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Large pathogen screening reveals first report of Megaselia scalaris (Diptera: Phoridae) parasitizing Apis mellifera intermissa (Hymenoptera: Apidae)
Journal of Invertebrate Pathology 137 (2016) 33–37
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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 b c
Laboratory of Applied Animal Biology, Faculty of Science, Badji Mokhtar University, Annaba, Algeria Laboratory of Agrozoology, Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium Faculty of Medicine, Badji Mokhtar University, Annaba, Algeria
a r t i c l e
i n f o
Article history: Received 22 December 2015 Revised 7 April 2016 Accepted 24 April 2016 Available online 26 April 2016 Keywords: Apis mellifera intermissa Colony health Pathogens Viruses Phorid fly Parasitism
a b s t r a c t As it is most likely that global warming will also lead to a shift in pollinator-habitats northwards, the study of southern species becomes more and more important. Pathogen screenings in subspecies of Apis mellifera capable of withstanding higher temperatures, provide an insight into future pathogen host interactions. Screenings in different climate regions also provide a global perspective on the prevalence of certain pathogens. In this project, we performed a pathogen screening in Apis mellifera intermissa, a native subspecies of Algeria in northern Africa. Colonies were sampled from different areas in the region of Annaba over a period of two years. Several pathogens were detected, among them Apicystis bombi, Crithidia mellificae, Nosema ceranae, Paenibacillus larvae, Lake Sinai Virus, Sacbrood Virus and Deformed Wing Virus (DWV). Our screening also revealed a phoroid fly, Megaselia scalaris, parasitizing honey bee colonies, which we report here for the first time. In addition, we found DWV to be present in the adult flies and 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 Bee pollination is indispensable for the world food supply, and honey bees are the most important commercial pollinators, providing valuable pollination services. The most widely used honey bee species, Apis mellifera, is native to Europe and Africa, and was introduced in other continents such as America and Australia for commercial pollination services. Most studies on honey bees therefore use the European honey bee Apis mellifera mellifera, which is most widespread. However A. mellifera has a wide range of subspecies (Franck et al., 2000). The study of these subspecies remains important, as these subspecies may harbor certain important characteristics which may be of importance in further breeding programs. It is generally accepted that global warming will lead to a shift in natural habitat (Root et al., 2003), and so cooler regions will become hotter and may therefore be more favorable to A. mellifera
⇑ Corresponding author at: Laboratory of Applied Animal Biology, Faculty of Science, Badji Mokhtar University, Annaba, Algeria. E-mail address: [email protected] (W. Loucif-Ayad). 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.
subspecies adapted to hotter climates; good examples are Apis mellifera intermissa, Apis mellifera sahariensis (Le Conte and Navajas, 2008). Climate change will most likely also have an impact on the numerous pathogens of honey bees, and the interactions with their host-species (Le Conte and Navajas, 2008). Migration of bee species and their pathogens northwards will lead to new encounters. It is therefore of outmost importance that we understand the current pathogen-bee interactions and prevalence in Southern regions where the subspecies of A. mellifera, adapted to a hot climate, is native. Pathogen screenings in different climate zones also enlarge our current insights into the omnipresence of different parasites and viruses in A. mellifera spp. One of the subspecies which is known for its ability to adapt to great variations in climate conditions and good cleaning behavior is A. mellifera intermissa (Adjlane and Haddad, 2014). This subspecies is native 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 therefore we conducted a large pathogen screening in Algeria to assess the prevalence of pathogens in A. mellifera intermissa. During our screening we screened for mites, microsporidia, protozoa and
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bacterial pathogens and viruses, as Apicystis bombi, Crithidia mellificae, Nosema ceranae, Paenibacillus larvae and Israeli Acute Paralysis Virus (IAPV), Slow Bee Paralysis Virus (SBPV), Deformed Wing Virus (DWV), Kashmir Bee Virus (KBV), Lake Sinai Virus (LSV), Sacbrood Virus (SBV), Acute Bee Paralysis Virus (ABPV) and Chronic Bee Paralysis Virus (CBPV) that are all described to occur as pathogens in A. mellifera spp. We also observed bee parasitism by a phoroid fly, Megaselia scalaris (Diptera: Phoridae). M. scalaris is a cosmopolitan and synanthropic scuttle fly, which acts as detrivore, parasite, facultative parasite and parasitoid. M. scalaris is known as a laboratory pest, infesting laboratory cultures of invertebrates such as cockroaches (Miller, 1979; Robinson, 1975), flies (Zwart et al., 2005), triatomines (Costa et al., 2007), mantids (Koch et al., 2013) and acarine ticks (Miranda-Miranda et al., 2011). Moreover, Macieira et al. (1983) and Rocha et al. (1984) reported the possibility of M. scalaris to act as a parasitoid in beehives of Melliponinae stingless bee species and colonies of European honey bees, however to date no records exist reporting this. We here report our large pathogen screening of A. mellifera intermissa in Algeria. We think these data will contribute to the growing knowledge concerning bee pathogens and their global spread and prevalence in different climate regions. Our data also provide information about pathogen influence in a subspecies of the European honey bee, known for its good cleaning behavior. 2. Material and methods 2.1. Sampling of honey bees and M. scalaris flies Asymptomatic hives from 18 apiaries located in 12 different geographical locations (Sidi Amar; El Hadjar; El bouni; Annaba; Seraidi; Ain berda; Cheurfa; Eulma; Berrahel; Oued el Aneb; Treat; Chetaibi) were sampled in the region of Annaba (36°540 0 N and 7°460 0 E), the extreme North-East of Algeria (Supplementary Fig. S1). At each locality, 2–3 apiaries were selected and bees were sampled from 2 to 4 hives. Per hive, an average of 60 bees and 10 larvae was sampled. This was done in two sampling efforts, performed in 2013 and 2014, and both were conducted in the period autumn to winter. For M. scalaris, during our experiment when honey bees were collected from hives and kept in closed boxes in the laboratory, fly parasitism was observed with flies emerging from the bees. We observed this several times during our two years sampling period. Larvae and adult flies, emerged from dead bees, were collected and stored in 90% ethanol until further use. 2.2. Nucleic acids extraction For honey bees, 30 bees were pooled and crushed using mortar and pestle in 9 ml of RLT buffer supplemented with b-mercaptoethanol (100/1; v/v) (RNeasy Mini Kit; Qiagen, Venlo, the Netherlands). After crushing, the exoskeletons were discarded and the liquid was centrifuged (2 min, 2000g). Then 0.5 ml supernatant was added to 1 ml of RLT buffer and stored at 80 °C until extraction. Before extraction, samples were thawed in an incubator at 37 °C for 10 min with shaking (300 rpm). After incubation, samples were centrifuged during 2 min at 2000g. For DNA extraction 200 ll of supernatant was mixed with 400 ll Lysis buffer G and 40 ll Proteinase K, and incubated for 1 h at 52 °C with shaking (400 rpm). Further extraction was done according to the manufacturer’s protocol (InvisorbÒ Spin Tissue Mini Kit, Protocol 1; Stratec, Berlin, Germany). RNA extractions were done starting with 200 ll of supernatant which was added to 200 ll of 70% ethanol. Further extraction was done according to the manufacturer’s protocol (RNeasy Mini Kit; Qiagen).
Honey bee larvae were pooled (n = 10) and crushed using mortar and pestle in 4.5 ml of RLT buffer supplemented with b-mercaptoethanol (100/1; V/V) (RNeasy Mini Kit, Qiagen) and stored at 80 °C until extraction. DNA extraction was performed as described above. The larval DNA was used to detect the causal agents of American foulbrood and European foulbrood. For M. scalaris, larval and adult samples were surface sterilized with 30% bleach before DNA and RNA extraction. DNA extraction of larvae and adult flies was done for single adults and pools of 3 larvae. These were crushed in 400 ll of lysis buffer G and 40 ll of proteinase K, then the samples were incubated for 1 h at 52 °C and further processed according to the manufacturer’s protocol (Invisorb spin tissue Mini Kit; Stratec). RNA extraction M. scalaris was done according to the manufacturer’s protocol’ (RNeasy Mini Kit; Qiagen), RNA was extracted for single adults and pools of 3 larvae. 2.3. PCR cDNA was synthesized using Oligo-dT primers and SuperScript II Reverse Transcriptase (Life Technologies; Merelbeke, Belgium) according to the manufacturer’s instructions. The cDNA was stored at 20 °C, until further use. Standard PCR was used for the detection of protozoa, fungi, bacteria, Dicistroviridae and DWV. Protocols and primer sequences are summarized in Supplementary material-Tables S1 and S2. PCR products were visualized on a 1.5% Agarose gel and stained with ethidium bromide. Detection of other viruses (SBV, LSV, SBPV and CBPV) and tracheal mite (Acarapis woodi) was done with CFX96TM Real-Time PCR detection system (Bio-Rad, Hercules, CA). Each reaction (20 ll) contained: 10 ll of GoTaqÒ qPCR Master Mix, (Promega, Madison, WI), 1 ll (10 lM) of forward primer, 1 ll (10 lM) of reverse primer and 8 ll of template DNA for A. woodi detection and 1/10 diluted cDNA for virus detection (Supplementary material-Table S3). Nuclease free water was used as a no template control and samples with Cq values above 35 were regarded as negative. Several positive samples of each detected pathogen were sent for Sanger sequencing (LGC Genomics, Luckenwalde Germany) in order to confirm the identity of the pathogens. 2.4. Negative strand detection of DWV Negative strand detection of DWV was performed on the phorid fly RNA as proof of a true infection. A multiplex ligation-dependent probe amplification (MLPA) was performed on the RNA extracts (described above) of larvae and adults as described by De Smet et al. (2012). Probes designed for the strand-specific detection of DWV as published by De Smet et al. (2012) were used to detect the negative strand. All the MLPA reagents were obtained from MRC-Holland (Amsterdam, the Netherlands). 3. Results and discussion In our large scale pathogen screening, we detected N. ceranae and C. mellificae as the most prevalent pathogens, followed by P. larvae and A. bombi. DWV was the most abundant virus in our screening, followed by LSV and SBV. Other viruses that we screened for (i.e. ABPV, IAPV, KBV, SBPV and CBPV), were not detected (Table 1). The microsporidian N. ceranae appeared to be present worldwide, including in northern Africa (Higes et al., 2009) where it was detected for the first time in Algeria. Our study showed a high rate of N. ceranae infections (81–86%), although the colonies which were sampled lacked the typical symptoms, i.e. high colony losses, which are often reported in highly infested apiaries (Genersch and Aubert, 2010; Genersch et al., 2010). According to Runckel et al.
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A.H. Menail et al. / Journal of Invertebrate Pathology 137 (2016) 33–37 Table 1 Infection rates (%) of honey bee pathogens in Algerian apiaries during autumn/winter 2013 and 2014.
Microsporidia Protozoa
Tracheal mites Bacteria⁄
Viruses
⁄
Pathogens
Autumn/ winter 2013 (Adults: n = 44) (Larvae: n = 39)⁄
Autumn/winter 2014 (Adults: n = 21) (Larvae: n = 20)⁄
Nosema ceranae Crithidia mellificae Apicystis bombi Acarapis woodi
86.36
80.95
38.64
47.62
Paenibacillus larvae Melissococcus plutonius DWV ABPV IAPV KBV SBV LSV SBPV CBPV
2.27 0
0 0
2.56
15
0 45.45 0 0 0 0 6.82 0 0
0 42.86 0 0 0 14.29 4.76 0 0
Number of larvae used to detect bacteria.
(2014), C. mellificae is associated to N. ceranae infections and their co-infection has a negative impact on the longevity of honey bee colonies. In agreement, in our screening we found that 87.1% of the colonies infected with C. mellificae were also infected with N. ceranae. C. mellificae has also been correlated with colony losses in the USA and Belgium (Cornman et al., 2012; Ravoet et al., 2013). In the present study, C. mellificae was detected at a high rate (39–48%) in A. mellifera intermissa, however as for N. ceranae, no colony losses were observed in C. mellificae-infected hives. Another protozoan pathogen, Apicystis bombi, is a general parasite of Bombus species (Lipa and Triggiani, 1996; Rutrecht and Brown, 2008). However, it has also been detected in A. mellifera at a very low rate. Our study confirmed the presence of this protozoan parasite in honey bees and this for the first time in A. mellifera intermissa. These results reflect this pathogen’s worldwide dispersion in honey bees and its arrival in Africa.
A
In our screening we also investigated bacterial pathogens and confirmed the presence of P. larvae in Algeria and North Africa in general (Hamdi et al., 2013; Adjlane et al., 2012). Adjlane et al. (2012) reported a prevalence of 45% of the screened colonies in Middle-North Algeria, based on microbiological and chemical techniques. We found a prevalence of P. larvae which was much lower (2.6% and 15% for the years 2013 and 2014, respectively). This could be explained by the different locations which were sampled, as our sampling was situated more in the north of the country. In contrast, M. plutonius, was not detected in our screening, although its presence has been reported in Algeria and Africa in general (Ellis and Munn, 2005). The absence of the tracheal mite A. woodi in our samples confirmed earlier studies (Ellis and Munn, 2005). Its absence may be related to the high amount of sunlight which inhibits its development and to the high level of hygienic behavior as displayed by A. mellifera intermissa (de Guzman, unpub. Sammataro et al., 2013; Kefuss, 1995). Among the viruses analyzed, only DWV, LSV and SBV were present in our screening. These findings reflect a relative low viral diversity in A. mellifera intermissa in contrast to other honey bee populations elsewhere (Antúnez et al., 2012; Bacandritsos et al., 2010; Garrido-Bailón et al., 2010; Petrovic´ et al., 2013). The high prevalence of DWV (45% in 2013 and 43% in 2014) was expected as wing deformities were observed in some apiaries, and a similar prevalence of DWV has been reported in the North of Algeria (Loucif-Ayad et al., 2013). In our screening, we detected SBV at a low rate (14.3%) only in our 2014 sampling, which could indicate that SBV is spreading in Algeria. This virus affects larval and adult stages without any clear pathological symptoms (Berenyi et al., 2006), and is one of the most widespread viruses (Ellis and Munn, 2005). Here we also report for the first time detection of LSV in A. mellifera intermissa, although at a low prevalence of 6.9% and 4.8% in our 2013 and 2014 sampling, respectively. During our sampling campaign we also did an interesting observation. We noticed that several bees were parasitized by a phorid fly species, and dipteran larvae emerged from these bees leaving the exoskeleton as remnants (Fig. 1, Panel B). Morphological as well as molecular species determination identified the phorid fly as M. scalaris. The detection of M. scalaris in the Algerian honey bee is to our knowledge the first record of honey bee parasitism by this fly species. Our findings confirm earlier speculations about the possible parasitism of A. mellifera by this phorid fly (Rocha
B
Fig. 1. A: Adult specimens of the phoroid fly, Megaselia scalaris, parasitizing A. mellifera intermissa. B: Top: arrows indicate larvae of M. scalaris, emerging from bee individuals of A. mellifera intermissa; Bottom: arrows indicate pupae of M. scalaris.
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et al., 1984). This discovery sheds light on the opportunistic behavior of this insect, i.e. feeding on wide array of invertebrate species. As M. scalaris was parasitizing a hive which was positive for DWV, we screened the larval and adult stages of the fly for this virus. We found the virus to be present in both adults and larvae. To clarify if both larvae and adults were truly infected with DWV, the detection of a replication intermediate, i.e. the negative strand of the virus, was performed. This negative strand of DWV could be detected in the larvae but not in the adult flies. These results indicate that DWV can truly infect M. scalaris, at least in the larval stages. To our knowledge this is also the first time this virus is reported to be present in M. scalaris and shown to truly infect a dipteran species. We believe that our findings further strengthen the hypothesis that flies of the Phoridae family could act as a vector for DWV, as several species of this family have been associated with this honey bee virus. Apocephalus borealis, was recorded as parasite of honey bee colonies in the USA, this species was also often infected with DWV (Core et al., 2012). A. borealis has also been detected in Belgium at a high prevalence of 31% (Ravoet et al., 2013), indicating this fly is also parasitizing honey bees outside the USA. More recently, a third species of phorid fly, Megaselia rufipes, has been identified as a facultative parasite of honey bees in Italy (Dutto and Ferrazzi, 2014). Although no DWV was found in M. rufipes, as the flies were not examined for viral presence, it is remarkable that these flies were also found in a colony which was affected by DWV. The detection of the negative strand of DWV by the strandspecific detection technique MLPA (De Smet et al., 2012) provides proof for a true infection of M. scalaris larvae, which is a strong indication that this fly could indeed act as a vector of DWV. Although we found no proof of DWV replication in the adult flies, it should be mentioned that only two adult specimens were screened, one of these was positive for DWV but no negative strand could be detected. It could be that we missed the detection of negative strand in adult flies, as our sample size was small. To fully confirm this hypothesis, where phorid flies act as DWV vector, further research is needed. These findings make the Phoridae a family of growing interest in the comprehensive studies carried out around the world to give answers to the colony collapse disorder and more generally to honey bee health. Aside from honey bees, the broad host range of these phorid flies also makes them a considerable danger to wild pollinators, as reported by Core et al. (2012). We therefore suggest that future pathogens screenings should also include Phoridae, and we encourage further research on the role of this fly family in pollinator health. 4. Conclusion Our study in A. mellifera intermissa, a subspecies of A. mellifera, adapted to a hot climate, revealed the presence of N. ceranae, P. larvae and DWV. Furthermore we present the first report of several pathogens, i.e. A. bombi, C. mellificae, LSV and SBV, in this honey bee subspecies. In this study we also report for the first time the parasitism of a phorid fly species, M. scalaris, in honey bees, and in addition, we found DWV to be present in adult flies and actively replicating in the fly larvae. We therefore suggest further research is needed toward the role of Phoridae species as a possible vector for DWV and in pollinator health in general. Acknowledgment The authors would like to thank the beekeepers for participating in this study. This research was supported by the Fund for Scientific Research (FWO-Vlaanderen, Brussels).
Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jip.2016.04.007. References Adjlane, N., Kechih, S., Doumandji, S., Haddad, N., 2012. Survey of American foulbrood in Apis mellifera intermissa colonies in mid-north region of Algeria. Uludag Bee J. 12 (3), 98–105. Adjlane, N., Haddad, N., 2014. The first data on hygienic behavior of Apis mellifera intermissa in Algeria. J. Biol. Earth Sci. 4, 1–5. Antúnez, K., Anido, M., Garrido-Bailón, E., Botías, C., Zunino, P., Martínez-Salvador, A., Martín-Hernández, R., Higes, M., 2012. Low prevalence of honey bee viruses in Spain during 2006 and 2007. Res. Vet. Sci. 93, 1441–1445. Bacandritsos, N., Granato, A., Budge, G., Papanastasiou, I., Roinioti, E., Caldon, M., Falcaro, C., Gallina, A., Mutinelli, F., 2010. Sudden deaths and colony population decline in Greek honey bee colonies. J. Invert. Pathol. 105, 335–340. 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Oviposition rate and relative viability of descendants from couples of Megaselia scalaris (Diptera: Phoridae) reared in different experimental conditions. Rev. Bras. Biol. 43, 223–228. Miller, P.L., 1979. Intraspecific phorid phoresy. Entomol. Monthly Mag. 114, 211– 214. Miranda-Miranda, E., Cossio-Bayugar, R., Martinez-Ibanez, F., Bautista-Garfias, C.R., 2011. Megaselia scalaris reared on Rhipicephalus (Boophilus) microplus laboratory cultures. Med. Vet. Entomol. 25, 344–347. Petrovic´, T., Vidanovic´, D., Stojanov, I., Lupulovic´, D., Lazic´, G., Plavša, N., Lazic´, S., 2013. First molecular detection of six honeybee viruses in Serbian apiaries. In: Proceedings of the 10th International Symposium. Modern Trends in Livestock Production, October 2–4 2013.
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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 b c
Laboratory of Applied Animal Biology, Faculty of Science, Badji Mokhtar University, Annaba, Algeria Laboratory of Agrozoology, Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium Faculty of Medicine, Badji Mokhtar University, Annaba, Algeria
a r t i c l e
i n f o
Article history: Received 22 December 2015 Revised 7 April 2016 Accepted 24 April 2016 Available online 26 April 2016 Keywords: Apis mellifera intermissa Colony health Pathogens Viruses Phorid fly Parasitism
a b s t r a c t As it is most likely that global warming will also lead to a shift in pollinator-habitats northwards, the study of southern species becomes more and more important. Pathogen screenings in subspecies of Apis mellifera capable of withstanding higher temperatures, provide an insight into future pathogen host interactions. Screenings in different climate regions also provide a global perspective on the prevalence of certain pathogens. In this project, we performed a pathogen screening in Apis mellifera intermissa, a native subspecies of Algeria in northern Africa. Colonies were sampled from different areas in the region of Annaba over a period of two years. Several pathogens were detected, among them Apicystis bombi, Crithidia mellificae, Nosema ceranae, Paenibacillus larvae, Lake Sinai Virus, Sacbrood Virus and Deformed Wing Virus (DWV). Our screening also revealed a phoroid fly, Megaselia scalaris, parasitizing honey bee colonies, which we report here for the first time. In addition, we found DWV to be present in the adult flies and 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 Bee pollination is indispensable for the world food supply, and honey bees are the most important commercial pollinators, providing valuable pollination services. The most widely used honey bee species, Apis mellifera, is native to Europe and Africa, and was introduced in other continents such as America and Australia for commercial pollination services. Most studies on honey bees therefore use the European honey bee Apis mellifera mellifera, which is most widespread. However A. mellifera has a wide range of subspecies (Franck et al., 2000). The study of these subspecies remains important, as these subspecies may harbor certain important characteristics which may be of importance in further breeding programs. It is generally accepted that global warming will lead to a shift in natural habitat (Root et al., 2003), and so cooler regions will become hotter and may therefore be more favorable to A. mellifera
⇑ Corresponding author at: Laboratory of Applied Animal Biology, Faculty of Science, Badji Mokhtar University, Annaba, Algeria. E-mail address: [email protected] (W. Loucif-Ayad). 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.
subspecies adapted to hotter climates; good examples are Apis mellifera intermissa, Apis mellifera sahariensis (Le Conte and Navajas, 2008). Climate change will most likely also have an impact on the numerous pathogens of honey bees, and the interactions with their host-species (Le Conte and Navajas, 2008). Migration of bee species and their pathogens northwards will lead to new encounters. It is therefore of outmost importance that we understand the current pathogen-bee interactions and prevalence in Southern regions where the subspecies of A. mellifera, adapted to a hot climate, is native. Pathogen screenings in different climate zones also enlarge our current insights into the omnipresence of different parasites and viruses in A. mellifera spp. One of the subspecies which is known for its ability to adapt to great variations in climate conditions and good cleaning behavior is A. mellifera intermissa (Adjlane and Haddad, 2014). This subspecies is native 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 therefore we conducted a large pathogen screening in Algeria to assess the prevalence of pathogens in A. mellifera intermissa. During our screening we screened for mites, microsporidia, protozoa and
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bacterial pathogens and viruses, as Apicystis bombi, Crithidia mellificae, Nosema ceranae, Paenibacillus larvae and Israeli Acute Paralysis Virus (IAPV), Slow Bee Paralysis Virus (SBPV), Deformed Wing Virus (DWV), Kashmir Bee Virus (KBV), Lake Sinai Virus (LSV), Sacbrood Virus (SBV), Acute Bee Paralysis Virus (ABPV) and Chronic Bee Paralysis Virus (CBPV) that are all described to occur as pathogens in A. mellifera spp. We also observed bee parasitism by a phoroid fly, Megaselia scalaris (Diptera: Phoridae). M. scalaris is a cosmopolitan and synanthropic scuttle fly, which acts as detrivore, parasite, facultative parasite and parasitoid. M. scalaris is known as a laboratory pest, infesting laboratory cultures of invertebrates such as cockroaches (Miller, 1979; Robinson, 1975), flies (Zwart et al., 2005), triatomines (Costa et al., 2007), mantids (Koch et al., 2013) and acarine ticks (Miranda-Miranda et al., 2011). Moreover, Macieira et al. (1983) and Rocha et al. (1984) reported the possibility of M. scalaris to act as a parasitoid in beehives of Melliponinae stingless bee species and colonies of European honey bees, however to date no records exist reporting this. We here report our large pathogen screening of A. mellifera intermissa in Algeria. We think these data will contribute to the growing knowledge concerning bee pathogens and their global spread and prevalence in different climate regions. Our data also provide information about pathogen influence in a subspecies of the European honey bee, known for its good cleaning behavior. 2. Material and methods 2.1. Sampling of honey bees and M. scalaris flies Asymptomatic hives from 18 apiaries located in 12 different geographical locations (Sidi Amar; El Hadjar; El bouni; Annaba; Seraidi; Ain berda; Cheurfa; Eulma; Berrahel; Oued el Aneb; Treat; Chetaibi) were sampled in the region of Annaba (36°540 0 N and 7°460 0 E), the extreme North-East of Algeria (Supplementary Fig. S1). At each locality, 2–3 apiaries were selected and bees were sampled from 2 to 4 hives. Per hive, an average of 60 bees and 10 larvae was sampled. This was done in two sampling efforts, performed in 2013 and 2014, and both were conducted in the period autumn to winter. For M. scalaris, during our experiment when honey bees were collected from hives and kept in closed boxes in the laboratory, fly parasitism was observed with flies emerging from the bees. We observed this several times during our two years sampling period. Larvae and adult flies, emerged from dead bees, were collected and stored in 90% ethanol until further use. 2.2. Nucleic acids extraction For honey bees, 30 bees were pooled and crushed using mortar and pestle in 9 ml of RLT buffer supplemented with b-mercaptoethanol (100/1; v/v) (RNeasy Mini Kit; Qiagen, Venlo, the Netherlands). After crushing, the exoskeletons were discarded and the liquid was centrifuged (2 min, 2000g). Then 0.5 ml supernatant was added to 1 ml of RLT buffer and stored at 80 °C until extraction. Before extraction, samples were thawed in an incubator at 37 °C for 10 min with shaking (300 rpm). After incubation, samples were centrifuged during 2 min at 2000g. For DNA extraction 200 ll of supernatant was mixed with 400 ll Lysis buffer G and 40 ll Proteinase K, and incubated for 1 h at 52 °C with shaking (400 rpm). Further extraction was done according to the manufacturer’s protocol (InvisorbÒ Spin Tissue Mini Kit, Protocol 1; Stratec, Berlin, Germany). RNA extractions were done starting with 200 ll of supernatant which was added to 200 ll of 70% ethanol. Further extraction was done according to the manufacturer’s protocol (RNeasy Mini Kit; Qiagen).
Honey bee larvae were pooled (n = 10) and crushed using mortar and pestle in 4.5 ml of RLT buffer supplemented with b-mercaptoethanol (100/1; V/V) (RNeasy Mini Kit, Qiagen) and stored at 80 °C until extraction. DNA extraction was performed as described above. The larval DNA was used to detect the causal agents of American foulbrood and European foulbrood. For M. scalaris, larval and adult samples were surface sterilized with 30% bleach before DNA and RNA extraction. DNA extraction of larvae and adult flies was done for single adults and pools of 3 larvae. These were crushed in 400 ll of lysis buffer G and 40 ll of proteinase K, then the samples were incubated for 1 h at 52 °C and further processed according to the manufacturer’s protocol (Invisorb spin tissue Mini Kit; Stratec). RNA extraction M. scalaris was done according to the manufacturer’s protocol’ (RNeasy Mini Kit; Qiagen), RNA was extracted for single adults and pools of 3 larvae. 2.3. PCR cDNA was synthesized using Oligo-dT primers and SuperScript II Reverse Transcriptase (Life Technologies; Merelbeke, Belgium) according to the manufacturer’s instructions. The cDNA was stored at 20 °C, until further use. Standard PCR was used for the detection of protozoa, fungi, bacteria, Dicistroviridae and DWV. Protocols and primer sequences are summarized in Supplementary material-Tables S1 and S2. PCR products were visualized on a 1.5% Agarose gel and stained with ethidium bromide. Detection of other viruses (SBV, LSV, SBPV and CBPV) and tracheal mite (Acarapis woodi) was done with CFX96TM Real-Time PCR detection system (Bio-Rad, Hercules, CA). Each reaction (20 ll) contained: 10 ll of GoTaqÒ qPCR Master Mix, (Promega, Madison, WI), 1 ll (10 lM) of forward primer, 1 ll (10 lM) of reverse primer and 8 ll of template DNA for A. woodi detection and 1/10 diluted cDNA for virus detection (Supplementary material-Table S3). Nuclease free water was used as a no template control and samples with Cq values above 35 were regarded as negative. Several positive samples of each detected pathogen were sent for Sanger sequencing (LGC Genomics, Luckenwalde Germany) in order to confirm the identity of the pathogens. 2.4. Negative strand detection of DWV Negative strand detection of DWV was performed on the phorid fly RNA as proof of a true infection. A multiplex ligation-dependent probe amplification (MLPA) was performed on the RNA extracts (described above) of larvae and adults as described by De Smet et al. (2012). Probes designed for the strand-specific detection of DWV as published by De Smet et al. (2012) were used to detect the negative strand. All the MLPA reagents were obtained from MRC-Holland (Amsterdam, the Netherlands). 3. Results and discussion In our large scale pathogen screening, we detected N. ceranae and C. mellificae as the most prevalent pathogens, followed by P. larvae and A. bombi. DWV was the most abundant virus in our screening, followed by LSV and SBV. Other viruses that we screened for (i.e. ABPV, IAPV, KBV, SBPV and CBPV), were not detected (Table 1). The microsporidian N. ceranae appeared to be present worldwide, including in northern Africa (Higes et al., 2009) where it was detected for the first time in Algeria. Our study showed a high rate of N. ceranae infections (81–86%), although the colonies which were sampled lacked the typical symptoms, i.e. high colony losses, which are often reported in highly infested apiaries (Genersch and Aubert, 2010; Genersch et al., 2010). According to Runckel et al.
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A.H. Menail et al. / Journal of Invertebrate Pathology 137 (2016) 33–37 Table 1 Infection rates (%) of honey bee pathogens in Algerian apiaries during autumn/winter 2013 and 2014.
Microsporidia Protozoa
Tracheal mites Bacteria⁄
Viruses
⁄
Pathogens
Autumn/ winter 2013 (Adults: n = 44) (Larvae: n = 39)⁄
Autumn/winter 2014 (Adults: n = 21) (Larvae: n = 20)⁄
Nosema ceranae Crithidia mellificae Apicystis bombi Acarapis woodi
86.36
80.95
38.64
47.62
Paenibacillus larvae Melissococcus plutonius DWV ABPV IAPV KBV SBV LSV SBPV CBPV
2.27 0
0 0
2.56
15
0 45.45 0 0 0 0 6.82 0 0
0 42.86 0 0 0 14.29 4.76 0 0
Number of larvae used to detect bacteria.
(2014), C. mellificae is associated to N. ceranae infections and their co-infection has a negative impact on the longevity of honey bee colonies. In agreement, in our screening we found that 87.1% of the colonies infected with C. mellificae were also infected with N. ceranae. C. mellificae has also been correlated with colony losses in the USA and Belgium (Cornman et al., 2012; Ravoet et al., 2013). In the present study, C. mellificae was detected at a high rate (39–48%) in A. mellifera intermissa, however as for N. ceranae, no colony losses were observed in C. mellificae-infected hives. Another protozoan pathogen, Apicystis bombi, is a general parasite of Bombus species (Lipa and Triggiani, 1996; Rutrecht and Brown, 2008). However, it has also been detected in A. mellifera at a very low rate. Our study confirmed the presence of this protozoan parasite in honey bees and this for the first time in A. mellifera intermissa. These results reflect this pathogen’s worldwide dispersion in honey bees and its arrival in Africa.
A
In our screening we also investigated bacterial pathogens and confirmed the presence of P. larvae in Algeria and North Africa in general (Hamdi et al., 2013; Adjlane et al., 2012). Adjlane et al. (2012) reported a prevalence of 45% of the screened colonies in Middle-North Algeria, based on microbiological and chemical techniques. We found a prevalence of P. larvae which was much lower (2.6% and 15% for the years 2013 and 2014, respectively). This could be explained by the different locations which were sampled, as our sampling was situated more in the north of the country. In contrast, M. plutonius, was not detected in our screening, although its presence has been reported in Algeria and Africa in general (Ellis and Munn, 2005). The absence of the tracheal mite A. woodi in our samples confirmed earlier studies (Ellis and Munn, 2005). Its absence may be related to the high amount of sunlight which inhibits its development and to the high level of hygienic behavior as displayed by A. mellifera intermissa (de Guzman, unpub. Sammataro et al., 2013; Kefuss, 1995). Among the viruses analyzed, only DWV, LSV and SBV were present in our screening. These findings reflect a relative low viral diversity in A. mellifera intermissa in contrast to other honey bee populations elsewhere (Antúnez et al., 2012; Bacandritsos et al., 2010; Garrido-Bailón et al., 2010; Petrovic´ et al., 2013). The high prevalence of DWV (45% in 2013 and 43% in 2014) was expected as wing deformities were observed in some apiaries, and a similar prevalence of DWV has been reported in the North of Algeria (Loucif-Ayad et al., 2013). In our screening, we detected SBV at a low rate (14.3%) only in our 2014 sampling, which could indicate that SBV is spreading in Algeria. This virus affects larval and adult stages without any clear pathological symptoms (Berenyi et al., 2006), and is one of the most widespread viruses (Ellis and Munn, 2005). Here we also report for the first time detection of LSV in A. mellifera intermissa, although at a low prevalence of 6.9% and 4.8% in our 2013 and 2014 sampling, respectively. During our sampling campaign we also did an interesting observation. We noticed that several bees were parasitized by a phorid fly species, and dipteran larvae emerged from these bees leaving the exoskeleton as remnants (Fig. 1, Panel B). Morphological as well as molecular species determination identified the phorid fly as M. scalaris. The detection of M. scalaris in the Algerian honey bee is to our knowledge the first record of honey bee parasitism by this fly species. Our findings confirm earlier speculations about the possible parasitism of A. mellifera by this phorid fly (Rocha
B
Fig. 1. A: Adult specimens of the phoroid fly, Megaselia scalaris, parasitizing A. mellifera intermissa. B: Top: arrows indicate larvae of M. scalaris, emerging from bee individuals of A. mellifera intermissa; Bottom: arrows indicate pupae of M. scalaris.
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et al., 1984). This discovery sheds light on the opportunistic behavior of this insect, i.e. feeding on wide array of invertebrate species. As M. scalaris was parasitizing a hive which was positive for DWV, we screened the larval and adult stages of the fly for this virus. We found the virus to be present in both adults and larvae. To clarify if both larvae and adults were truly infected with DWV, the detection of a replication intermediate, i.e. the negative strand of the virus, was performed. This negative strand of DWV could be detected in the larvae but not in the adult flies. These results indicate that DWV can truly infect M. scalaris, at least in the larval stages. To our knowledge this is also the first time this virus is reported to be present in M. scalaris and shown to truly infect a dipteran species. We believe that our findings further strengthen the hypothesis that flies of the Phoridae family could act as a vector for DWV, as several species of this family have been associated with this honey bee virus. Apocephalus borealis, was recorded as parasite of honey bee colonies in the USA, this species was also often infected with DWV (Core et al., 2012). A. borealis has also been detected in Belgium at a high prevalence of 31% (Ravoet et al., 2013), indicating this fly is also parasitizing honey bees outside the USA. More recently, a third species of phorid fly, Megaselia rufipes, has been identified as a facultative parasite of honey bees in Italy (Dutto and Ferrazzi, 2014). Although no DWV was found in M. rufipes, as the flies were not examined for viral presence, it is remarkable that these flies were also found in a colony which was affected by DWV. The detection of the negative strand of DWV by the strandspecific detection technique MLPA (De Smet et al., 2012) provides proof for a true infection of M. scalaris larvae, which is a strong indication that this fly could indeed act as a vector of DWV. Although we found no proof of DWV replication in the adult flies, it should be mentioned that only two adult specimens were screened, one of these was positive for DWV but no negative strand could be detected. It could be that we missed the detection of negative strand in adult flies, as our sample size was small. To fully confirm this hypothesis, where phorid flies act as DWV vector, further research is needed. These findings make the Phoridae a family of growing interest in the comprehensive studies carried out around the world to give answers to the colony collapse disorder and more generally to honey bee health. Aside from honey bees, the broad host range of these phorid flies also makes them a considerable danger to wild pollinators, as reported by Core et al. (2012). We therefore suggest that future pathogens screenings should also include Phoridae, and we encourage further research on the role of this fly family in pollinator health. 4. Conclusion Our study in A. mellifera intermissa, a subspecies of A. mellifera, adapted to a hot climate, revealed the presence of N. ceranae, P. larvae and DWV. Furthermore we present the first report of several pathogens, i.e. A. bombi, C. mellificae, LSV and SBV, in this honey bee subspecies. In this study we also report for the first time the parasitism of a phorid fly species, M. scalaris, in honey bees, and in addition, we found DWV to be present in adult flies and actively replicating in the fly larvae. We therefore suggest further research is needed toward the role of Phoridae species as a possible vector for DWV and in pollinator health in general. Acknowledgment The authors would like to thank the beekeepers for participating in this study. This research was supported by the Fund for Scientific Research (FWO-Vlaanderen, Brussels).
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