picked at random and transferred to new cages. Adult mosquitoes were fed with 10% sugar water. A live rabbit was used for blood feeding of female mosquitoes (1 h every day). Surviving females and their eggs were recorded every 24 h until the death of the last female. Survival, longevity and fecundity of the females were calculated using the data obtained by methods outlined in Krebs (1985). This procedure was carried out for 3 generations. Offspring from the F₁ generation were used in establishment of the F₂ generation and similarly F₂ offspring were used in establishment of the F₃ generation. Differences in main daily fecundity and developmental cycle were compared using the Tukey test. Survival curves were compared by means of the log rank test for the survivors of the exposed generations and the control group.

RESULTS

Sex ratio and developmental cycle: Sex ratio results (Table 1) indicated that in the treatment group female ratios were slightly higher than male ratios. The sex ratios in the LC₂₀ treatment group fluctuated but the last generation sex ratios were same as the 1st generation ratios. Sex ratio in the F₁ generation of the LC₇₀ group was 1:1.13 but in all other generations for both treatment concentrations sex ratios were close to 1:1. Sex ratio of the control group was 1:1.40.

Developmental time of An. superpictus from F₁-F₃ generation exposed to LC₂₀ did not differ significantly (p>0.05) between the groups. LC₂₀ and control group development time were nearly identical. LC₇₀ group results were significantly higher (p<0.05) than LC₂₀ group and control group results.

Oviposition: According to our results, both oviposition and pre- and postoviposition periods in females showed differences between the control and treatment groups (Table 2). The preoviposition period in the treatment groups was in general longer than that in the control group but the difference was not statistically significant. Oviposition period in the LC₂₀ group varied between 38 and 56 days and fluctuated from F₁-F₃. Oviposition period for the two treatment groups showed a significant increase in the F₂ generation (LC₂₀: 56, LC₇₀: 54).

Table 1:	Mean developmental time in days and male:female ratio of Anopheles superpictus exposed to sublethal concentrations of B.t.i. for 3 generations

Table 2:	Periods of the pre-oviposition, oviposition, postoviposition and longevity of female Anopheles superpictus that emerged from surviving larvae after sublethal concentrations of B.t.i. for 3 treatment periods

Table 3:	Population parameters and rate of hatching, pupae and emergence of Anopheles superpictus surviving from larvae exposed to sublethal concentrations of B.t.i. for 3 generations

Postoviposition period varied between the 2 treatment groups and was very variable and no directional change could be determined. In general, longevity increased in all treatment groups for all generations.

Growth parameters, fecundity, hatching, pupation and emergence rate: The results showed a decrease in Gross Reproductive Rate (GRR) with increasing concentrations.

In all generations individuals showed greater GRR values at LC₂₀ when compared with LC₇₀. In both treatment groups, there was an initial drop in GRR in the F₁ generation (LC₂₀: 10114, LC₇₀: 9634), followed by an increase in the F₂ generation, reaching values above or similar (LC₂₀: 14376, LC₇₀: 9791) to those of the control group (10145). However, in the F₃ generation GRR values again dropped below those of the control group for both treatment lines (Table 3).

Mean generation time showed a decrease in both treatment groups, while this drop occurred in the F₃ generation in the LC₂₀ group (34.01). It occurred in the F₂ generation in the LC₇₀ group (36.31).

The Net Reproductive Rate (NRR) substantially fell from F₁-F₃ for both treatment lines. However, F₂ generation values were higher than or similar to those of the control group.

Although, GRR and NRR values changed with treatment and showed a decreasing trend, the finite and intrinsic growth rate did not show any differences between generations or treatment lines.

Hatching rates were substantially reduced in all generations for both treatment groups, when compared to the control group. Although, no trend in hatching rate was observed in the LC₂₀ group, in the LC₇₀ group there was 1st a decrease (F₂: 62%) and then an increase (78%) in hatching rate from F₁-F₃. Pupation rates were not correlated with hatching rates. Pupation rates decreased from F₁-F₃ in the LC₂₀ group and decreased from F₁-F₂ and increased from F₂-F₃ in the LC₇₀ group. The change in direction of emergence rates for both treatment lines was similar to that seen for pupation rates.

Fecundity: Mean daily fecundity did not show any significant (p<0.05) differences between generations (F₁-F₃) or treatment groups (LC₂₀, LC₇₀) or between generations and controls. Although, we did not determine any significant differences between the control and treatment groups, mean daily fecundities observed in both lines for all generations were lower than those in the control group. In addition, mean daily fecundity of the LC₇₀ line was slightly lower than that of the LC₂₀ line and control line (Table 4).

Fig. 1:	Survivorship curves of Anopheles superpictus that emerged from larvae surviving LC20 concentrations of Vectobac 12 AS for 3 treatment generations and the control group

Fig. 2:	Survivorship curves of Anopheles superpictus that emerged from larvae surviving LC70 concentrations of Vectobac 12 AS for 3 treatment generations and the control group

Table 4:	Mean daily fecundity of female Anopheles superpictus that emerged from larvae surviving treatment with (three generation) 2 different sublethal concentrations of Vectobac 12 AS and the control group

Survival: The survival curves of the treatment groups (all generations) and control group (Fig. 1 and 2) were compared by means of the log-rank method. The only significant differences (p<0.05) in the LC₂₀ group were between F₂ and F₁ and between the F₃ generation and the control group. In the LC₇₀ group there were no significant differences between the F₁, F₂ and F₃ generations.

DISCUSSION

According to the results, some life parameters were adversely affected more than others. Development cycle was influenced by increasing B.t.i. concentrations. The effects of a low sublethal concentration (LC₂₀) were not significant (p>0.05) but a high concentration (LC₇₀) had serious effects on developmental time, prolonging the maturation period. In other words, exposure to a low concentration of B.t.i. may shorten the duration of the development cycle (results were statistically not significant), whereas, a high concentration (LC₇₀) may prolong the duration of the development cycle. However, Flores et al. (2004) found somewhat different results showing that exposure to low concentrations of B.t.i. significantly shortened the duration of the developmental cycle and exposure to high concentrations caused no apparent significant differences.

Sex ratios were influenced by B.t.i. application but there was no clear difference between the generations and treatment groups except for in the LC₇₀ F₁ generation. The effects are biased towards a reduction in the proportion of females compared to the control group. Based on the results obtained here, it can be concluded that the sex ratio did not differ between generations or among individuals exposed to different sublethal concentrations of B.t.i. However, sex ratios in all exposure groups differed from those of the control group. Flores et al. (2004) obtained a reduction in female ratios after treatment with B.t.i. This indicates that treatment of populations with B.t.i. could lead to a reduction in reproductive population size. At the same time, studies on the effects of chemical insecticides on sex ratio indicate a distortion towards males (Priyalakshmi et al., 1999). All preliminaryobservations and results shown here indicate that the effects of chemical and biological insecticides on sex ratios are nearly the same.

Results also revealed that there was an extension in total ovipositional period compared to the control group. This effect increased with increasing B.t.i. concentration, but was only observed for generations in the LC₇₀ line. Postovipositional results were not correlated with the results obtained during the ovipositional period. This period varied widely. As a result, Vectobac causes an increase in female longevity but this is not related to B.t.i. concentration. Flores et al. (2004) showed an extension of the preovipositional period but the control group preoviposition period was shorter than that of the treatment groups in that study. At the same time, they showed an extension of longevity.

Net and gross reproductive rates decreased with B.t.i. exposure. The reduction was greater with increasing B.t.i. concentrations but this trend was not observed in the 2nd generation. We cannot fully explain this result but it may be a response by the population to continuous insecticide pressure resulting from B.t.i. exposure. The reduction in the GRR and R₀ with increasing B.t.i. concentration indicates lower reproductive potential in females. In this study, this was reflected by a decline in total fecundity. While, results showed a decline in total fecundity, Foo and Yap (1982) could not show any significant differences between control group and B.t.i. H-14 treated groups for fecundity, but Zahiri and Mulla (2006) reported a reduction in oviposition values by B.t.i. and B.s. in tests with a range of concentrations from 0.1-2.0 mg L^-1. Flores et al. (2004) showed a decline in GRR with increasing B.t.i. concentrations and reported lower reproductive potential in females with increasing B.t.i. concentrations. They also, indicated that this was reflected by a decline in total fecundity resulting from an exposure to concentrations higher than LC₅₀. According to Prilayakshmi et al. (1999), chemical insecticides (fenitrothion, deltamethrin and cypermethrin) have the same effects.

The generation time determined with sublethal concentrations is shorter than that of the control group except for the LC₂₀ F₁ generation but is not significantly higher than the control group. Results for generation time indicate a reduction in values from generation to generation. Results obtained from the 1st generation were nearly identical to those from the control group, but generation time decreased substantially in the F₂ and F₃ generations for both treatment lines. This resulted in a daily reduction in population size although finite growth rates did not show any significant differences between generations. Flores et al. (2004) found an increase in generation time in Ae. aegypti lines that were treated with a sublethal concentration.

Although, GRR and R₀ values varied between generations, the intrinsic growth rate showed no significant differences between generations, concentrations, or the control group.

LC₇₀ group survival curves showed no significant difference from those of the control group and no significant difference between generations. However, the LC₂₀ group showed significant differences from the control group and also between the F₂ and F₁ and F₂ and F₃ generations. Zahiri and Mulla (2006) found that survival rates of larvae decreased with increasing B.t.i. exposure. Their study demonstrated that egg raft deposits are adversely affected by an increase in B.t.i. concentrations and also demonstrated that females die before they can deposit their whole compliment of eggs. Flores et al. (2004) found significant differences among the exposed individuals in all treatment groups, but they did not show any differences between the control group and groups treated with LC₅₀ or LC₇₀ concentrations.

Mean daily fecundity was adversely affected by sublethal concentrations. These effects increased with increasing B.t.i. concentrations but were not statistically significant.

Concentrations of B.t.i. used in this study were not higher than the normal application rates used in mosquito control studies. We noted some adverse effects of B.t.i. concentrations on some biological parameters. These results suggest additional advantages of B.t.i. for use in control programs. These findings along with the findings of Flores et al. (2004) and Zahiri and Mulla (2006) have revealed the extra potential of this agent as a larvicide.

ACKNOWLEDGEMENTS

We are grateful to Sinan Kaynas for taking part in the experiment and Salim Calis for helping with the An. superpictus rearing procedure.