Assessment of Lethal, Sublethal, and Transgenerational Effects of Beauveria bassiana on the Demography of Aedes albopictus (Culicidae: Diptera)

Dengue fever is one of the most rapidly spreading arthropod-borne diseases. Diurnal vectorial properties of Aedes albopictus contribute to the dispersion of the dengue viruses. Frequent and injudicious use of synthetic insecticides led to the evolution of resistant phenotypes in Ae. albopictus which necessitates the search for an alternative of current control strategies. Developing a long-lasting and environmentally safe tactic based on knowledge of ecology and population dynamics of Ae. albopictus is critical. Therefore, with a view towards biological control and ecology, the effect of entomopathogenic fungi (EPF) Beauveria bassiana on filial and first filial generations of Ae. albopictus were studied. Investigations showed 87.5% adulticidal activity leading to altered fecundity and adult longevity in a filial generation. The lethal (LC50) and sublethal (LC20) concentrations of B. bassiana were applied to filial generation (F0) to study demographic parameters in the first filial generation (F1). Results showed reduced net reproductive rates (Ro) intrinsic rate of increase (r), and mean generation time (T) compared to uninfected controls. Prolonged larval and pupal duration were observed followed by reduced longevity of male and female adults. Fecundity in the first filial generation was significantly changed with the lethal and sublethal concentrations of B. bassiana. Thus, it is concluded that B. bassiana has the potential to play a vital role in integrated mosquito management strategies.


Introduction
Mosquito-borne diseases have been a primary concern for the human population for a long time. Due to Aedes albopictus, more than 100 nations in tropical and subtropical regions around the world are confronting the dangers of dengue fever, yellow fever, and chikungunya [1]. Diurnal and repeated blood-feeding behavior of Ae. albopictus makes it more dangerous than other mosquito species, it also facilitate the rapid transmission of diseases [2]. Moreover, past injudicious use of synthetic insecticides against larvae and adults of mosquito led to the evolution of resistant phenotypes [3], environmental contamination, and human health risks. Specifically, indoor use of synthetic insecticides against medically-important pests like mosquito negatively impact human health [4,5]. Non-target and broad spectrum insecticides negatively impact biodiversity [6][7][8] and life cycles of non-target insects [9][10][11]. All these issues contribute for the need of research for alternative control methods which would be Insects 2020, 11,178 2 of 17 long-lasting and safe for the environment and humans. As biological control agents, entomopathogenic fungi (EPF) are cosmopolitan and can be used safely against insect pests [12][13][14][15][16][17][18][19][20][21]. In recent studies, EPF have also shown promising adverse effects on different mosquito species [22][23][24].
EPF can be used more efficiently if the impacts of the life cycle of Ae. albopictus are better known. Population dynamics and population ecology of insects play a vital role in the development of long-lasting control with EPF [22,25,26]. Life table analysis of Ae. albopictus can help to determine the best time and stage upon which EPF should be applied for management [27,28].
Life table studies for Ae. albopictus have been conducted with a focus on immature stages but the adult populations (male especially) are overlooked [29]. Life table analysis without the inclusion of the male population cannot predict valid demographic parameters; hence age stage two-sex life tables are used [30][31][32]. The age stage two-sex life table, developed by Chi [33] subsequently and mathematically proven [34], can differentiate the stage and male populations.
The current study was planned with Ae. albopictus, and assessments of Beauveria bassiana isolated from two different sources were done. The best isolate was used to examine transgenerational effects on demographic parameters of filial offspring and first generations offspring by utilizing the age stage two-sex life table, which will guide us towards the effective use of B. bassiana at the most impactful time and stage of Ae. albopictus. This will contribute to development of a useful, and eco-friendly tool for integrated pest management of this urban pest.

Rearing of Ae. albopictus
Ae. albopictus (Foshan strain) eggs were collected from Guangdong Center for Disease Control and Prevention Guangzhou, China (2017). Eggs were brought on disinfected media (jars cleaned with ethanol 90%, air dried 30 mints) to the Laboratory of Bio-Pesticide Innovation and Application of Guangdong Province, College of Agriculture, South China Agricultural University, Guangzhou, China. Filter papers containing the eggs were placed in glass beakers (200 mL) containing water for hatching under controlled (Temperature: 28 ± 2 • C, Relative Humidity:64 ± 5% and photoperiod 11:13 h light:dark) laboratory conditions (pathogen-free environment) [35]. Two-day old post-hatching larvae were transferred in 1000 mL glass jars (15 × 10 cm). Well-ground fish food (Godzilla, CST945) was provided to larvae as food [36]. Pupae were collected and placed in a separate glass beaker (200 mL) which was placed in white cloth cage (30 × 30 cm) for adult emergence [22]. Male and female adults were provided a 10% sugar solution (w/v) while egg-laying female adults were blood-fed on mice with ethical approval (SCAU-AEC-2010-04-16) by the Guangdong Province Administration Office of Laboratory Animals. Conical shaped wet filter paper fitted in a 200 mL glass beaker (filled with distilled water) was used as an oviposition site.
Isolates were passaged multiple times to prevent aging [38]. A two-week-old potato dextrose agar grown monoconidial culture maintained at 25 • C was used in suspension formation. For stock solutions, disinfected spatulas were used to harvest the conidia in 0.05% Tween-80 (Sigma-P1754) diluted in distilled water [39]. Stock solutions were stored at 4 • C for further use [40,41]. Serial dilutions were made to provide the desired concentrations from the stock solution.

Screening of Beauveria bassiana Isolates
Two isolates of B. bassiana (Bb-01 and Bb-10) were screened out against the adult (2 days old males and females) of Ae. albopictus. Five concentration (3 × 10 8 , 3 × 10 7 , 3 × 10 6 ,3 × 10 5 , 3 × 10 4 spores/mL) were prepared for both isolates, whereas 0.05% Tween-80 (Sigma-P1754) diluted in distilled water was taken as tween control and distilled water was a general control [39]. Plastic jars (10 × 10 × 10 cm) were sprayed with fungal concentrations (10 mL) and air-dried for two hours. Ten adults were added to the jar of each concentration of every isolate. The whole experiment was repeated four times. Sugar solution (10%) was provided to male and female while the additional blood meal was provided to females for egg-laying. All experiments were conducted under controlled laboratory conditions as described above. Data regarding adult mortality for 7 days (24 h interval) [12,13,22]. Adults showing no movement were considered dead.

Selection of Beauveria bassiana Isolate
The LC 50 was calculated for both isolates of B. bassiana (POLO-PC software). The fungal isolate with the lowest LC 50 was chosen for further experimentation with the age stage two-sex life table.

Assay of Blood-Fed Females
A selected isolate of B. bassiana was again tested on blood-fed and non-blood-fed females, five concentrations (3 × 10 8 , 3 × 10 7 , 3 × 10 6 , 3 × 10 5 , 3 × 10 4 spores/mL) were prepared and applied with the same methodology as described above. Females used in experimentation were 2 days old (24 h starved) and concentrations were applied 8 h post blood-feeding. A total of 1000 newly emerged adults (1:1) were selected for bioassays. Males and females (250 each) were separated with an electric aspirator and subjected to Lethal (LC 50 ) and sublethal (LC 20 ) concentrations separately, in treated jar assays (500 adults, 1:1) water diluted 0.05% Tween-80 (Sigma-P1754) was used as control. Mortality was observed every 24 h for 7 days, after the seven days of treatment the remaining adults of filial generation (F 0 ) were copulated (1pair/cage) in plastic cages covered with a white cloth. Blood meals were provided every four days until the death of females (Scholte et al. 2006). Glass beakers with wet filter paper were used as ovipositional sites. Fecundity and longevity were observed until the death of every individual of the filial generation [42].

Transgenerational Effect of Beauveria bassiana on First Filial Generation (F1)
Eggs laid by filial generations, 100 eggs were collected from each group (LC 50 , LC 20 , and control). Eggs were placed individually on disinfected plastic trays (150 mL distilled water) without fungal exposure in pathogen-free environment. Powered fish food (Godzilla, CST945) was individually given to larvae as food. The transgenerational effects were studied from larva to adult stage. Data were recorded every 12 h until the death of all individuals. On the emergence of adults, individuals of the first filial generation were paired and shifted in cages for seeking the data regarding longevity and fecundity.

Statistical Analysis
The calculation of lethal and sublethal concentrations was conducted by using POLO-PC software [43]. Mortality data were analyzed by one-way ANOVA. Means were separated by using Tukey's HSD test in Minitab 16 software at a 5% level of significance. Life table parameters like development, fecundity and, longevity were obtained by using the age-stage two-sex life table [33,[44][45][46]. The bootstrap technique (n = 100,000) was used for the calculation of standard errors for life table parameters [47]. The program (TWO SEX-MS Chart) for age-stage two-sex life table analysis was designed in visual basics for the window operating system and can be obtained from the following link [33,46] (http://140.120.197.173/Ecology/prod02.html) (Chung Hsing University).
In age stage two-sex life table net reproductive rate (R 0 ), which shows a total number of offspring by an individual throughout its life was calculated via the equation while l x , the probability of a newly laid egg surviving to age x can be calculated as m x is mean fecundity of individuals at age x can be obtained from the following equation The intrinsic rate of increase (r) was evaluated utilizing the iterative bisection strategy and adjusted with the Euler-Lotka condition with the age-indexed (Goodman 1982).
The finite rate was calculated as λ = e r Length of time that a population needs to increase to R 0 -fold of its population size at the stable age-stage distribution is called as mean generational time, and is calculated as e xj , the length of time that an individual of age x and stage j is expected to live could be obtained from the equation below [44] The reproductive value (v xj ) was calculated according to [48,49] and was calculated as Insects 2020, 11, 178 5 of 17

Selection of Beauveria Bassiana Isolate
Lethal (LC50) and sublethal (LC20) doses for both isolates (Bb-01, Bb-10) of B. bassiana were calculated from pre-experimentation data ( Table 1). The isolate with the lowest LC50 was selected for further studies; hence B. bassiana isolate Bb-01 met the desired criteria.

Assay of Blood-Fed Females
Blood-fed and non-blood fed females of Ae. albopictus were exposed to five concentrations of B. bassiana (Bb-01) by contact assay in the jar. Non-significant results were seen ( Figure 2) between blood-fed and non-blood fed females. Blood meal did not affect the adulticidal activity of B. bassiana (Bb-01).

Selection of Beauveria bassiana Isolate
Lethal (LC 50 ) and sublethal (LC 20 ) doses for both isolates (Bb-01, Bb-10) of B. bassiana were calculated from pre-experimentation data ( Table 1). The isolate with the lowest LC 50 was selected for further studies; hence B. bassiana isolate Bb-01 met the desired criteria.

Assay of Blood-Fed Females
Blood-fed and non-blood fed females of Ae. albopictus were exposed to five concentrations of B. bassiana (Bb-01) by contact assay in the jar. Non-significant results were seen ( Figure 2) between blood-fed and non-blood fed females. Blood meal did not affect the adulticidal activity of B. bassiana (Bb-01).

Influence of Beauveria Bassiana on Longevity and Fecundity of Filial Generation (F0)
After the treatment of Lethal (LC50) and sublethal (LC20) concentrations of B. bassiana (Bb-01), the effect of longevity and fecundity of Ae. albopictus (filial generation, F0) were seen ( Table 2). For the filial generation exposed to LC50 and LC20 of Bb-01, male longevity was reduced to 21.33 and 26.09 days respectively, while maximum male longevity was seen in controls (29.91 ± 1.20). Female longevity also showed a similar tread to male longevity (days), where LC50 of Bb-01 presented the lowest longevity (22.22 ± 1.21) followed by LC20 of Bb-01 (27.65 ± 1.77) as compared to the control (30.07 ± 0.41). A significant reduction in fecundity (eggs/female) was also observed. Exposure to LC50 of Bb-01 results in the fewest number of eggs (189.31 ± 8.11) followed by the LC20 exposure (230.47 ± 9.32) and controls (357.33 ± 9.30).    (Table 2). For the filial generation exposed to LC 50 and LC 20 of Bb-01, male longevity was reduced to 21.33 and 26.09 days respectively, while maximum male longevity was seen in controls (29.91 ± 1.20). Female longevity also showed a similar tread to male longevity (days), where LC 50 of Bb-01 presented the lowest longevity (22.22 ± 1.21) followed by LC 20 of Bb-01 (27.65 ± 1.77) as compared to the control (30.07 ± 0.41). A significant reduction in fecundity (eggs/female) was also observed. Exposure to LC 50 of Bb-01 results in the fewest number of eggs (189.31 ± 8.11) followed by the LC 20 exposure (230.47 ± 9.32) and controls (357.33 ± 9.30).

Effect of Beauveria bassiana on First Filial Generation (F1)
For evaluation of transgenerational changes in the first filial generation of Ae. albopictus, an age stage two-sex life table was used. Basic parameters of life table such as developmental time, longevity, and fecundity are presented in Table 3. Egg hatching of 100% was observed with both control and sub-lethal (LC 20 ) concentration of B. bassiana, while 95% of egg hatching was observed after treatment of lethal (LC 50 ) concentration of Bb-01. An opposite trend was observed in egg duration, where control (2.00 ± 0.01 days) and sublethal concentration (LC 20 ) of B. bassiana (2.00 ± 0.01) showed prolongs the egg hatching duration, while early egg hatching (days) was seen in lethal (LC 50  Population parameters calculated with the help of the age stage two-sex life table are shown in Table 4. The intrinsic rate of increase (r) was inversely related to concentration, which varied from 0.27 to 0.25 and 0.23 in the control, LC 20 , and LC 50 respectively. Mean finite rate of increase (λ) had a significant difference (per day) between control (1.32 ± 0.01), LC 20 (1.29 ± 0.02) and LC 50 (1.26 ± 0.06). The net reproduction rate (R 0 ) (offspring/individual) was high in control (133.0 ± 16.21) and then gradually decrease significantly in LC 20 (89.16 ± 9.31) and LC 50 (50.62 ± 8.31). Significant differences were also observed between mean generation times (T), 17.83 days for control and 17.80 and 17.09 days in LC 20 and LC 50 respectively (p < 0.05).  The age stage survival rate (Sxj) signifies that in the first filial generation overall life span of Ae. albopictus in the control group was longer and was reduced after treatment of LC 20  Age stage reproductive value (Vxj) shows the maximum reproductive value of a stage in life span, adult females from the control group showed the highest peak of reproductive value as compared to lethal and sublethal concentrations of B. bassiana ( Figure 5). Daily reproduction (Figure 6) also showed a significant difference in the mean number of eggs (p < 0.05). Egg-laying started 2 days post blood meal; in the beginning, controlled females showed a maximum number of eggs followed by females of LC 20 and LC 50 . With time reduction in a mean number of eggs was seen in treated females.  The age stage survival rate (Sxj) signifies that in the first filial generation overall life span of Ae. albopictus in the control group was longer and was reduced after treatment of LC20 and LC50 of B. bassiana ( Figure 3A-C, respectively). A similar trend was also observed in age-stage life expectancy (Exj) where treated group individuals had a lower life expectancy and overall life span ( Figure 4A-C).
Age stage reproductive value (Vxj) shows the maximum reproductive value of a stage in life span, adult females from the control group showed the highest peak of reproductive value as compared to lethal and sublethal concentrations of B. bassiana ( Figure 5). Daily reproduction ( Figure  6) also showed a significant difference in the mean number of eggs (p < 0.05). Egg-laying started 2 days post blood meal; in the beginning, controlled females showed a maximum number of eggs followed by females of LC20 and LC50. With time reduction in a mean number of eggs was seen in treated females     shown as Figure 3A, while 3B and 3C showed the age-stage life expectancy (Exj) after exposure to sub-lethal (LC20) and lethal (LC50) concentrations of a selected isolate of B. bassiana (Bb-01) respectively. Life stages are shown in distinctive colors.   :5A (control), 5B (LC20), and 5C (LC50). Ae. albopictus age stage reproductive value (Vxj) of the control group is shown as Figure 3A, while 3B and 3C showed the age stage reproductive value (Vxj) after exposure to sub-lethal (LC20) and lethal (LC50) concentrations of a selected isolate of B. bassiana (Bb-01) respectively. Life stages are shown in distinctive colors.

Discussion
The life table of tracheal arthropods (insects) is directly related to the control of insects. Increased knowledge of insect stages, survival, and reproduction, can lead us towards promising and longlasting control of insect pests [36,[50][51][52][53][54][55]. Two virulent isolates of B. bassiana (extracted from different soils) were screened against Ae. albopictus with both isolates showing noteworthy adulticidal activity. The isolate extracted from soil of (Bb-01) in Pakistan showed more virulence than the isolate from the

Discussion
The life table of tracheal arthropods (insects) is directly related to the control of insects. Increased knowledge of insect stages, survival, and reproduction, can lead us towards promising and long-lasting control of insect pests [36,[50][51][52][53][54][55]. Two virulent isolates of B. bassiana (extracted from different soils) were screened against Ae. albopictus with both isolates showing noteworthy adulticidal activity. The isolate extracted from soil of (Bb-01) in Pakistan showed more virulence than the isolate from the soil of cold area (Bb-10) soil. Bb-01 provided 87.5% adult mortality which also goes in favor of previous studies where Ae. albopictus and Culex spp. showed 80-90% adult mortality after treatment of EPF [23,40,56,57]. Due to the least LC 50 and high adulticidal activity, Bb-01 was selected for further studies to assess its transgenerational effects on the filial and first filial generation of Ae. albopictus.
Insect chitin made cuticle is the main line of defense against EPF. In the case of medical insects like mosquito, complete control (Mortality) via EPF is not so easy. While EPF causes mortality in two weeks, an essential aspect is the effect of EPF on life parameters of mosquito. B. bassiana produces secondary products like beauvericin, bassianin, bassianolide, beauverolides, beauveriolides, tenellin, oosporein, and oxalic acid which evade the humoral and immune system of the insect [58,59]. Hyphae of B. bassiana absorbs the sugar contents from the hemolymph of insect which contributes towards insect weakness and disrupts various biological parameters of insect pests [60,61]. The current study strengthens this argument because egg hatching, larval duration, pupal duration, adult longevity, and fecundity in a filial and first filial generation were affected significantly. Lower egg hatching was observed due to the treatment of lethal concentration (LC 50 ) from Bb-01 which goes in favor of previous studies in Aedes spp and S. litura (Fabricius 1775) where EPF causes a reduction in egg hatching [62][63][64].
EPF penetrates the cuticle and affects the fat body of the insect depleting energy resource which directly affects the insect stadium [65][66][67], which resulted in prolonged larval and pupal duration. A similar trend was observed in the current study, where the lethal and sublethal concentration of Bb-01 showed long larval and pupal duration. Prolonged larval and pupal duration has also been reported in past studies of Musca domestica (Linnaeus 1758) and Culex spp. [22,23,68].
In epidemiological models for diseases of Ae. albopictus, adult control is the most important thing [69][70][71]. Short longevity of Ae. albopictus adults will help in the control of vector-borne diseases [72]. Our findings showed reduced male and female longevity in filial and first filial generation Insects 2020, 11, 178 13 of 17 due to the treatment of lethal and sublethal concentrations of Bb-01, previously Ae. albopictus and Ae. aegypti had also shown a significantly short life span after infection of EPF [40,41,69]. Anopheles spp. also showed low adult survival after the treatment of EPF [69,[73][74][75][76]. A similar trend was observed in Rhynchophorus ferrugineus (Olivier) and Trogoderma granarium, where 20-30% reduced longevity was observed after treatment of EPF [77,78].
The infection of EPF fluctuates the body temperature of females, resulting in the loss of appetite, which led to reduced fecundity [79]. In current studies, the concentration of Bb-01 was inversely proportional to the mean number of eggs from females of Ae. albopictus. In past studies reduction in blood-feeding [80], retardation in fecundity has been significantly reported due to infection of EPF [41]. Musca domestica and S. litura showed less mean number of eggs after the treatment of EPF [18,68]. B. bassiana showed a significant effect on all demographic parameters of Ae. albopictus in a filial and first filial generation.

Conclusions
This study is first of his kind and provides basic yet important time-specific and age-specific information for a better understanding of Ae. albopictus population dynamics under the influence of B. bassiana. From our investigation, the conclusion can be drawn that B. bassiana has lasting effects on the developmental parameters of Ae. albopictus. Mainly the impact of the Bb-01 on adult longevity and female fecundity was significantly affected. Considering the impact of B. bassiana on transgenerational parameters of filial and first filial generation, it can be integrated with effective dengue vector control strategies.