Winter Hive Debris Analysis Is Significant for Assessing the Health Status of Honeybee Colonies (Apis mellifera)

Abstract

Honeybee diseases are one of the most significant and most common causes of honeybee colonies' weakness and death. An early diagnosis of subclinical infections is necessary to implement precautionary and control measures. Sampling debris from hive bottom boards is simple, non-invasive, and cheap. In this study, we collected winter debris samples in apiaries located in the continental part of Croatia. We used molecular methods, PCR and qPCR, for the first time to analyze those samples. Laboratory results were compared with the health condition and strength of honeybee colonies at an apiary in spring. Our study successfully identified the presence and quantity of various pathogens, including the presence of Vairimorpha spp. (Nosema spp.), quintefied Paenibacillus larvae, Acute Bee Paralysis Virus (ABPV), Black Queen Cell Virus (BQCV), Deformed Wing Virus (DWV), and Sacbrood Virus (SBV). However, our analysis did not detect Melissococcus plutonius, Crithidia mellificae, Lotmaria passim, and Aethina tumida. Samples of winter debris were also examined for the presence and quantification of the V. destructor mites, and their natural mite fall was observed in spring. Honeybee colonies were simultaneously infected by an average of four to six pathogens. Some observed honeybee colonies developed characteristic symptoms, while others did not survive the winter.

Keywords: Acute Bee Paralysis Virus; Aethina tumida; Black Queen Cell Virus; Crithidia mellificae; Deformed Wing Virus and Sacbrood Virus; Lotmaria passim; Melissococcus plutonius; PCR/qPCR; Paenibacillus larvae; Vairimorpha spp. (Nosema spp.); Varroa destructor; honeybee colony (Apis mellifera); samples of winter hive debris.

Figure 1

Examination results for the presence and quantification of P. larvae (a) and C. mellificae (b) using real-time PCR methods.

Figure 2

The results of ABPV, BQCV, DWV and SBV detection by specific RT-qPCRs in the tested samples of hive debris. Legend: Negative sample: no viral RNA was detected in tested samples: less than 1 copy of virus RNA in 5 μL of tested RNA; Very weak positive sample: between 1 and 100 copies of virus RNA were detected in 5 μL of tested RNA; Weak positive sample: between 101 and 1000 copies of virus RNA were detected in 5 μL of tested RNA; Positive sample: between 1001 and 100,000 copies of virus RNA were detected in 5 μL of tested RNA; Strong positive sample: more than 100,001 copies of virus RNA were detected in 5 μL of tested RNA.

Figure 3

The summarised results of detected percentages of ABPV, BQCV, DWV and SBV, by specific RT-qPCRs in the tested samples of hive debris. Legend: Negative sample: no viral RNA was detected in tested samples: less than 1 copy of virus RNA in 5 μL of tested RNA; Very weak positive sample: between 1 and 100 copies of virus RNA were detected in 5 μL of tested RNA; Weak positive sample: between 101 and 1000 copies of virus RNA were detected in 5 μL of tested RNA; Positive sample: between 1001 and 100,000 copies of virus RNA were detected in 5 μL of tested RNA; Strong positive sample: more than 100,001 copies of virus RNA were detected in 5 μL of tested RNA.

Figure 4

An overview of the identified pathogens in debris samples taken from bottom boards of hives.

Figure 5

The proportion of parasitic mites V. destructor in one gram of hive debris sample, taken in the winter period at all observed apiaries.

Figure 6

The number of counted parasitic mites V. destructor from each observed hive and from hive bottom board debris samples, collected in winter and spring.