The effect of two distinct viral-enriched inocula on the immune response and chemical cues in honey bee pupae

Abstract The combination of Varroa destructor (Varroa) and the viruses it vectors is a major driver of honey bee (Apis mellifera) colony losses. Hygienic behavior and individual immunity enable bees to cope with some pests and parasites. Hygienic response of adult bees is driven by olfactory cues emanating from diseased brood. In this study, we tested the effect of viral inoculation on the chemical profile and immune response of pupae, independently of Varroa. We injected pupae with various doses of an enriched inocula of either a deformed wing virus (DWV) B-A recombinant or Israeli acute paralysis virus (IAPV) and followed their development and survival for five days. At the end of the five-day period, volatile profiles, viral loads, and expression of seven immune genes were assessed. Both IAPV and the DWV loads increased to equivalent high levels irrespective of the initial dose applied. Notably, greater rates of mortality (60% loss) were observed with the highest IAPV dose when compared to the lowest IAPV dose (15% loss). The DWV inoculum, caused limited mortality but nonetheless inhibited pupae development. The IAPV injected pupae showed evidence of a dose dependent DWV amplification from either a DWV variant in the original IAPV inoculum or from an endogenous source of DWV in the pupae themselves. IAPV-injected pupae had lower expression of immune genes than DWV-injected pupae, suggesting IAPV inhibits the pupae immune response. Overall, among the seven tested immune genes six were upregulated with only vago downregulated, suggesting inhibition of the RNAi pathway following infection. Chemical cues of mock and untreated pupae were similar, but notably different from the virus-injected pupae for both inocula. Our findings show that Varroa-independent virus inoculated pupae produce unique virus-specific chemical cues; the ultimate consequence of such a change might lead to virus specific bee behavioral responses. Authors summary This study investigated how two major honey bee viruses, Deformed Wing Virus (DWV) and Israeli Acute Paralysis Virus (IAPV), affect pupae independent of the Varroa mite, which typically spreads them. This study was conducted in the laboratory on incubated pupae isolated from the colony and injected with two viral preparations (inocula). We tested the impact after five-days incubation. The results show that both viruses reach high levels in pupae, though IAPV inoculum proved far more deadly (up to 60% mortality) and appeared to suppress the pupae’s immune response, possibly even amplifying co-occurring DWV. While less lethal, the DWV inoculum still inhibited normal development. Interestigly, the virus-infected pupae produced distinct, virus-specific chemical cues—unique volatile profiles different from healthy controls. Since adult honey bees use olfactory cues to detect and remove diseased brood (hygienic behavior), these findings suggest that the viruses themselves generate the “scent of sickness,” which could trigger colony-wide behavioral responses and offer a vital target for enhancing the bees’ natural defenses against these major drivers of colony loss. Competing Interest Statement The authors have declared no competing interest.