Journal of Invertebrate Pathology 137 (2016) 33–37 Contents lists available at ScienceDirect Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/jip Large pathogen screening reveals first report of Megaselia scalaris (Diptera: Phoridae) parasitizing Apis mellifera intermissa (Hymenoptera: Apidae) ⇑ Ahmed Hichem Menail a,1, Niels Piot b,1, Ivan Meeus b, Guy Smagghe b, Wahida Loucif-Ayad a,c, a Laboratory of Applied Animal Biology, Faculty of Science, Badji Mokhtar University, Annaba, Algeria b Laboratory of Agrozoology, Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium c Faculty of Medicine, Badji Mokhtar University, Annaba, Algeria article info abstract Article history: As it is most likely that global warming will also lead to a shift in pollinator-habitats northwards, the Received 22 December 2015 study of southern species becomes more and more important. Pathogen screenings in subspecies of Revised 7 April 2016 Apis mellifera capable of withstanding higher temperatures, provide an insight into future pathogen host Accepted 24 April 2016 interactions. Screenings in different climate regions also provide a global perspective on the prevalence of Available online 26 April 2016 certain pathogens. In this project, we performed a pathogen screening in Apis mellifera intermissa, a native subspecies of Keywords: Algeria in northern Africa. Colonies were sampled from different areas in the region of Annaba over a per- Apis mellifera intermissa iod of two years. Several pathogens were detected, among them Apicystis bombi, Crithidia mellificae, Colony health Pathogens Nosema ceranae, Paenibacillus larvae, Lake Sinai Virus, Sacbrood Virus and Deformed Wing Virus Viruses (DWV). Our screening also revealed a phoroid fly, Megaselia scalaris, parasitizing honey bee colonies, Phorid fly which we report here for the first time. In addition, we found DWV to be present in the adult flies and Parasitism replicating virus in the larval stages of the fly, which could indicate that M. scalaris acts as a vector of DWV. Ó 2016 Elsevier Inc. All rights reserved. 1. Introduction subspecies adapted to hotter climates; good examples are Apis mellifera intermissa, Apis mellifera sahariensis (Le Conte and Bee pollination is indispensable for the world food supply, and Navajas, 2008). Climate change will most likely also have an honey bees are the most important commercial pollinators, provid- impact on the numerous pathogens of honey bees, and the ing valuable pollination services. The most widely used honey bee interactions with their host-species (Le Conte and Navajas, 2008). species, Apis mellifera, is native to Europe and Africa, and was intro- Migration of bee species and their pathogens northwards will lead duced in other continents such as America and Australia for com- to new encounters. It is therefore of outmost importance that we mercial pollination services. Most studies on honey bees therefore understand the current pathogen-bee interactions and prevalence use the European honey bee Apis mellifera mellifera, which is most in Southern regions where the subspecies of A. mellifera, adapted widespread. However A. mellifera has a wide range of subspecies to a hot climate, is native. Pathogen screenings in different climate (Franck et al., 2000). The study of these subspecies remains impor- zones also enlarge our current insights into the omnipresence of tant, as these subspecies may harbor certain important character- different parasites and viruses in A. mellifera spp. One of the sub- istics which may be of importance in further breeding programs. species which is known for its ability to adapt to great variations It is generally accepted that global warming will lead to a shift in climate conditions and good cleaning behavior is A. mellifera in natural habitat (Root et al., 2003), and so cooler regions will intermissa (Adjlane and Haddad, 2014). This subspecies is native become hotter and may therefore be more favorable to A. mellifera to North-West Africa and occurs in Algeria, Morocco and Tunisia between the Atlas and the Mediterranean sea and Atlantic ocean coast. Studies on this subspecies of A. mellifera are scarce, and ⇑ Corresponding author at: Laboratory of Applied Animal Biology, Faculty of therefore we conducted a large pathogen screening in Algeria to Science, Badji Mokhtar University, Annaba, Algeria. assess the prevalence of pathogens in A. mellifera intermissa. During E-mail address: [email protected] (W. Loucif-Ayad). our screening we screened for mites, microsporidia, protozoa and 1 Both authors contributed equally to this work. http://dx.doi.org/10.1016/j.jip.2016.04.007 0022-2011/Ó 2016 Elsevier Inc. All rights reserved. 34 A.H. Menail et al. / Journal of Invertebrate Pathology 137 (2016) 33–37 bacterial pathogens and viruses, as Apicystis bombi, Crithidia Honey bee larvae were pooled (n = 10) and crushed using mor- mellificae, Nosema ceranae, Paenibacillus larvae and Israeli Acute tar and pestle in 4.5 ml of RLT buffer supplemented with Paralysis Virus (IAPV), Slow Bee Paralysis Virus (SBPV), Deformed b-mercaptoethanol (100/1; V/V) (RNeasy Mini Kit, Qiagen) and Wing Virus (DWV), Kashmir Bee Virus (KBV), Lake Sinai Virus stored at À80 °C until extraction. DNA extraction was performed (LSV), Sacbrood Virus (SBV), Acute Bee Paralysis Virus (ABPV) and as described above. The larval DNA was used to detect the causal Chronic Bee Paralysis Virus (CBPV) that are all described to occur agents of American foulbrood and European foulbrood. as pathogens in A. mellifera spp. We also observed bee parasitism For M. scalaris, larval and adult samples were surface sterilized by a phoroid fly, Megaselia scalaris (Diptera: Phoridae). M. scalaris with 30% bleach before DNA and RNA extraction. DNA extraction of is a cosmopolitan and synanthropic scuttle fly, which acts as detri- larvae and adult flies was done for single adults and pools of 3 lar- vore, parasite, facultative parasite and parasitoid. M. scalaris is vae. These were crushed in 400 ll of lysis buffer G and 40 ll of pro- known as a laboratory pest, infesting laboratory cultures of inver- teinase K, then the samples were incubated for 1 h at 52 °C and tebrates such as cockroaches (Miller, 1979; Robinson, 1975), flies further processed according to the manufacturer’s protocol (Invi- (Zwart et al., 2005), triatomines (Costa et al., 2007), mantids sorb spin tissue Mini Kit; Stratec). RNA extraction M. scalaris was (Koch et al., 2013) and acarine ticks (Miranda-Miranda et al., done according to the manufacturer’s protocol’ (RNeasy Mini Kit; 2011). Moreover, Macieira et al. (1983) and Rocha et al. (1984) Qiagen), RNA was extracted for single adults and pools of 3 larvae. reported the possibility of M. scalaris to act as a parasitoid in bee- hives of Melliponinae stingless bee species and colonies of 2.3. PCR European honey bees, however to date no records exist reporting this. We here report our large pathogen screening of A. mellifera cDNA was synthesized using Oligo-dT primers and SuperScript intermissa in Algeria. We think these data will contribute to the II Reverse Transcriptase (Life Technologies; Merelbeke, Belgium) growing knowledge concerning bee pathogens and their global according to the manufacturer’s instructions. The cDNA was stored spread and prevalence in different climate regions. Our data also at À20 °C, until further use. provide information about pathogen influence in a subspecies of Standard PCR was used for the detection of protozoa, fungi, bac- the European honey bee, known for its good cleaning behavior. teria, Dicistroviridae and DWV. Protocols and primer sequences are summarized in Supplementary material-Tables S1 and S2. PCR 2. Material and methods products were visualized on a 1.5% Agarose gel and stained with ethidium bromide. Detection of other viruses (SBV, LSV, SBPV 2.1. Sampling of honey bees and M. scalaris flies and CBPV) and tracheal mite (Acarapis woodi) was done with CFX96TM Real-Time PCR detection system (Bio-Rad, Hercules, CA). l l Ò Asymptomatic hives from 18 apiaries located in 12 different Each reaction (20 l) contained: 10 l of GoTaq qPCR Master l l geographical locations (Sidi Amar; El Hadjar; El bouni; Annaba; Mix, (Promega, Madison, WI), 1 l (10 M) of forward primer, l l l Seraidi; Ain berda; Cheurfa; Eulma; Berrahel; Oued el Aneb; Treat; 1 l (10 M) of reverse primer and 8 l of template DNA for A. Chetaibi) were sampled in the region of Annaba (36°5400 N and woodi detection and 1/10 diluted cDNA for virus detection (Supple- 7°4600 E), the extreme North-East of Algeria (Supplementary mentary material-Table S3). Nuclease free water was used as a no Fig. S1). At each locality, 2–3 apiaries were selected and bees were template control and samples with Cq values above 35 were sampled from 2 to 4 hives. Per hive, an average of 60 bees and 10 regarded as negative. Several positive samples of each detected larvae was sampled. This was done in two sampling efforts, per- pathogen were sent for Sanger sequencing (LGC Genomics, Lucken- formed in 2013 and 2014, and both were conducted in the period walde Germany) in order to confirm the identity of the pathogens. autumn to winter. For M. scalaris, during our experiment when honey bees were 2.4. Negative strand detection of DWV collected from hives and kept in closed boxes in the laboratory, fly parasitism was observed with flies emerging from the bees. Negative strand detection of DWV was performed on the phorid We observed this several times during our two years sampling per- fly RNA as proof of a true infection. A multiplex ligation-dependent iod. Larvae and adult flies, emerged from dead bees, were collected probe amplification (MLPA) was performed on the RNA extracts and stored in 90% ethanol until further use. (described above) of larvae and adults as described by De Smet et al. (2012).