Horizontal transmission of Beauveria bassiana spores using infected males and inoculation device: impact on survival and fecundity of Ceratitis capitata (Diptera: Tephritidae)

The mode of transmission of fungus spores (horizontal transmission or assisted auto-dissemination) directly influences the effectiveness of a fungal pathogen when used as a control agent. Fungal infections cause physiological alterations leading to the host's death. During this process, the fungus uses the energy reserves in the hemolymph of insects, affecting the development and performance of individuals and, therefore, the demographic features of their populations. In this work, we evaluated topical inoculation and a passive dissemination device in the transmission of Beauveria bassiana (Balsamo) Vuillemin conidia to Ceratitis capitata Wiedemann (Diptera: Tephritidae). Survival and fecundity were negatively affected by the action of the fungus, and mortality was influenced by the inoculation method. Inoculated sterile males were as competitive as untreated males and reduced the fecundity and survival of females. We conclude that the pathogenic action of B. bassiana reduces the survival and fecundity parameters of C. capitata infected by horizontal transmission, while the behavioral response of treated sterile males is similar to that of sterile-fertile untreated males. We discuss the potential use of this strategy as part of the pest management of C. capitata.


Introduction
In natural ecosystems, entomopathogenic fungi (EPF) play an important role in the regulation of insect populations (Goettel et al., 2010) and therefore have great potential as biocontrol agents (Dimbi et al., 2003). Infection by EPF begins with the adherence of the conidium and penetration through the cuticle, followed by the consumption of nutrients present in the insect's hemolymph by the fungus. During mycelial growth, toxins are produced as secondary metabolites, which may cause physiological and behavioral 1 3 Vol:. (1234567890) alterations (Anderson et al., 2011;Jin et al., 2015;Khachatourians & Qazi, 2008;Molnár et al., 2010;Xia et al., 2002). The fecundity of infected hosts can be strongly affected during the invasion process, and thus may represent an additional component in the suppression of pest populations (Mkiga et al., 2020;Shoukat et al., 2020). Baverstock et al. (2010) mention that fungal spore transmission may occur by horizontal transmission among individuals of the same species or by assisted auto-dissemination (using artificial autoinoculation devices). Both modes of transmission have been able to cause fungal infections in some target fruit fly pests (e.g., Dimbi et al., 2013;Patt et al., 2015;Quesada-Moraga et al., 2008;Sookar et al., 2014;Toledo et al., 2017;Yousef et al., 2018). In both cases, the horizontal transmission rate has been the key factor determining the spread of the pathogen within host populations (Baverstock et al., 2010;Steinkraus, 2006).
Beauveria bassiana (Balsamo) Vuillemin is a facultative fungus with a wide host range found in both temperate and tropical zones (Shah & Pell, 2003;Zimmermann, 2007). The pathogenicity of B. bassiana has been evaluated in the fruit fly genus Anastrepha spp. (De la Rosa et al., 2002;Toledo et al., 2007) and in Ceratitis capitata Wiedemann (Diptera: Tephritidae) (Dimbi et al., 2003;Ekesi et al., 2002;Flores et al., 2013;Toledo et al., 2006Toledo et al., , 2017. Dias et al. (2022a) note that B. bassiana is one of the most promising and versatile fungus species that can function as a biocontrol agent. Montoya et al. (2020) evaluated this species with the concurrent use of dissemination devices of fungus conidia and the Sterile Insect Technique (SIT), observing an increase in infected wild fruit flies, and thus proposed an additive or synergistic effect of both technologies.
The Mediterranean fruit fly, C. capitata, is a highly polyphagous, multivoltine species that infects more than 353 host fruit species around the world (Papadopoulos, 2008). Currently, this pest has an extensive distribution in tropical and temperate zones, limiting the growth and development of international fresh fruit trade (Diamantidis et al., 2011;Dias et al., 2022b;Malacrida et al., 2007;Stewart & Johanson, 1999;Vera et al., 2002). In Mexico, the SIT is applied to control C. capitata transient entries using an integrated pest management (IPM) framework. The sterile insects are provided by the mass-rearing facility located in Metapa, Chiapas, where the insects have been inoculated with B. bassiana conidia to infect wild flies in the border with Guatemala. The use of dissemination devices to disperse EPF conidia has been another strategy employed to control pest populations (Flores et al., 2013;Toledo et al., 2017). The selected EPF strains must allow enough time for interactions among conspecifics before the insect's death. However, how the infection process affects the sexual competitiveness of males, as well as the resulting fitness of the female conidia recipients, is still not well known. Thus, the objectives of the present work were to determine the effect of the horizontal transmission of B. bassiana on the survival, fecundity, and fertility of C. capitata individuals using inoculated adults and dissemination devices. We also determined the impact of the fungus infection on sterile male performance as vectors of EPF. The above will contribute to a better understanding of pathogen-host interactions, and thus reinforce these ecologically oriented control strategies within IPM.

Materials and methods
Biological material. Males (sterile, fertile) and fertile females of C. capitata were provided by the Moscamed facility (SADER-SENASICA) located in Metapa, Chiapas, Mexico. After emergence, each sex was kept separately in Plexiglas cages (30 × 30 × 30 cm) with water and food (a 3: 1 mixture of sugar: hydrolyzed protein [MP Biomedicals, LLC, Santa Ana, California, USA]) until they reached sexual maturity. Laboratory conditions were maintained at 25 ± 1 °C, 60 ± 10% R.H, and a photoperiod of 12:12 h light: dark. All tests were carried out inside a cage 3 m in diameter × 2 m high made of saran screen (20 by 20 mesh) in an isolated room inside the Moscamed facility to reinforce biological safety measures.
The B. bassiana strain was formulated with 2 × 10 9 conidia per g with Celite 400 as inert material (Laboratorio de Organismos Benéficos, Hongos, Insectos y Nematodos, Talisman, Chiapas, Mexico). According to Toledo et al. (2017), this strain produces 99.6% mortality, a median survival time of 3.8 ± 0.1 d, CL 50 and CL 90 of 8.2 × 10 5 and 1.6 × 10 7 , respectively. The viability of the fungus was determined before each test according to the microculture technique (Jiménez, 1992) and with observations of germinated conidia at 40 × using an optical microscope (Dialux 20 EB; Leitz, Wetzlar, Germany). The conidia were considered viable when the length of the germ tube was greater than the diameter. The evaluations were carried out with batches of conidia with a recorded viability of ≥ 90%.
Inoculation methods. Adults of C. capitata were infected with B. bassiana conidia either topically or using the inoculation device method: 1) Topical inoculation. We applied 0.05 g of the B. bassiana formulation to 50 fertile or sterile male or female C. capitata in a 50 ml Falcon conical centrifuge tube (29 mm diameter × 115 mm height), which was shaken gently for 10 s for a homogeneous inoculation of all individuals. 2) Inoculation with PVC device. Insects were inoculated using the technique developed by Maniania (1994) for tsetse flies. A device was constructed using a PVC tube (9 cm in diameter × 10 cm high) with two perforations of 1 cm in diameter in the lower part, and 100 × 10 mm Petri dish bottoms were used as lids on both ends. The interior of the tube was lined with yellow plush fabric (felt), similarly to the dissemination device developed by Toledo et al. (2017), which was homogeneously impregnated with 2 g of the conidia formulation. Groups of 50 females or males were placed inside test tubes (25 × 150 mm) and then introduced through the perforations on the lower part of the PVC device. The insects were recovered after 10 min, which was enough time to ensure contact with the fungal conidia. Inoculated adults were separated by sex and kept in Plexiglas cages (30 × 30 × 30 cm) for 20 min to allow them to remove excess conidia by grooming.
The effect of B. bassiana conidia on the fecundity, fertility, and survival of C. capitata adults was determined through the following tests: bassiana on C. capitata was determined by direct inoculation of females or by horizontal transmission through inoculated males. The combinations evaluated were: inoculated males + inoculated females, inoculated males + non-inoculated females, non-inoculated males + inoculated females. Fungal conidia were applied to adults either by topical inoculation or with the PVC device. A treatment with non-inoculated males + non-inoculated females was included as a control treatment. This resulted in seven treatments, three combinations × two inoculation methods + control.
For each treatment, 10 fertile fly pairs were placed in plastic containers of 1.75 L (17.3 cm high, 21.2 × 11.3 cm) provided with water and food (3:1 mixture of sugar: hydrolyzed protein). The container was placed in a horizontal position and kept under laboratory conditions for 10 days. As oviposition substrate, the opening of the plastic container was covered with a piece of white lycra fabric (Dorian Gray Likra Pantyhose). Laid eggs fell into a petri dish with tap water placed below the container opening. Each container was one experimental unit, and each repetition was carried out with a different production batch of C. capitata. We performed five replicates per treatment for horizontal transmission. b) Inoculation of sterile males. The effect of infected sterile males on the survival, fecundity, and fertility of fertile females was compared with that of inoculated fertile males and the interaction between fertile and inoculated sterile males. The treatments were (1) inoculated sterile males and (2) non-inoculated sterile males, both interacting with fertile males. The topical method was used to apply the conidia to both sterile and fertile males.
According to each treatment, 10 males and 10 females were placed in plastic containers of 1.75 L as described above. In the fertile-sterile male treatments, 5 sterile males (inoculated or healthy) and 5 fertile males were placed with 10 fertile females. Each container was an experimental unit with six replicates per treatment, each one with a different production batch of C. capitata. Fecundity, fertility, mortality, and mycosis were estimated as follows: Fecundity and egg fertility. Eggs were collected in a Petri dish bottom (150 × 20 mm) with 200 ml of sterile water placed under the oviposition substrate. For each treatment, eggs were recovered daily using a piece of black organza cloth (10 × 10 cm) and were aligned and counted using a stereomicroscope (Nikon, SMZ645, USA). A sample of 100 eggs was placed in an incubation chamber (plastic Petri dish of 100 × 10 mm, where a piece of black cloth was placed on a piece of 2-mm-thick moistened filter paper). After four days, the hatched eggs were counted using a stereomicroscope with a 4 × objective. Egg fertility was expressed as percentage of hatched eggs.
Mortality and fungal sporulation. The number of dead adults in each treatment was recorded daily. Dead adults were disinfected for 20 s in a 1% sodium hypochlorite solution and then washed three times with sterile distilled water to eliminate saprophytic microorganisms. Subsequently, the specimens were placed in a humid chamber (sterile wet filter paper in 100 × 10 mm plastic Petri dishes) sealed with Parafilm (Bemis Company, Inc. Neenah, WI, 54956) and kept under laboratory conditions. After five days, the flies were examined under a stereomicroscope to verify the presence of mycosis.
Statistical analysis. In each test, survival curves over the course of the ten days of evaluation were compared using a log-rank test. Fecundity, number of eggs per live female per day, was log-transformed to obtain homoscedasticity prior to performing an analysis of variance (ANOVA), and mean comparisons were performed with a Tukey test (α = 0.05). Fertility, expressed as the proportion of hatched eggs, was analyzed with a GLM with binomial distribution and a logit link function. Treatment means were compared by contrasts with a significance level of 95%.
Horizontal transmission of B. bassiana was calculated as the proportion of non-inoculated fruit flies that died and developed mycoses after interaction with inoculated adults. The efficacy of both methods of inoculation by sex to transfer conidia was analyzed using a generalized linear model (GLM) with binomial distribution and a logit link function. In the evaluation with sterile males, the ability of sterile and fertile males to infect females was compared using a GLM with binomial distribution and a logit link function. The treatments were compared by orthogonal contrasts (α = 0.05). All analyzes were performed in JMP® 11.0.0 (JMP® SAS Institute, http:// www. jmp. com).

a) Inoculation method
Survival of females (χ 2 = 217.98, d.f. = 6, P < 0.001) and males (χ 2 = 189.34, d.f. = 6, P < 0.001) differed significantly between treatments. Survival of directly inoculated females registered a maximum of 20% at 5 days. In treatments with inoculated males, the survival of non-inoculated females showed a gradual reduction throughout the 10 days. In the control treatment, the mortality was lower than 20% after 10 days. The survival curve of females did not differ between topical inoculation and the use of an inoculation device (Fig. 1).
The number of eggs per female was higher at day 3 and 4 in each treatment, with the control treatment having the highest number of eggs (Fig. 1). Fecundity was significantly affected by treatment (F = 18.58, d.f. = 6, 24, P < 0.001). The control treatment had the highest fecundity, and the treatments with directly inoculated females showed the lowest fecundity. When comparing the inoculation methods, the treatments with topical inoculation showed lower fecundity than those with the device method ( Table 1).
The fertility of C. capitata was significantly affected in treatments with the fungus spore (χ 2 = 399.59, d.f. = 6, P < 0.001), with the control having the highest proportion of hatched eggs. The treatments with directly inoculated males showed the lowest egg hatching percentage. The treatments with topical inoculation of males showed the lowest fertility values (Table 1).
The number of eggs per female per day showed the highest peak between days 3 and 4 in each treatment. In the treatments with non-inoculated males, the number of eggs per female exhibited a gradual decrease, while there was greater variation after day 6 in the treatments with inoculated males (Fig. 1). The total fecundity for the entire period was significantly lower in treatments with inoculated males than in treatments with noninoculated males (F = 17.25, d.f. = 1.25, P < 0.001).
The egg fertility of C. capitata was significantly affected by treatment (χ 2 = 724.7, d.f. = 3, P < 0.001). Inoculation of  fertile males significantly reduced the percentage of hatched eggs. When inoculated or non-inoculated sterile males interacted with fertile males and females, egg fertility was lower than with non-inoculated sterile males (Table 2). Horizontal transmission to females did not differ significantly between the three treatments that included males inoculated with B. bassiana, with a mycosis percentage of 73.3% in each treatment (Table 2).

Discussion
As expected, the survival and fecundity of C. capitata were affected by the infection of B. bassiana.
The use of inoculated sterile males or dissemination devices of fungus conidia resulted in the horizontal transmission of B. bassiana to untreated insects. Both inoculation methods allowed the successful interaction between treated and untreated insects, allowing thus a horizontal transmission between them, which provides a better understanding of how infections by fungi, such as B. bassiana, affect the survival and reproduction of contaminated insects. This undoubtedly will lead to the implementation of more effective and timelier IPM strategies (Shoukat et al., 2020) for the control of fruit fly populations. Our results support the proposal to simultaneously use the SIT and EPF as in Montoya et al. (2020). Furthermore,   (Toledo et al., 2017;Yousef et al., 2018). Our results showed a range of 65-80% horizontal transmission of B. bassiana conidia to untreated C. capitata adults. These results agree with Chergui et al. (2020), who recorded a maximum of 51.67 and 73.33% mortality on the 15th day of a contact bioassay in males and females, respectively. The success of EPF as a control agent depends mainly on the possibility of transmitting the infection among conspecifics, as well as on the behavioral response of the insect species during the host-pathogen interaction (Baverstock et al., 2010;Dimbi et al., 2013). In the case of fruit flies, fungal transmission is enhanced by the mating behavior displayed by the adults. Fruit fly males form leks (i.e., groups of males calling for matings [Shelly, 2018;Thaochan & Ngampongsai, 2018]) and compete with each other to copulate with the females present. This behavior generates several interactions that increase the chances of successful fungal transmission among conspecifics. We observed that the horizontal transmission of conidia was higher with the topical method than with the dissemination device, which could be due to the topical method resulting in a larger number of conidia per fly than the device method. Chergui et al. (2020) indicate that different methods of B. bassiana application produce different results. According to the criteria by Kaneshiro et al. (1993), a larger amount of conidia could generate female rejection of sterile males, although the excess of EPF could be reduced by grooming (Baverstock et al., 2010). Moreover, grooming allows conidia to be deposited in preferred gathering sites for adults, for example, during the lek formation (Arita & Kaneshiro, 1985, 1989Thaochan & Ngampongsai, 2018).
Survival of treated adults was reduced from 7 to 10 days. During this period, the females can oviposit before dying; however, their reproductive potential was greatly reduced by EPF infection (see Toledo et al., 2007). Daily fecundity was not affected during the onset of fungal infection, but total fecundity was significantly reduced throughout the 10-day period, potentiated by early mortality of infected females. We observed that fecundity was lower in directly infected females than in females infected by inoculated fertile males, which also agrees with Chergui et al. (2020), who noted that different application methods would produce different results. However, these findings still support the proposal to include the use of fungal spore dissemination devices in the framework of IPM, where mortality accumulated with other control strategies such as SIT  or parasitoids such as biocontrol agents (Martínez -Barrera et al., 2019, 2020) among other alternatives, will render better pest suppression.
The effect of B. bassiana of reducing the fecundity, fertility, and survival of C. capitata females in this study was due to the physiological alterations derived from the pathogenic infection Usman et al., 2021). For example, the depletion of sugar and other compounds in the insect hemolymph by EPF (e.g., Jin et al., 2015;Peng et al., 2015;Xia et al., 2002) has serious consequences on the fitness parameters of the host insects . The observed reduction in the oviposition rate and fertility of infected females has been highly associated with the action of EPF, which negatively influences insect populations as a resulting compensation for their delayed mortality (Dimbi et al., 2013). The adverse effects on fecundity can also be caused by the antifeedant activity of B. bassiana during the invasive process (Ekesi, 2001). Our results showed that fecundity was lower in directly infected females than in females infected by inoculated fertile males. We also observed a negative effect on egg hatching when fertile males were inoculated than when inoculated sterile males interacted with non-inoculated fertile males.
The use of insects as vectors of biocontrol agents has been proposed as a strategy to improve biological control programs through the horizontal transmission of pathogens (Vickers et al., 2004;Llácer et al., 2013;Diouf et al., 2022). The application of this strategy in fruit fly programs allows the release of inoculated sterile males to disseminate conidia into wild populations of C. capitata (Flores et al., 2013;Toledo et al., 2017). Recently, Diouf et al. (2022) introduced the term "boosted SIT" to refer to the use of sterile insects as vectors of biocides to trigger an epizootic in wild populations. For successful results, quality parameters, such as dispersion and competitiveness, of sterile males must remain unaffected after inoculation (Novelo-Rincón et al., 2009;Ramírez y Ramírez et al., 2022), at least long enough to interact with a wild population.
In conclusion, the inoculation of B. bassiana conidia using a dissemination device or the topical method was effective in causing mycosis in adult C. capitata, and the horizontal transmission of fungal spores significantly reduced the fecundity, egg fertility, and survival of C. capitata females. These results support the proposal of including this strategy in IPM programs for the suppression of Mediterranean fruit fly populations.