Effects of Deformed Wing Virus Infection on Expressions of Immune- and Apoptosis-Related Genes in Western Honeybees (Apis mellifera)

Abstract

Honeybees are globally threatened by several pathogens, especially deformed wing virus (DWV), as the presence of DWV in western honeybees is indicative of colony loss. The high mortality rate is further exacerbated by the lack of effective treatment, and therefore understanding the immune and apoptosis responses could pave an avenue for the treatment method. In this study, DWV was directly injected into the white-eyed pupae stage of western honeybees (Apis mellifera). The DWV loads and selected gene responses were monitored using the real-time PCR technique. The results showed that honeybee pupae that were injected with the highest concentration of viral loads showed a significantly higher mortality rate than the control groups. Deformed wings could be observed in newly emerged adult bees when the infected bees harbored high levels of viral loads. However, the numbers of viral loads in both normal and crippled wing groups were not significantly different. DWV-injected honeybee pupae with 104 and 107 copy numbers per bee groups showed similar viral loads after 48 h until newly emerged adult bees. Levels of gene expression including immune genes (defensin, abaecin, and hymenoptaecin) and apoptosis genes (buffy, p53, Apaf1, caspase3-like, caspase8-like, and caspase9-like) were analyzed after DWV infection. The expressions of immune and apoptosis genes were significantly different in infected bees compared to those of the control groups. In the pupae stage, the immune genes were activated by injecting DWV (defensin and hymenoptaecin) or Escherichia coli (defensin, abaecin, and hymenoptaecin), a positive control. On the contrary, the expression of apoptosis-related genes (buffy, caspase3-like, caspase8-like, and caspase9-like genes) was suppressed at 96 h post-infection. In DWV-infected newly emerged adult bees, abaecin, hymenoptaecin, Apaf1, and caspase8-like genes were upregulated. However, these genes were not significantly different between the normal and crippled wing bees. Our results suggested that DWV could activate the humoral immunity in honeybees and that honeybee hosts may be able to protect themselves from the virus infection through immune responses. Apoptosis gene expressions were upregulated in newly emerged adult bees by the virus, however, they were downregulated during the initial phase of viral infection.

Interaction between Thiamethoxam and Deformed Wing Virus Type A on Wing Characteristics and Expression of Immune and Apoptosis Genes in Apis mellifera

“…Viral infection within the host via viral entry, replication, and spreading can induce the antiviral innate immune responses [ 66 ]. Upregulation of several AMP genes, including abaecin , hymenoptaecin , and defensin , was also shown in other studies where honey bees were infected with DWV-A [ 56 ]. Treatment with thiamethoxam led to the downregulation of abaecin and defensin genes in crippled wings adult honey bees.…”

“…Our study demonstrated that the effects of treatment with only thiamethoxam induce increased wing deformity in newly emerged adult honey bees. Moreover, numerous studies have also demonstrated that DWV is also the cause of crippled wings in honey bees [ 56 , 59 ]. Our result showed that honey bees exposed to thiamethoxam and DWV-A stressors had a high percentage of crippled wings as newly emerged adult honey bees.…”

“…AMPs play a crucial role in the insect immune system and contribute to individual and social immunity [ 50 , 61 , 62 ]. Previous studies have indicated that the expressions of AMP genes in honey bees were upregulated after the invasion of pathogens, including microsporidian Nosema [ 63 ], Paenibacillus larvae [ 64 ], viruses [ 56 ], and ectoparasitic mites [ 65 ]. Viral infection within the host via viral entry, replication, and spreading can induce the antiviral innate immune responses [ 66 ].…”

“…Evidence from previous studies suggested that virus infection induced apoptosis in insects and that the infection was mitigated by the elimination of the infected cells [ 70 , 71 , 72 ]. Honey bees injected with DWV had suppressed the expression of caspases in the pupal stage, which likely promoted the virus survival in hosts [ 56 ]. Honey bee co-exposure groups showed a strong alteration of immune gene expressions and downregulation of two apoptosis genes.…”

“…Relative quantification in real-time PCR was determined in antimicrobial peptides (AMPs), and apoptosis-related genes [ 50 , 56 ]. Ribosomal protein subunit 5 (RPS5) and β -actin were used as housekeeping genes for all primers shown in Table S1 .…”