Megaselia scalaris (Diptera: Phoridae), an Opportunist Parasitoid of Honey Bees in Cameroon

Contemporary variation and declines in honey bee colonies in most parts of the world have been attributed to several factors among which pests, pathogens and diseases remain preeminent (Goulson et al. 2015). Among these pests, phorid flies, previously considered of minor economic importance, are becoming increasingly prominent following reports of their contribution to widespread colony declines (Core et al. 2012; Dutto & Ferrazzi 2014). These dipteran parasitoids pose a threat to honey bees as well as other social bees such as bumble bees (Core et al. 2012; Otterstatter et al. 2002). Apocephalus borealis Brues is known to be a potential vector of deformed wing virus and Nosema ceranae (Microspora: Nosematidae) in honey bees (Core et al. 2012). Melaloncha sp. Brues attacks both honey and stingless bees resulting in severe infestations popularly referred to as ‘Autumn disease’ in South America due to the season in which they normally occur (Knutson & Murphy 1990). Phorid fly species in the genus Megaselia has been reported to parasitise slowmoving or deformed bees (Knutson & Murphy 1990; Dutto & Ferrazzi 2014; Core et al. 2012). Although M. scalaris is known to occur in Africa and infest honey bees (Disney 2008), their impact on bee health in Cameroon is poorly documented (Pirk et al. 2016). This is likely due to absence of surveillance. As part of a honey bee pest surveillance project we provide here both morphological and genetic evidence of M. scalaris infestations of honey bee colonies in Cameroon. We examined a total of 120 colonies in 21 apiaries in Cameroon distributed across the savannas in the North West (NW) Region (n = 65 colonies), the dense evergreen forests in the South West (SW) Region (n = 23) and the degraded evergreen forests in the East Region (n = 32) (Fig. 1). These regions are among the major beekeeping areas of Cameroon (Ingram & Njikeu 2011). All available colonies were sampled in apiaries with £10 colonies, while 10 colonies were randomly sampled in apiaries with >10 colonies. In each colony, approximately 30 worker honey bees were aspirated from the inner walls and entrance of the hive and placed in 50 ml centrifuge tube. The tubes were sealed tightly to kill the bees by asphyxiation. The death bee specimens were held at room temperature (25 ± 2.0 °C) within the same tubes, recapped with fine mesh netting (~0.25 mm) that excluded phorid flies and allowed sufficient air exchange for any developing parasites. Emergent fly larvae were allowed to pupate and their development followed until adult emergence (Fig. 2). Adult flies were collected and preserved in 95 % ethanol (DROP-CHIM, Cameroon) for subsequent molecular identification. Flies were tentatively identified as M. scalaris by a taxonomist (J.K. Mugambi) at the National Museums of Kenya, Nairobi. Genomic DNA was extracted from the individual whole fly samples using the ISOLATE II Genomic DNA kit (Bioline, U.K.) as per the manufacturer’s instructions. DNA barcodes were generated from selected phorid specimens to confirm the morphological identification. The barcode primers LCO1490 and HCO2198 (Folmer et al. 1994) were used to amplify a ~700 bp region of the partial mitochondrial cytochrome oxidase subunit 1 (CO1) gene. Amplification was carried out using the Mytaq HS Mix, 2x kit (Bioline, U.K.). Polymerase chain reaction (PCR) was performed in a 25 μl mixture containing 10–50 ng genomic DNA template, 20 μM of each primer (forward and reverse), 9.5 μl of RNase free water and 12.5 μl of MyTaq HS Mix, 2x buffer in a T100TM thermal cycler (Bio Rad). The cycling conditions were as follows; 95 °C for 1 min, followed by 35 cycles of 95 °C for 20 s, 45 °C for 20 s, 72 °C for 15 s and final extension of 72 °C for 7 min. Amplicons were resolved on a 1 % agarose gel stained with ethidium bromide against a 1 kb DNA