A scientific note on detection of honeybee viruses in the darkling beetle (Alphitobius diaperinus, Coleoptera: Tenebrionidae), a new pest in Apis cerana cerana colonies

Apidologie, Mar 2016

Zhiguo Li, Shaokang Huang, Wei-fone Huang, Haiyang Geng, Yazhou Zhao, Meng Li, Yanping Chen, Songkun Su

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A scientific note on detection of honeybee viruses in the darkling beetle (Alphitobius diaperinus, Coleoptera: Tenebrionidae), a new pest in Apis cerana cerana colonies

Apidologie A scientific note on detection of honeybee viruses in the darkling beetle (Alphitobius diaperinus , Coleoptera: Tenebrionidae), a new pest in Apis cerana cerana colonies Zhiguo LI 2 Shaokang HUANG 2 Wei-fone HUANG 2 Haiyang GENG 2 Yazhou ZHAO 1 Meng LI 2 Yanping CHEN 0 Songkun SU 2 0 USDA-ARS, Bee Research Laboratory , Beltsville, MD 20705 , USA 1 Institute of Apiculture, Chinese Academy of Agricultural Sciences , Beijing 100093 , China 2 College of Bee Science, Fujian Agriculture and Forestry University , Fuzhou 350002 , China viruses / Apis cerana / Alphitobius diaperinus / honeybees - The darkling beetle, also known as the lesser mealworm (Alphitobius diaperinus Panzer, Coleoptera: Tenebrionidae), exhibits a cosmopolitan distribution in poultry houses and piggeries (McAllister et al. 1995). A. diaperinus originates from sub-Saharan Africa, and this species has adapted to moist and warm poultry facilities (Geden and Hogsette 1994). A. diaperinus is a nocturnal omnivore considered as a serious pest of poultry facilities; low light environments, such as poultry houses and piggeries, provide an optimum habitat for A. diaperinus (Esquivel et al. 2012). Larvae and adults can damage poultry houses by tunnelling into insulation materials (Esquivel et al. 2012). The lesser mealworm beetle can also act as a reservoir of poultry pathogens, including viruses, bacteria and fungi (McAllister et al. 1995; Esquivel et al. 2012). Our study described the lesser mealworm inhabiting honeybee (Apis cerana cerana ) hives. A. diaperinus beetles were first found in our experimental apiary during our hive inspection in daytime twice a week. We further surveyed two more apiaries to further confirm that A. diaperinus is a new pest in A. c. cerana colonies. The three honeybee (A. c. cerana ) apiaries were located in Fuzhou, Fujian Province, Southeast China (latitude N26°08’, longitude E119°28’). The honeybees were kept in a four-frame hive with Langstroth frames, and all of the colonies examined in the study were queenright and strong in population. A. diaperinus beetles were seen inhabiting the surface area of the solid bottom boards of hives, the cracks and joints of the bottom boards and the frames (top and bottom bars) of combs. Two to eighteen beetles were found in each inhabited hive. The number of beetles inhabiting the three different apiaries significantly differed, as revealed by nonparametric Kruskal–Wallis test (P < 0.05). The beetle parasitism rates in the three different apiaries were 34.5 % (10/29), 30.8 % (4/13) and 36.7 % (11/30), respectively. The beetle larvae were also observed in the cracks and crevices of the bottom boards of the hives. Some beetles were resting inside the empty cells of the combs. Other beetles were distributed in clusters in the shadow of the hives. They tended to avoid light and rested underneath the fallen hive debris when the inner cover of the hive was opened. However, beetle eggs were not observed in the hives. The adult lesser mealworms collected from A. c. cerana colonies were further used to examine whether they can act as reservoirs of common honeybee viruses. The adult lesser mealworms were individually surfacesterilised with 10 % sodium hypochlorite for 10 min and then used for RNA extraction with TRIzol (Invitrogen) in accordance with the manufacturer’s instructions. Total RNA (3 μg) was reverse-transcribed to cDNA by using the PrimescriptTM first-strand cDNA synthesis kit (Takara). The synthesised cDNA was subjected to PCR amplification of seven common honeybee viruses (online resource Table S1). The PCR was performed with the same amplification program as in our previous study (Li et al. 2014). The expected PCR products were directly sequenced to further confirm the RT-PCR based results. The sequences obtained in this study were submitted to GenBank, and the accession numbers were received (online resource Figure S1). The virus sequences obtained from the beetles and from the honeybees (Apis mellifera ) retrieved from GenBank were phylogenetically analysed in MEGA6 software (Tamura et al. 2013). A tagged RT-PCR was employed to detect the negative-stranded transcripts of Black queen cell virus (BQCV) and Israeli acute paralysis virus (IAPV) in adult lesser mealworms to investigate whether these honeybee viruses can replicate in the adult lesser mealworms. The total RNA and the tagged primers used in previous studies were used to synthesise the cDNA of BQCV and IAPV, respectively (Kukielka et al. 2008; Li et al. 2014). The cDNA was then cleaned with a MinElute® reaction cleanup kit (Qiagen) to remove short oligonucleotides and inorganic ions and to improve the specificity of PCR. The purified cDNA was then used to detect the replication intermediates of BQCV and IAPV; PCR was performed in accordance with previously described methods by using a tag primer (Yue and Genersch 2005) as sense primers and virusspecific primers as antisense primers (Kukielka et al. 2 (...truncated)


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Zhiguo Li, Shaokang Huang, Wei-fone Huang, Haiyang Geng, Yazhou Zhao, Meng Li, Yanping Chen, Songkun Su. A scientific note on detection of honeybee viruses in the darkling beetle (Alphitobius diaperinus, Coleoptera: Tenebrionidae), a new pest in Apis cerana cerana colonies, Apidologie, 2016, pp. 759-761, Volume 47, Issue 6, DOI: 10.1007/s13592-016-0430-1