Studying the Effect of the Symbiotic Bacterium Wolbachia on Some Biological Indicators of the Trichogramma brassicae

INTRODUCTION

What is being discussed in agricultural management today is reducing the use of chemical pesticides, preserving the environment, supporting natural enemies, and ultimately producing healthy food. Therefore, biological control specialists are always looking for solutions to improve and increase the efficiency of natural enemies in different regions and ecosystems. Among the factors that ensure the success of a natural enemy before release is the recognition of measurable characteristics in a suitable population or ecotype [1-3]. Among these criteria, the sex ratio created by the female wasp is an important qualitative criterion, and since female parasitoid wasps are the main factor in killing the pest, hypotheses have been made regarding the advantage of using species or populations of single-sex parasitoid wasps in the release against a pest [4, 5].

Trichogramma wasp, with more than 180 species, is the largest and best-known genus of the Trichogrammatidae family. Due to its ease of mass reproduction and the possibility of using its various species as parasitoids of lepidopteran eggs that damage crops, it has the highest level of release in the world among natural enemies of pests in terms of application [6, 7]. After the introduction of female-producing species of Trichogramma wasp in the 1990s, many studies were conducted on the factors that cause and the advantages of using unisexual Trichogramma wasps [5]. The factor of unisexual reproduction of bees (thelytokous parthenogenesis) can have a genetic or microbial origin that causes the development of females from unfertilized eggs. In the Trichogramma cacociae (Marcal), the main cause of genetic female reproduction has been identified [8]. Wolbachia bacteria are known to induce parthenogenesis in more than 18 species of Trichogramma bees and there is also the possibility of horizontal transmission within and between species [9].

The results of numerous studies conducted to determine the different effects of the symbiotic bacteria Wolbachia on host fitness indicate very different effects of this bacterium on biological, olfactory, and morphological indices of Trichogramma bees, including fertility, lifespan, parasitism rate, exit percentage, dispersal power, host recognition, leg length, etc. Some of the results related to the topic will be discussed [10-16].

In this study, while adding a bisexual population resulting from the removal of bacteria from the body of infected bees using antibiotics and determining the functional response of three unisexual and bisexual populations of Trichogramma brassicae, the result of storing parasitized eggs of the mealybug was examined and compared by the aforementioned populations.

MATERIALS AND METHODS

Collection and identification of ecotypes of Trichogramma bee reproductive material

To obtain the reproductive population of Trichogramma bees, parasitic eggs were collected from weeds, including Xanthium stramarium L. (Asterales: Asteraceae), and corn and rice fields. After transferring the parasitic eggs to a constant temperature room and leaving the bees, each of the strains separately from the collection location and host type at a temperature of 60 ± 10 °C, a relative humidity of 60 ± 10%, and a period of 16 hours of light and 8 hours of darkness on the eggs. Sterilized Ephestia kuehniella (Lep.: Pyralidae) were reared, and each of them was placed individually in test tubes (12 × 75 mm) before the bee hatched from the emergence of female bees. Immediately after the emergence of female bees, paper strips containing fresh Ephestia eggs were provided to each of them, and the type of virginity in virgin bees was determined by the isofemale method.

By observing female reproductive individuals in each of the populations, a pure line was created and its breeding continued in appropriate and controlled conditions. To prove the possibility of female bees being produced by Wolbachia bacteria, first, female bees were fed with a solution of water, honey, and antibiotics [12]. And again, their reproduction method was checked after 3-4 generations. In case of returning to the bisexual state, there is a high possibility of infection with bacteria-inducing virginity, and for the final confirmation, the molecular method and the specific primer of the wsp gene were used [17]. To identify bees, in addition to using the key related to morphological traits, the molecular identification method based on the ITS2 region was also used [18].

Creation of bisexual and reproductive populations infected with Wolbachia bacteria

In this research, to evaluate the effect of Wolbachia bacteria and compare some characteristics of bisexual and female populations, three purified Trichogramma brassicae lines with the abbreviations A (Asexual), B (Bisexual), and CB (Cured Bisexual) were created by single material method. Line A was infected with PI-Wolbachia. In population B, there was bisexual reproduction and the bisexual line CB was created using antibiotic treatment for four generations of female bees infected with the bacteria (A) with water and honey impregnated with the antibiotic tetracycline hydrochloride (SIGMA) at a ratio of 50 mg/ml [1] and were grown in laboratory conditions. It was continued at two temperatures of 20 and 25 °C. To eliminate and prevent the possible effects of antibiotics, all experiments were carried out in generations four and later.

Functional response test

To evaluate the functional response of Trichogramma brassicae bee populations, concentrations of 2, 5, 10, 20, 50, 65, and 85 fresh eggs of Euphtheria that were sterilized by UV rays were randomly distributed and scattered on a white cardboard strip (10 × 40 mm) were glued. For each density, female bees with a lifespan of less than 24 hours (in the case of the bisexual population, mated bees were used) in 18 replicates separately and separately for each population in test tubes (16 × 100 mm) were placed containing a certain density of host eggs. The samples were simultaneously placed in a constant temperature room (25 ± 1 °C, 60 ± 10% relative humidity, 16 hours of light, and 8 hours of darkness). After 24 hours, the whole bee insects were removed from the tubes and the samples were kept in the same environmental conditions until the parasitized eggs turned black, and finally, the number of parasitized eggs per density was recorded for each population.

The analysis of functional responses consisted of two separate steps [19, 20]. In the first step, the shape of the curve or the type of reaction function of logistic regression of the ratio of parasite eggs (N_a) to the initial density of host eggs (N₀) was determined based on the polynomial equation. Then, P₀-P₃ parameters were estimated with the CATMOD procedure in SAS software [21]. In the second step, using non-linear least square regression (NLIN procedure with DUD technique in SAS software), search power parameters or attack coefficient (a) and acquisition time (T_h) were estimated. To compare the parameters estimated in different populations, a pairwise comparison was done [21].

The maximum attack rate was also calculated based on the ratio of the total test time to the time of reaching a host [22]. In many researches related to the functional response, the parameters of the attack coefficient and the acquisition time have a more comparative aspect, which in this research was also used to evaluate the effect of Wolbachia bacteria on the parasitism behavior of bisexual bees (B and CB) and unisexual bees (A) of Trichogramma brassicae bees populations were compared two by two.

Determining the exit percentage of bees stored in the cold

To evaluate the effect of the storage period on the output of each of the A, B, and CB lines grown at two temperatures of 20 and 25 °C, paper strips containing fresh Ephastia eggs were provided to each of them separately for 24 hours. Then parasitized eggs were kept in 20 glass test tubes with dimensions of 16 × 100 mm under the conditions of a bee breeding environment. After reaching the stage of pre-pupae or white-eyed pupa (approximately 10 days at 20 °C and 5 days at 25 °C), the tubes containing eggs are kept in a cold room (8 °C and total darkness) for 1, 2, 4, 5, 6, and 7 weeks were placed. After the storage period, the tubes were taken out of the cold room and transferred to a temperature of 25 °C. After the departure of the bees, while checking the sex ratio, the departure percentage of each of the A, B, and CB populations was determined and recorded.

Statistical test

For the statistical comparison of the results, after transforming the data into the square root inverse sine method, the factorial test was used based on a completely random design in the SAS software (version 6.12). Finally, the averages were compared with each other by Tukey's test.

RESULTS AND DISCUSSION

Identification of parasitoid wasps and symbiotic bacteria

In addition to the identification of the parasitoid wasp with the scientific name. Trichogramma brassicae Bezd, its bacterial contamination was also confirmed. The sequences related to the wsp gene of the Wolbachia bacterium and the ITS2 region of the Trichogramma brassicae bee infected with this bacterium have been recorded.

Functional response test

The results of logistic regression and the estimated values ​​for the three parts of the curve of the proportion of eggs parasitized by each of the Trichogramma brassicae populations are reflected in Table 1. Given that the estimated numbers for the linear part of the curve are negative, the functional response of all three populations was determined to be of type II. The functional response curve of the bisexual (B) and bacterially infected (A) and bisexual antibiotic-treated (CB) populations to the increase in host egg density is presented in Figure 1 and the degree of data fit to the model (r2 or coefficient of determination) is presented in Table 2, which shows that the model is well fitted to the data. The search power (a) and access time (T_h) parameters for the bees of the three populations A, B, and CB were obtained as 0.2393 h^-1, 0.7862 h^-1, 0.3016 h^-1, 0.7684 h^-1, 0.2525 h^-1, and 0.7701 h^-1, respectively. As is clear from this table, zero is located in the 95% confidence interval of the D_a and D_Th parameters, which indicates that the treatments do not differ significantly from each other in terms of search power and access time.

Table 1. Estimated values ​​by logistic regression in different parts of the functional response curve of bacterially infected (A), naturally bisexual (B), and antibiotic-treated (CB) female populations of the bee Trichogramma brassicae to increasing Ephestia kuehniella egg density.

Table 2. Functional response parameters of bacterially infected (A), naturally bisexual (B), and antibiotic-treated (CB) populations of Trichogramma brassicae to different densities of Ephestia kehniella eggs, estimated by the Disk equation.

Percentage of exit of bees stored in cold

Analysis of variance of the total data, which was a combination of three factors of bee population (three populations), rearing temperature (20 and 25 °C), and storage time (1-7 weeks), showed that the treatments had a significant difference at the one percent probability level (P = 0.0001, F_41,168 = 41.34). Also, the difference between the treatments related to each of the factors was significant and there was an interaction between them (P = 0.0001). According to the results obtained in this study, the percentage of the exodus of bees infected with bacteria (A), naturally bisexual (B), and antibiotic-treated bisexual (CB) was not significantly different from each other, regardless of the effect of rearing temperature, and all three were placed in the same group (Table 3). Theoretically, the highest parasitism or maximum attack rate by female-producing unisexual, naturally bisexual, and antibiotic-treated bisexual populations was determined to be 30.52, 31.23, and 31.16 host eggs per day, respectively, and the numerical value of this index in the naturally bisexual population B was higher than that of the two populations CB and A.

Table 3. Percentage of hatching of parasitized Ephestia kuehniella eggs by bacterially infected (A), naturally bisexual (B), and antibiotic-treated (CB) female populations of Trichogramma brassicae after storage under cold storage conditions (Mean ± SE).