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)