Immune Response and Hemolymph Microbiota of Apis mellifera and Apis cerana After the Challenge With Recombinant Varroa Toxic Protein

Abstract The honey bee is a significant crop pollinator and key model insect for understanding social behavior, disease transmission, and development. The ectoparasitic Varroa destructor mite put threats on the honey bee industry. A Varroa toxic protein (VTP) from the saliva of Varroa mites contributes to the toxicity toward Apis cerana and the deformed wing virus elevation in Apis mellifera. However, the immune response and hemolymph microbiota of honey bee species after the injection of recombinant VTP has not yet been reported. In this study, both A. cerana and A. mellifera worker larvae were injected with the recombinant VTP. Then the expressions of the honey bee immune genes abaecin, defensin, and domeless at three time points were determined by qRT–PCR, and hemolymph microbial community were analyzed by culture-dependent method, after recombinant VTP injection. The mortality rates of A. cerana larvae were much higher than those of A. mellifera larvae after VTP challenge. VTP injection induced the upregulation of defensin gene expression in A. mellifera larvae, and higher levels of abaecin and domeless mRNAs response in A. cerana larvae, compared with the control (without any injection). Phosphate buffer saline (PBS) injection also upregulated the expression levels of abaecin, defensin, and domeless in A. mellifera and A. cerana larvae. Three bacterial species (Enterococcus faecalis, Staphylococcus cohnii, and Bacillus cereus) were isolated from the hemolymph of A. cerana larvae after VTP injection and at 48 h after PBS injections. Two bacterial species (Stenotrophomonas maltophilia and Staphylococcus aureus) were isolated from A. mellifera larvae after VTP challenge. No bacterial colonies were detected from the larval hemolymph of both honey bee species treated by injection only and the control. The result indicates that abaecin, defensin, and domeless genes and hemolymph microbiota respond to the VTP challenge. VTP injection might induce the dramatic growth of different bacterial species in the hemolymph of the injected larvae of A. mellifera and A. cerana, which provide cues for further studying the interactions among the honey bee, VTP, and hemolymph bacteria.

host of Varroa mites was A. cerana, which are normally not threatened by this mite due to a stable host-parasite bond established over a long evolutionary time and a mixture of the defense system in A. cerana that limits the Varroa population growth (Peng et al. 1987, Oldroyd 1999. The new host A. mellifera is damaged by Varroa mite and becomes wing deformed by deformed wing virus (DWV) usually carried by the mite. Varroa toxic protein (VTP) was identified from the saliva of Varroa mites (Zhang and Han 2018). The recombinant VTP killed A. cerana worker larvae and pupae in the absence of DWV, but was safe for A. mellifera individuals, and resulted in elevated DWV titers and the subsequent development of deformed wing adults (Zhang et al. 2018). However, no information is available on the immune response of the honey bees to the VTP.
Insect symbiotic bacteria can stimulate the immune system of their insect host and thereby raise the efficiency of pathogen defense (Evans 2006, De Souza et al. 2009). In general, antimicrobial peptides (AMPs) are crucial effectors for insect's innate immune system (Wu et al. 2018). Honey bees differ in their ability to protect themselves against V. destructor infestation (Rinderer et al. 2014). Insect gut microbiota are affected by physiological (diet, metabolism, immune system, and gut anatomy) and biological (interactions, transmission, and bottlenecks) processes of insects (Bonilla-Rosso and Engel 2018). Gut bacteria play significant roles in health and strength, contribute extremely to host immunity, improve nutrient deficient diets, degrade recalcitrant food ingredients, and protect the host from parasites and pathogens (Dillon and Dillon 2004, Mazmanian et al. 2005, Engel and Moran 2013, Rooks and Garrett 2016, Tomasova et al. 2016, Lee et al. 2018.
Insect hemolymph is recognized as a key mediator of nutritional and immunological homeostasis and is generally considered to be almost microbe free in healthy insects (Lemaitre and Hoffmann 2007). Now more evidence indicates that various nonpathogenic microorganisms can stably or transiently inhabit hemolymph in a diversity of insects (Blow and Douglas 2019). The most-reported hemolymph microorganisms are bacteria of the genus Spiroplasma (Phylum Tenericutes, Family Mollicutes) widely associated with insects in the Hymenoptera, Diptera, Lepidoptera, Hemiptera, and Coleoptera orders (Herren et al. 2014), and members of the Enterobacteriaceae (γ-proteobacteria) in aphids, specifically Serratia symbiotica and the sister taxa Hamiltonella defensa and Regiella insecticola (Henry et al. 2013, Zytynska et al. 2016. But hemolymph microbiota in honey bees has not been reported so far.
In this study, the expressed and purified VTP was injected into the fifth-stage larvae of A. cerana and A. mellifera, to determine the immune response and hemolymph microbiota changes of the honey bee larvae at different time intervals.

Honey Bees and Varroa Mites
Apis cerana and A. mellifera colonies were maintained with the standard beekeeping practice in Guangdong Institute of Applied Biological Resource, Guangzhou, China. Freshly capped larvae (the fifth stage) of A. mellifera and A. cerana were randomly collected from colonies without chalkbrood and foulbrood symptoms according to the published method (Kanbar andEngels 2003, Zhang andHan 2018). Varroa destructor mature female mites were collected from worker pupae in A. mellifera hives, using a soft camel hairbrush. The colonies were not been treated with acaricides. Approximately 20 mites were placed in sterile Petri dishes (diameter = 9 cm) and used for RNA extraction and bioassays within 1 h.

Virus and Microsporidia Loads
Viruses and microsporidia loads were examined from honey bee by RT-PCR amplification using the sequences of 16 bee viruses available from NCBI (acute bee paralysis (5 U/μl,Sangon Biotech Co.,Ltd.,Shanghai,China), and 1 µl template DNA were performed in a C1000 TM Thermal Cycler PCR system (Bio-Rad, Hercules, CA), with the following parameters: initial denaturation at 95°C for 3 min; followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 58°C for 30 s, and extension at 60°C for 30 s; and a final extension at 72°C for 10 min. The amplicons of the expected bands were verified from a 1% agarose gel and visualized after SYBR green staining with an imaging system (Sagecreation Science co., Beijing, China). The PCR product was purified using a gel extraction kit (Promega, Madison, WI) and subcloned into a pGEM-T Easy Vector (Promega) before transformation into Escherichia coli DH5αcompetent cells (Takara, Kyoto, Japan). Five independent positive clones were selected using a blue-white screen and sequenced in both directions using an Applied Biosystems 3730 automated sequencer (Applied Biosystems, Foster City, CA) at Sangon Biotech Co., Ltd (Shanghai, China).

Larvae Injection With Recombinant VTP
VTP gene was obtained from the Varroa mites, and the expression and purification of recombinant VTP were conducted as previously described (Zhang and Han 2018). The purified recombinant VTP was injected into the fifth-stage larvae according to the described method (Zhang and Han 2018). Briefly, a 0.2-μl aliquot of the purified recombinant-VTP at a concentration of 0.2 μg/ml was injected into the hemocoel of an A. mellifera or A. cerana larva near the end of the abdomen, by using pulled glass capillary needles in conjunction with a Harvard micro-injector system (IM-31, Narishige, Tokyo, Japan). Negative controls, such as uninjected bees (=CK) and bees injected with only sterile PBS (=PBS) or only the needle puncture, were established, with three replicates per each treatment.
After injection, larvae were reared in 48-well culture plates under regulated conditions (34 ± 1°C temperature, 80% relative humidity, and 16:8 [L:D] h dark photoperiod) without microbial infection and bled larvae were discarded. From each treatment, 10 live larvae were randomly collected 12, 24, or 48 h post-treatment, frozen in liquid nitrogen, and stored at −80°C for RNA extraction.

Expression Profile of Larval Immune Genes After VTP Injection
Three immune genes (abaecin [Gene Id. GB18323], domeless [Gene Id. GB16422], and defensing [Gene Id. GB19392]) were used to evaluate the immune response of the injected larvae by using qRT-PCR with the primers in Table 2. Housekeeping β-actin gene (GenBank Accession No. AB023025) was used in the internal reference to normalize the target gene expression. Each reaction (20 µl volume) contained 1 µl (10 ng) cDNA template, 0.8 µl 10 µM forward/reverse primers, 10 μl 2× FastStart Essential DNA Green Master (Roche, Shanghai, China), and 7.4 µl RNase-free water. qRT-PCR was performed on a Rotor Gene Q Real Time Thermal Cycler (Qiagen, Hilden, Germany), with the following parameters: initial denaturation for one cycle at 95°C for 10 min, followed by 40 cycles at 95°C for 15 s, and 60°C for 30 s. The qRT-PCR was repeated three times, and the independent RNA sample preparation consisted of three technical replicates.

Microbiota Analysis of the Larval Hemolymph After VTP Injection
The A. mellifera and A. cerana live larvae were collected after 12, 24, or 48 h post-treatments of VTP injection, PBS injection, injection only (needle puncture), or control (without any treatment). Prior to honey bee larvae dissection, the larvae were disinfected to remove external microbes with 75% ethanol and then rinsed three times with sterile ultra-pure water (Engel and Moran 2013). The larval abdomen was faced up in a sterile Petri plate. After a small hole was carefully made using a sterile sharp-billed tweezers, the hemolymph (usually 30-60 µl from each larva) was collected with a pipette in a sterile tube. Three replicates with 10 larvae for each replicate were established for each treatment.
To identify the bacterial isolates from the plates, genomic DNA of each isolate was extracted, and bacterial 16S rRNA gene was amplified with the general bacterial primers 28F 5′-GAGTTTGATCNTGGCTCAG-3′ and 1392R 5′-ACGGGCGGTGTGTRC-3′ (Mattila et al. 2012, Moran et al. 2012). The PCR mixture contained 5 µl of 10× Pfu buffer, 4 µl of dNTP mixture (2.5 mM), 1 µl of each primer (10 µM), 2 µl of deionized formamide, 1 µl of MgCl 2 (25 mM), 1 µl of genomic DNA, and 0.5 µl of Pfu DNA polymerase in a total volume of 50 µl. PCR amplification was performed in Gradient thermocycler (Applied Biosystems). The PCR condition was 10 min at 95°C, followed by 25 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 45 s, and 2 min for elongation at 72°C; and a final extension step of 72°C for 10 min. PCR products of different bacterial isolates were sequenced by Sangon Biotech Co., Ltd., (Shanghai, China). The resulting sequences were compared with the data set in NCBI GenBank.

Data Analysis
The relative value of the gene expression was analyzed by the comparative CT method (2ˉ ∆∆CT ; Livak and Schmittgen 2001). All the graphs were performed using Prism 6.0 for Windows (GraphPad, La Jolla, CA, www.graphpad.com). The abundance of nucleic acid in each sample was normalized by dividing the calculated abundance of the gene of interest by the abundance of actin gene. Data were statistically analyzed using one-way ANOVA with SPSS 16.0 software (SPSS Inc., Chicago, IL), followed by Tukey's multiple comparison post hoc test. Differences among means were considered significant at P< 0.05.

Virus Occurrence From the Larvae
No visible clinical symptoms or pathogen colonies (such as American foulbrood or chalkbrood) were found in the honey bee larvae. For the detection of 17 viruses and 2 microsporidia parasites in the larvae, the RT-PCR was conducted according to the primers in Table  1. DWV, BQCV, CWV, and MV were detected from more than 60% of the checked larvae of A. mellifera (Table 3). LSV and SBV were detected from most of the A. cerana larvae. Interestingly, the most common virus for A. mellifera and A. cerana was IAPV, which was detected from both honey bee species (Table 3).

Mortality of Honey Bee Larvae After VTP Injection
The mortality rates of the injected and uninjected A. mellifera and A. cerana larvae after 12, 24, and 48 h were shown in Fig. 1. Compared with the controls, survival rates of A. mellifera larvae did not show significant difference after 12, 24, or 48 h of VTP injection. The survival rates of A. cerana larvae after 12, 24, and 48 h of VTP injection were significantly decreased by 22, 36, and 52%, respectively. It seemed that VTP challenge significantly decreased the survival rates of A. cerana larvae but did not influence the mortality rates of A. mellifera larvae.

VTP Protein Expression and Purification
The full-length VTP sequence from V. destructor mite consisted of 952 base pairs and contained 405 open reading frame nucleotides encoding a 134 amino acid polypeptide. The predicted molecular mass of the VTP amino acid sequence was 14.6 kDa, and predicted theoretical isoelectric point was 5.27, in accordance with previously published results (Zhang and Han 2018). The recombinant plasmid pET-28a-VTP transformants were expressed in E. coli BL21 (DE-3) cells. The expression of the recombinant protein was induced by IPTG under growth conditions of 25°C (Fig. 2). The expected bands were detected at approximately 30 kDa. The fusion protein was purified with a Ni 2+ -NTA agarose gel column and reexamined using a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel. The purified protein (>95% purity) of VTP showed the estimated molecular weight of a single band (Fig. 2).

The Expression Profiles of Immune Genes
The expression patterns of abaecin, domeless, and defensin genes in the challenged to A. mellifera and A. cerana larvae were obtained after 12, 24, and 48 h by qRT-PCR ( Fig. 3A-F). In response to VTP injection, the expression patterns of abaecin in A. mellifera larvae showed no significant changes among the treatment and the controls. The transcript level of abaecin gene was the highest expression level after 12, 24, and 48 h of PBS injection (Fig. 3A). The mRNA level of defensin gene was significantly higher at 12 h than that at 24 h (F = 3.133, P = 0.275, F= 4.164, P = 0.330) after VTP injection, but decreased at 48 h treatment (Fig. 3B). Compared with the controls, the mRNA levels of domeless gene expression did not significantly differ at three time points after VTP injection in A. mellifera larvae. Meanwhile, the mRNA level of domeless gene expression at 12 h after PBS injection was highly (F = 14.062, P = 0.296) expressed in A. mellifera larvae (Fig. 3C).
In the A. cerana larvae, the abaecin transcript was highly upregulated at 24 h after VTP injection (F = 6.093, P = 0.363; F = 0.163, P = 0.214) and at 48 h after puncture injection or at 12 h after PBS injection ( Fig. 3D and F). The defensin mRNA level of the A. cerana larvae was significantly higher at 12 and 48 h after PBS injection, but no significant differences in the defensin mRNA levels were found from the larvae challenged with VTP and the controls (Fig. 3E). The domeless expressions were significantly upregulated at 12 h after puncture injection, at 12 and 48 h after PBS injection, or at 24 h after VTP injection, but downregulated at 48 h after VTP injection compared with the controls (no injection,   puncture injection, and PBS injection). So the purified recombinant VTP could increase or decrease the expression levels of abaecin, defensin, and domeless genes at different time intervals, suggesting that the VTP might be involved in the response of honey bee immune genes.

The Changes in Hemolymph Microbiota of the Larvae After VTP Injection
The hemolymph microbial community from the A. mellifera and A. cerana larvae were analyzed by the culture-based method. From the colonies clearly identical in size, color, and morphology, five representative bacterial species were selected and identified from the VTP-injected larvae of A. mellifera and A. cerana, based on 16S rRNA sequences (Fig. 4). Three bacterial species (Enterococcus faecalis, Staphylococcus cohnii, and Bacillus cereus) were isolated from TSA or HIA plates in both anaerobic and aerobic conditions from the hemolymph of A.cerana larvae at three time points after VTP injection and at 48 h after PBS injections. Two bacterial species (Stenotrophomonas maltophilia and Staphylococcus aureus) were isolated on HIA and TSA plates under aerobic and anaerobic conditions from A. mellifera larvae at three time points after VTP injection. No bacterial colonies were detected on any plates from the larval hemolymph of both honey bee species treated by injection only and the control (without any injection). The loads of these bacterial species were variable from 1 to over 200 colonies on a plate. The results indicated that VTP injection induced the growth of S. maltophilia and S. aureus in the hemolymph of the injected A. mellifera larvae, and VTP or PBS injection stimulated the bacterial growth of E. faecalis, S. cohnii and B. cereus in the hemolymph of the injected larvae in A. cerana.

Discussion
Honey bees are infected by different pathogens and parasites, such as viruses, bacteria, protozoa, and different mites. In this study, more virus species were detected from A. mellifera larvae than those in A. cerana larvae. Multiple virus infections were common in both honey bee species. The occurrence of DWV, BQCV, CWV, MV and IAPV in A. mellifera has been reported in several studies (Carreck et al. 2010, Ai et al. 2012, Mordecai et al. 2016. The higher DWV virus prevalence in A. mellifera than in A. cerana suggested that the western honey bees potentially act as carriers of honey bee viruses (Diao et al. 2019). The present results confirmed that VTP influences the immune reaction and causes dramatic changes of the bacterial community in the hemolymph of A. cerana, and A. mellifera larvae. Honey bee's defense pathways such as the transmembrane signal transducing pathway (Toll), immune deficiency (Imd), Janus kinase/ signal transducers and activators of transcription (JAK/STAT), and intracellular signaling pathways (JNK) are involved in the inducible defense (Brutscher andFlenniken 2015, Erban et al. 2019). Honey bee larvae and drones are protected by a powerful innate immune system in the hemolymph (Gätschenberger et al. 2013). Energy stores and metabolic enzymes are regulated when the hemolymph of honey bees responded to Paenibacillus larvae (Chan et al. 2009). The transient synthesis of the three antimicrobial peptides (AMPs) hymenoptaecin, defensin1, and abaecin in the hemolymph of A. mellifera larvae were found after the larvae were challenged with sterile PBS and Escherichia coli cells, respectively (Randolt et al. 2008). In this study, the expression patterns of abaecin, defensin, and domeless genes induced by VTP injection, PBS injection, and injection only were determined by qRT-PCR at three time intervals. VTP injection induced the defensin response in A. mellifera larvae, and abaecin and domeless response in A. cerana larvae, compared with the control. PBS injection also induced abaecin, defensin, and domeless response in A. mellifera and A. cerana larvae. These results contradicted the finding of abaecin and defensin suppression by Varroa mite during bee development (Gregory et al. 2005) and are more constant with the report that specific honey bee immune genes were upregulated in developing brood during Varroa parasitism (Aronstein et al. 2012, Gregorc et al. 2012. Five core bacterial species (Snodgrassella alvi, Gilliamella apicola,Bifidobacterium) and four other bacterial species (Frischella perrara, Bartonella apis, Parasaccharibacter apium, and Gluconobacter) were found from the gut of honey bees by culture-dependent and -independent methods (Babendreier et al. 2007, Bottacini et al. 2012, Donaldson et al. 2016, Kwong and Moran 2016. Compared with the core species, four other species were less abundant and unstable in the gut of honey bees. The bacterial community was influenced by the quarantine disease American foulbrood caused by Paenibacillus larvae in the worker bee , and by the parasite V. destructor (Pakwan et al. 2018) and pathogens Nosema and Lotmaria passimin adult bees . The Varroa mites are reservoirs of the pathogenic bacteria in the apicultures (Hubert et al. 2015). The culturable bacteria associated with V. destructor were oxalotrophic ones in the Proteobacteria and Actinobacteria (Maddaloni and Pascual 2015). Gram-positive bacteria (Bacillus and Microbacterium genera) and Gram-negative bacteria (Brevundimonas and Rhizobium genera) were also detected on the Varroa body surface based on the culture-dependent method (Vanikova et al. 2015). Furthermore, it was found that bacterial communities in V. destructor and Tropilaelaps mercedesae were dominated by Enterobacteriaceae and Enterococcus (Pakwan et al. 2018). Although gut microbiota was not studied in this paper, E. faecalis, S. cohnii, and B. cereus from the hemolymph of A. cerana larvae, and S. maltophilia and S. aureus from the hemolymph of A. mellifera larvae were for the first time detected after these larvae were injected with purified recombinant VTP. However, no bacterial colonies were found from the larval hemolymph of both honey bee species treated by injection only and the control without any injection. These results indicated that different bacterial species were present in the hemolymph of both honey bee species and VTP injection stimulated the bacterial growth in the larval hemolymph. How the bacteria existed in the hemolymph remained unclear. It seemed impossible that these bacteria were introduced into the hemolymph by the injection itself because no bacteria were detected from the larvae challenged by injection only. Maybe the loads of these unseen bacteria in the hemolymph of the control larvae were too low to be detected by the present culture method.
In the present study, E. faecalis, S. cohnii, and B. cereus were isolated from the hemolymph of A. cerana larvae. Surprisingly, apart from VTP, sterile PBS injection also induced the bacterial loads in the larval hemolymph. It seemed that bacteria in the hemolymph of A. cerana larvae are sensitive to overgrow by foreign materials. Enterococcus faecalis was reported from A. mellifera (Gaggia et al. 2015. Staphylococcus cohnii and B. cereus were also isolated from A. mellifera honey bee (Kačániová et al. 2020). However, it was uncertain that these reported bacterial species were associated with the hemolymph of A. mellifera honey bee. The bacteriocin-producing E. faecalis was tested against different spoilage and pathogenic micro-organisms, including Paenibacillus larvae (Jaouani et al. 2014). Paenibacillus larvae infection enriched the abundance of E. faecalis in the whole worker bee . As a biocontrol agent, B. cereus from honey samples and other apiarian sources was used to inhibit the bacterium P. larvae in Fig. 3. Relative expression of abaecin, defensin, and domeless in Apis mellifera and Apis cerana treated with VTP, PBS, and needle puncture. Different letters on the bars indicate that the means are significantly different among treatments according to the Tukey's test. Asterisks above bars indicate significant differences between the treatment and the corresponding control (P < 0.05).
A. mellifera bees (Alippi and Reynaldi 2006). Whether mite infection induced increasing E. faecalis and/or B. cereus loads to inhibit P. larvae disease in the hemolymph needs further study.
Stenotrophomonas maltophilia and S. aureus were found from the hemolymph of A. mellifera larvae. However, this species resides in a broad range of environments and is commonly identified only as multidrug-resistant opportunistic pathogens of humans (Looney et al. 2009), in soils or in association with plants (Ryan et al. 2009), and also associated with multiple insect species, including the diamondback moth Plutella xylostella (Indiragandhi et al. 2007), the red turpentine beetle Dendroctonus valens (Morales-Jiménez et al. 2012), the 12-toothed pine bark beetle Ips sexdentatus (Sevim et al. 2012), the Asian malaria mosquito Anopheles stephensi (Hughes et al. 2016), the peach fruit fly Bactrocera zonata (Naaz et al. 2016), the muga silkworm Antheraea assamensis (Gandotra et al. 2018), and the wings of the Colorado potato beetle Leptinotarsa decemlineata (Kang et al. 2020). Stenotrophomonas maltophilia was also isolated from the honeydew of Indian lac insect Kerria lacca (Shamim et al. 2018) and from the stable fly Stomoxys calcitrans larvae presenting antifungal activity against Beauveria bassiana (Moraes et al. 2014). Furthermore, S. maltophilia associated with Delia antiqua larvae can inhibit B. bassiana infection (Zhou et al. 2019). The functions of S. maltophilia in the hemolymph of A. mellifera honey bees upon VTP induction should be an interesting topic for further investigation. S. aureusis a human wound pathogen which can be counteracted by lactic acid bacterial symbionts in honey bees (Olofsson et al. 2016). However, this bacterial species was also detected from the digestive gut of adult worker honey bees (Fasasi 2018). Why this species also existed in the larval hemolymph of A. mellifera bees remains unknown.
In summary, in this study, VTP injection induced the response of the immune genes (abaecin, defensin, and domeless) and the changes of the hemolymph microbiota of A. mellifera and A. cerana larvae. The mortality rates of A. cerana larvae were much higher than those of A. mellifera larvae after VTP challenge. Three bacterial species (E. faecalis, S. cohnii, and B. cereus) and two bacterial species (S. maltophilia and S. aureus) were for the first time detected from the hemolymph of A. cerana and A. mellifera larvae after VTP injection, respectively.