Dynamics of the presence of israeli acute paralysis virus in honey bee colonies with colony collapse disorder

Abstract

The determinants of Colony Collapse Disorder (CCD), a particular case of collapse of honey bee colonies, are still unresolved. Viruses including the Israeli acute paralysis virus (IAPV) were associated with CCD. We found an apiary with colonies showing typical CCD characteristics that bore high loads of IAPV, recovered some colonies from collapse and tested the hypothesis if IAPV was actively replicating in them and infectious to healthy bees. We found that IAPV was the dominant pathogen and it replicated actively in the colonies: viral titers decreased from April to September and increased from September to December. IAPV extracted from infected bees was highly infectious to healthy pupae: they showed several-fold amplification of the viral genome and synthesis of the virion protein VP3. The health of recovered colonies was seriously compromised. Interestingly, a rise of IAPV genomic copies in two colonies coincided with their subsequent collapse. Our results do not imply IAPV as the cause of CCD but indicate that once acquired and induced to replication it acts as an infectious factor that affects the health of the colonies and may determine their survival. This is the first follow up outside the US of CCD-colonies bearing IAPV under natural conditions.

Figure 1

Loads of Israeli Acute Paralysis Virus (IAPV) and Deformed Wing Virus (DWV genomic copies in honey bee samples from CCD- and IAPV- bearing colonies over time. (a) IAPV. (b) DWV. The viral genomic copy number was determined by RT-qPCR as described in Materials and Methods. Colored bars, different colonies. Each bar represents the average viral load (±SD) of a pool of 10 bees (three replicates). (a) The levels of virus varied significantly per colony (colony 3 different from the rest) and per month. (September significantly lower than the rest; April and December significantly higher than the rest) Colony F = 5.8, d.f. = 4,16, p < 0.01; Month F = 17.2 d.f. = 4,16, p < 0.01, (GLM, Post-hoc test: LSD).

Figure 2

IAPV replication in honey bee samples from CCD-colonies. Number of copies of positive- and negative-sense RNA strands measured by RT-qPCR as described in Materials and Methods. Sample dates and colonies are indicated at the x-axis. All tested pairs +/− were significantly different (t-test, p < 0.05).

Figure 3

Infectivity of IAPV extracted from CCD-colonies. Virus-free pupae (white-eye) from a healthy colony were inoculated with IAPV-extracted from naturally infected bees from CCD- and 17-colonies. X-axis, amplification-fold of IAPV genomic copies at 72 h after injection of IAPV-extracted at various time points in the year, y-axis. (Dependent t-test, t = 3.27215, p < 0.01).

Figure 4

IAPV-virion proteins are synthesized in larvae injected with IAPV extracts of bees from CCD-colonies. Immunoblot analysis of protein extracted from virus-free larvae injected with IAPV extracts from colony samples (indicated above the figure). Panels (a) and (b), injection of IAPV samples from July 2011 and February 2012, respectively. All viral samples were separated by PAGE and immunoblotted with anti-VP3 antiserum. Arrows, virion proteins recognized by the serum. Molecular size markers are indicated on the right. mi, control mock-injected larvae with extracts derived from healthy bees.

Figure 5

Honey bee population in IAPV-positive, CCD- and healthy control colonies. Adult (black) and brood (gray). CCD-colonies, 7, 8 and 10. Non-CCD colony, 17. Control colonies, 16 and 18. Colony numbers are indicated in each plot.