Effect of Ascosphaera apis Infestation on the Activities of Four Antioxidant Enzymes in Asian Honey Bee Larval Guts

Ascosphaera apis infects exclusively bee larvae and causes chalkbrood, a lethal fungal disease that results in a sharp reduction in adult bees and colony productivity. However, little is known about the effect of A. apis infestation on the activities of antioxidant enzymes in bee larvae. Here, A. apis spores were purified and used to inoculate Asian honey bee (Apis cerana) larvae, followed by the detection of the host survival rate and an evaluation of the activities of four major antioxidant enzymes. At 6 days after inoculation (dpi) with A. apis spores, obvious symptoms of chalkbrood disease similar to what occurs in Apis mellifera larvae were observed. PCR identification verified the A. apis infection of A. cerana larvae. Additionally, the survival rate of larvae inoculated with A. apis was high at 1–2 dpi, which sharply decreased to 4.16% at 4 dpi and which reached 0% at 5 dpi, whereas that of uninoculated larvae was always high at 1~8 dpi, with an average survival rate of 95.37%, indicating the negative impact of A. apis infection on larval survival. As compared with those in the corresponding uninoculated groups, the superoxide dismutase (SOD) and catalase (CAT) activities in the 5- and 6-day-old larval guts in the A. apis–inoculated groups were significantly decreased (p < 0.05) and the glutathione S-transferase (GST) activity in the 4- and 5-day-old larval guts was significantly increased (p < 0.05), which suggests that the inhibition of SOD and CAT activities and the activation of GST activity in the larval guts was caused by A. apis infestation. In comparison with that in the corresponding uninoculated groups, the polyphenol oxidase (PPO) activity was significantly increased (p < 0.05) in the 5-day-old larval gut but significantly reduced (p < 0.01) in the 6-day-old larval gut, indicating that the PPO activity in the larval guts was first enhanced and then suppressed. Our findings not only unravel the response of A. cerana larvae to A. apis infestation from a biochemical perspective but also offer a valuable insight into the interaction between Asian honey bee larvae and A. apis.


Introduction
Honey bees are of great importance thanks to their pollination of numerous wildflowers and agricultural crops; their production of api-products, such as honey, royal jelly, propolis, beeswax, and bee pollen; and their scientific applications as research models [1]. However, as a kind of representative eusocial insect, honey bees are prone to infections by various pathogens and parasites, such as bacteria, fungi, viruses, and Varroa mite [2,3]. Among these, Ascosphaera apis, an obligate fungal pathogen of honey bee broods, causes chalkbrood disease and results in a dramatic decline in colony strength and productivity alone or in combination with other biotic or abiotic factors, which has given rise to severe losses for the apicultural industry [4]. For insects, including honey bees, the midgut is 6-day-old larva and further elucidated the miRNA-regulated mechanism of A. apis invasion. However, biochemical research regarding A. cerana-A. apis interaction is very limited at present, hindering a deeper understanding of the mechanism underlying responses of A. cerana to A. apis infestation.
In the current work, A. apis spores were purified and used to inoculate the A. cerana 3-day-old larvae, followed by a calculation of the host survival rate and an evaluation of the activities of four antioxidant enzymes, specifically SOD, CAT, GST, and polyphenol oxidase (PPO), in the guts of both A. apis-inoculated and uninoculated larvae. The findings from the present study could not only clarify the effect of A. apis invasion on the host survival rate and the activities of the aforementioned four antioxidant enzymes of great importance in larval guts but also offer a valuable understanding of A. cerana larval responses to A. apis and host-pathogen interactions.

Fungi and Bee Larvae
A. apis was previously isolated from chalkbrood mummies and conserved at the Honey Bee Protection Laboratory of the College of Animal Sciences (College of Bee Science), Fujian Agriculture and Forestry University, Fuzhou, China [2,28,29]. A. cerana worker larvae were derived from 3 strong colonies reared in the apiary of the College of Animal Sciences (College of Bee Science), Fujian Agriculture and Forestry University, Fuzhou, China.

Purification of A. apis Spores
A. apis stored at 4 • C was transferred to PDA medium and cultured at 33 ± 0.5 • C in a constant temperature and humidity chamber (Jingke, Shanghai, China). After 10 days of culturing, white mycelia were removed, and black fruiting bodies were harvested and transferred to an RNAase-free EP tube, following our previously established protocol [30,31]. Next, 1 mL of sterile water was added to the EP tube, followed by complete grinding. Then, the grinding fluid was centrifuged at 25 • C, 7000× g for 3 min. The supernatant was removed, and 1 mL of sterile water was added, followed by centrifugation at 25 • C, 7000× g for 3 min. The centrifugation was repeated twice to clean the spores, which were then stored at 4 • C until use.

Experimental Inoculation and Survival Rate Calculation
Honey bee larvae were reared and inoculated with A. apis spores by following our previously described method [23]. In brief, (1) the larvae diet was prepared following Feng et al. [32], preheated to 35 • C, and added to 6-well culture plates. (2) PCR amplification was performed to detect the honey bee colonies reared in the apiary, and three colonies with negative results were selected as experimental colonies. The 2-day-old larvae were carefully transferred to 6-well culture plates using a Chinese graft, and after 24 h, the larvae were transferred to 48-well culture plates (1 larva/well) and placed in an incubator (35 • C, 95% RH). (3) The purified A. apis spores were subjected to gradient dilution and mixed with the diet, with a final concentration of 1 × 10 7 spore/mL. The 3-day-old treatment group larvae were fed the diet containing spores (0 days after inoculation, 0 dpi), while 3-day-old larvae in the control groups were fed the diet without spores; the diet was changed daily. There were 3 biological replicas of this experiment. The dead larvae in both treatment and control groups were recorded and removed every 24 h until 8 dpi, followed by survival rate calculations.

Evaluation of the SOD Activity
Following the method described by Li et al. [10], the larval gut SOD activity was evaluated using the Insect SOD ELISA Kit (MLBIO, Shanghai, China). Briefly, the gut samples were fully ground with a high-throughput tissue grinder (MEIBI, Hangzhou, China); the grinding fluid was transferred to a sterile EP tube; 750 mL of 1 × PBS solution was added, followed by centrifugation at 1000× g for 10 min; the supernatant was incubated on ice; 50 mL of the standard sample was added to the standard well, while 40 mL of sample diluent and 10 mL of grinding fluid were added into the sample well, followed by gentle shaking. Meanwhile, a blank well was set and sealed with a film, and the microtiter plate was incubated at 37 • C for 30 min. The reaction solution was then discarded, and washing buffer was added to the wells. The solution was removed after standing for 30 s, and the operation was repeated 5 times. Then 50 mL of enzyme standard reagent was added to each standard well and sample well, and 50 mL each of chromogenic A and B were added. After gentle shaking, the solution was placed at 37 • C for 10 min; 50 mL of termination solution was added into each well to terminate the reaction; finally, the OD value at 450 nm from each well was measured by a Thermo Scientific Varioskan LUX (ThermoFisher, Waltham, MA, USA). This experiment included three biological replicas.
The specific antioxidant enzyme activity was expressed as units of enzyme activity per milligram of protein. Data were analyzed and plotted by GraphPad Prism 8 software (GraphPad Software, San Diego, CA, USA). Experimental data were presented as mean ± SD and subjected to Student's t-tests.

Examination of CAT and GST Activities
According to the method described by Li et al. [10], the larval gut CAT activity was examined with an Insect CAT ELISA Kit (MLBIO, Shanghai, China). GST activity was checked using an Insect GST ELISA Kit (MLBIO, Shanghai, China) by following the described method by Li et al. [10]. The operation and calculation methods were the same as in Section 2.4.

Detection of PPO Activity
On basis of the method described by Li et al. [33], the larval gut PPO activity was evaluated using the Polyphenol Oxidase (PPO) Activity Assay Kit (Solarbio, Beijing, China). The gut samples (each 0.1 g) in the six groups mentioned above were transferred to sterile EP tubes, and 1 mL of extraction solution (0.05 M sodium phosphate (pH 7.0), 4% (w/v) insoluble PVP, and 0.5% (w/v) Triton X-100) was added. Next, the gut tissues were thoroughly ground using a high-throughput tissue grinder, followed by centrifugation at 8000× g for 10 min. The supernatant was transferred to a new EP tube and placed on ice for measurement. The assay tube and control tube reaction systems were prepared, placed in a 25 • C water bath for 10 min, and then quickly transferred to a 100 • C metal bath for 10 min. The reaction system was mixed thoroughly and centrifuged at 5000× g for 10 min, and the supernatant was transferred to a new EP tube and placed on ice. The assay and control OD values were detected at 410 nm and respectively named A assay and A control, and the difference between them was named ∆A. PPO activity was calculated as follows: PPO (U/g) = 120 × ∆A ÷ W (∆A = A assay − A control), where W is the sample mass in grams.

Verification of A. cerana Larvae Infection by Inoculation with A. apis Spores
In the present study, a prominent symptom of chalkbrood disease was observed in larvae inoculated with A. apis spores-white mycelia first penetrated from the posterior end of the larva at 4 dpi, extended to the anterior end, and eventually covered the entire larval body surface ( Figure 1A). In addition, as shown in Figure 1B, agarose gel electrophoresis indicated that fragments with the expected sizes (about 217 bp) could be amplified from the A. apis-inoculated larval guts and A. apis spores but could not be amplified from the uninoculated larval guts and sterile water. Collectively, these results confirmed the infection of A. cerana larvae by inoculation with A. apis spores.
In the present study, a prominent symptom of chalkbrood disease was observed in larvae inoculated with A. apis spores-white mycelia first penetrated from the posterior end of the larva at 4 dpi, extended to the anterior end, and eventually covered the entire larval body surface ( Figure 1A). In addition, as shown in Figure 1B, agarose gel electrophoresis indicated that fragments with the expected sizes (about 217 bp) could be amplified from the A. apis-inoculated larval guts and A. apis spores but could not be amplified from the uninoculated larval guts and sterile water. Collectively, these results confirmed the infection of A. cerana larvae by inoculation with A. apis spores.

Survival Rate of A. cerana Larvae after A. apis Spores Infection
It was detected that the survival rate of A. cerana larvae in the A. apis-inoculated group was 97.92%, 83.33%, and 33.33% at 1 dpi~3 dpi, respectively; additionally, the larval survival rate sharply decreased to 4.16% at 4 dpi and to 0 at 5 dpi (shown in Figure 2). Comparatively, the survival rate of larvae in the uninoculated group was always high at 1 dpi~8 dpi, with an average survival rate of 95.37%, as shown in Figure 2.

Survival Rate of A. cerana Larvae after A. apis Spores Infection
It was detected that the survival rate of A. cerana larvae in the A. apis-inoculated group was 97.92%, 83.33%, and 33.33% at 1 dpi-3 dpi, respectively; additionally, the larval survival rate sharply decreased to 4.16% at 4 dpi and to 0 at 5 dpi (shown in Figure 2). Comparatively, the survival rate of larvae in the uninoculated group was always high at 1 dpi-8 dpi, with an average survival rate of 95.37%, as shown in Figure 2.
In the present study, a prominent symptom of chalkbrood disease was observed in larvae inoculated with A. apis spores-white mycelia first penetrated from the posterior end of the larva at 4 dpi, extended to the anterior end, and eventually covered the entire larval body surface ( Figure 1A). In addition, as shown in Figure 1B, agarose gel electrophoresis indicated that fragments with the expected sizes (about 217 bp) could be amplified from the A. apis-inoculated larval guts and A. apis spores but could not be amplified from the uninoculated larval guts and sterile water. Collectively, these results confirmed the infection of A. cerana larvae by inoculation with A. apis spores.

Survival Rate of A. cerana Larvae after A. apis Spores Infection
It was detected that the survival rate of A. cerana larvae in the A. apis-inoculated group was 97.92%, 83.33%, and 33.33% at 1 dpi~3 dpi, respectively; additionally, the larval survival rate sharply decreased to 4.16% at 4 dpi and to 0 at 5 dpi (shown in Figure 2). Comparatively, the survival rate of larvae in the uninoculated group was always high at 1 dpi~8 dpi, with an average survival rate of 95.37%, as shown in Figure 2.

Effect of A. apis Infection on GST Activity in A. cerana Larval Guts
In this current work, as compared with that in the corresponding control groups, the GST activity was significantly increased (p < 0.05) in the 4-day-old (8.69 ± 1.40 IU/L) and 5-day-old (7.67 ± 0.12 IU/L) larval guts, whereas reduced (p > 0.05) in the 6-day-old (7.50 ± 0.08 IU/L) larval gut ( Figure 5).

Discussion
Previously, there was no documentation of the course of chalkbrood disease that occurred in A. cerana larvae. It was reported that A. apis spores were consumed by A. mellifera larvae via food sharing and germinated in the midgut lumen, with the disappearance of the diaphragm between the midgut and hindgut at the prepupal stage [34]; the fungal spores and food debris then swarmed into the hindgut lumen, where the mycelia rapidly grew in contact with O2, followed by the penetration of the peritrophic membrane, gut wall, and body wall, eventually covering the entire larva, with a thick layer of white mycelia [4,24]. In the current work, the A. apis spores were purified and mixed with the diet to feed 3-day-old larvae of A. cerana, and no apparent symptoms of chalkbrood disease were detected at 1-3 dpi; however, mycelia penetrated from the posterior end of the larva at 4 dpi and then extended to the anterior end, finally covering the entire larval body surface ( Figure 1A). This course of chalkbrood disease was similar to that of A. mellifera larvae infected by A. apis [35], which was in line with the fact that A. apis was an exclusive Figure 5. GST activity in A. cerana 4-, 5-, and 6-day-old larval guts infected by A. apis. The experimental data are shown as mean ± SD and were subjected to Student's t-tests, ns: p > 0.05, *: p < 0.05, **: p < 0.01. AcCK1, AcCK2, and AcCK3 respectively represent the uninfected 4-, 5-, and 6-day-old larval guts, whereas AcT1, AcT2, and AcT3 respectively represent the A. apis-infected 4-, 5-, and 6-day-old larval guts. Figure 5. GST activity in A. cerana 4-, 5-, and 6-day-old larval guts infected by A. apis. The experimental data are shown as mean ± SD and were subjected to Student's t-tests, ns: p > 0.05, *: p < 0.05, **: p < 0.01. AcCK1, AcCK2, and AcCK3 respectively represent the uninfected 4-, 5-, and 6day-old larval guts, whereas AcT1, AcT2, and AcT3 respectively represent the A. apis-infected 4-, 5-, and 6-day-old larval guts.

Discussion
Previously, there was no documentation of the course of chalkbrood disease that occurred in A. cerana larvae. It was reported that A. apis spores were consumed by A. mellifera larvae via food sharing and germinated in the midgut lumen, with the disappearance of the diaphragm between the midgut and hindgut at the prepupal stage [34]; the fungal spores and food debris then swarmed into the hindgut lumen, where the mycelia rapidly grew in contact with O2, followed by the penetration of the peritrophic membrane, gut wall, and body wall, eventually covering the entire larva, with a thick layer of white mycelia [4,24]. In the current work, the A. apis spores were purified and mixed with the diet to feed 3-day-old larvae of A. cerana, and no apparent symptoms of chalkbrood disease were detected at 1-3 dpi; however, mycelia penetrated from the posterior end of the larva at 4 dpi and then extended to the anterior end, finally covering the entire larval body surface ( Figure 1A). This course of chalkbrood disease was similar to that of A. mellifera larvae infected by A. apis [35], which was in line with the fact that A. apis was an exclusive fungal pathogen of bee larvae. In addition, agarose gel Figure 6. PPO activity in A. cerana 4-, 5-, and 6-day-old larval guts infected by A. apis. The experimental data are shown as mean ± SD and were subjected to Student's t-tests, ns: p > 0.05, *: p < 0.05, **: p < 0.01. AcCK1, AcCK2, and AcCK3 respectively represent the uninfected 4-, 5-, and 6-day-old larval guts, whereas AcT1, AcT2, and AcT3 respectively represent the A. apis-infected 4-, 5-, and 6-day-old larval guts.

Discussion
Previously, there was no documentation of the course of chalkbrood disease that occurred in A. cerana larvae. It was reported that A. apis spores were consumed by A. mellifera larvae via food sharing and germinated in the midgut lumen, with the disappearance of the diaphragm between the midgut and hindgut at the prepupal stage [34]; the fungal spores and food debris then swarmed into the hindgut lumen, where the mycelia rapidly grew in contact with O 2 , followed by the penetration of the peritrophic membrane, gut wall, and body wall, eventually covering the entire larva, with a thick layer of white mycelia [4,24]. In the current work, the A. apis spores were purified and mixed with the diet to feed 3-day-old larvae of A. cerana, and no apparent symptoms of chalkbrood disease were detected at 1-3 dpi; however, mycelia penetrated from the posterior end of the larva at 4 dpi and then extended to the anterior end, finally covering the entire larval body surface ( Figure 1A). This course of chalkbrood disease was similar to that of A. mellifera larvae infected by A. apis [35], which was in line with the fact that A. apis was an exclusive fungal pathogen of bee larvae. In addition, agarose gel electrophoresis showed that fragments of expected sizes (about 217 bp) could be amplified from A. apis-inoculated larval guts and A. apis spores, but not from the uninoculated larval guts and sterile water ( Figure 1B). In summary, these results together confirmed the A. apis infection of A. cerana larvae after spore inoculation under lab conditions, which gave rise to chalkbrood disease. This offered solid experimental evidence for further study on interactions between A. cerana larvae and A. apis.
In the present study, we observed that the survival rate of A. cerana larvae after A. apis inoculation was 97.92%, 83.33%, 33.33% at 1~3 dpi, which sharply decreased to 4.16% at 4 dpi and to 0 at 5 dpi, whereas that of the uninoculated larvae was always high at 1~8 dpi (95.37% on average) (Figure 2). This indicated that the increased infection time of A. apis negatively influenced larval survival, following the pathogenesis mentioned above. To the best of our knowledge, this is the first experimental evidence of the survival rate of A. cerana larvae infected by A. apis.
In insects, protective and detoxifying enzymes are critical in maintaining normal physiological functions and biochemical metabolisms [8,10,36]. Among these, SOD and CAT can remove oxidative and toxic molecules such as O 2and hydroxyl radical OHproduced by exogenous compounds [11]. Li et al. [10] reported that both SOD and CAT activities were significantly reduced in the A. m. ligustica 3-day-old worker larvae at 96 h after inoculation with A. apis spores. In this current work, we found that as compared with those in the corresponding uninfected groups, the SOD and CAT activities in the 5-and 6-day-old A. apis-infected larval guts were significantly decreased, as shown in Figures 3 and 4, similar to the finding in the A. m. ligustica larvae infected by A. apis [10]. However, the SOD and CAT activities in the 4-day-old larval guts were reduced, but there was no significant difference between the A. apis-infected and uninfected groups (Figures 3 and 4), showing little influence from the two aforementioned antioxidant enzymes given that there was only low-level spore germination and mycelial growth in the early stage of A. apis infection. Collectively, these results demonstrated that A. apis infestation could negatively influence the SOD and CAT activities of honey bee larvae, which may be a strategy adopted by A. apis during the long-term coevolution and interactions with A. cerana larvae. After feeding the fourth-and fifth-instar larvae of Hyphantria cunea with the leaves of Bacillus thuringiensis transgenic poplar, Ding et al. [37] detected that the activities of both SOD and CAT displayed an increase-decrease trend. After inoculating Nilaparvata lugens Stål with Metarhizium flavoviride spores, Zhang et al. [36] found that the SOD and CAT activities continuously increased as infection time prolonged. Taken together, these results indicated that the SOD and CAT activities exhibited different trends in various insects responding to pathogen infections.
As a key component of the antioxidant enzyme system, GST is involved in the detoxification process of exogenous compounds in honey bees [12,13]. Yan et al. [38] discovered that infection by highly pathogenic strains of entomo pathogenic nematode significantly altered the GST activity in the Anoplophora glabripennis larvae, presenting an overall increasedecrease-increase trend. Huang et al. [39] observed that the GST activity was significantly higher in Plutella xylostella L. parasitized by Diadegma semiclausum than that in the control group. In the present study, we found that the GST activity in 4-and 5-day-old larval guts after A. apis infestation was significantly increased, as shown in Figure 5. Thus, A. cerana larvae were likely to enhance GST activity in response to the oxidative stress caused by A. apis infestation. However, it was detected that the GST activity in 6-day-old larval guts after A. apis infestation was reduced, without a significant difference between the A. apis-infected and uninfected groups (Figure 5), to a certain extent reflecting complex host-pathogen interaction and A. apis-caused attenuation of the GST activity.
In insects, PPO is engaged in melanin formation, keratinization, and wound healing and also exerts a pivotal function in host immune defense [40,41]. The activity and the content of PPO are often used as indicators for evaluating insect immunity [36,41]. Li et al. [42] documented that the PPO activity in infected Apriona germari larvae hemolymph increased to a maximum at 2.5 days after Beauveria bassiana infection and then decreased at 3 days after infection. Wertheim et al. [43] reported that the expression level of the PPO3 gene in Drosophila was significantly upregulated at 48-72 h after the parasitic wasp challenge. In Nilaparvata lugens Stål, there was a significant elevation of PO content at 72 h after infection with Metarhizium flavoviride [36]. In this work, we observed increased PPO activity in the 4-day-old larval gut, which was significantly elevated in the 5-day-old larval gut after A. apis infestation. The results showed that with the accumulation of spores and mycelia in the larval gut, the host reinforced the PPO activity to combat the A. apis infestation. Intriguingly, the PPO activity was significantly decreased in the gut tissue of 6-day-old larvae infected by A. apis ( Figure 6). This indicated that at the late stage of infection, the PPO activity in the larval gut was suppressed because of the growing fungal stress. Taken together, these findings were suggestive of complex interactions between A. cerana larvae and A. apis.

Conclusions
In a nutshell, the A. apis spore inoculation of A. cerana larvae gave rise to chalkbrood disease, which decreased the host survival rate. The A. apis infestation affected the activities of SOD, CAT, GST, and PPO (Figure 7), indicative of a host-adopted defense strategy mediated by antioxidant enzymes and complex host-pathogen interactions.
increased to a maximum at 2.5 days after Beauveria bassiana infection and then decreased at 3 days after infection. Wertheim et al. [43] reported that the expression level of the PPO3 gene in Drosophila was significantly upregulated at 48-72 h after the parasitic wasp challenge. In Nilaparvata lugens Stål, there was a significant elevation of PO content at 72 h after infection with Metarhizium flavoviride [36]. In this work, we observed increased PPO activity in the 4-day-old larval gut, which was significantly elevated in the 5-day-old larval gut after A. apis infestation. The results showed that with the accumulation of spores and mycelia in the larval gut, the host reinforced the PPO activity to combat the A. apis infestation. Intriguingly, the PPO activity was significantly decreased in the gut tissue of 6-day-old larvae infected by A. apis ( Figure 6). This indicated that at the late stage of infection, the PPO activity in the larval gut was suppressed because of the growing fungal stress. Taken together, these findings were suggestive of complex interactions between A. cerana larvae and A. apis.

Conclusions
In a nutshell, the A. apis spore inoculation of A. cerana larvae gave rise to chalkbrood disease, which decreased the host survival rate. The A. apis infestation affected the activities of SOD, CAT, GST, and PPO (Figure 7), indicative of a host-adopted defense strategy mediated by antioxidant enzymes and complex host-pathogen interactions. Author Contributions: R.G. and D.C. designed this research; K.Z. and Z.F. contributed to the writing of the article; K.Z., Z.F., X.F., Z.W., S.W., S.G., X.G., H.Z., X.J., P.Z., Q.L., and M.C. conducted experiments and data analyses. R.G. and D.C. supervised the study and the preparation of the manuscript. All authors have read and agreed to the published version of the manuscript.