Nosema Bombi: a Pollinator Parasite with Detrimental Fitness Effects
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Journal of INVERTEBRATE PATHOLOGY Journal of Invertebrate Pathology 96 (2007) 118–124 www.elsevier.com/locate/yjipa Nosema bombi: A pollinator parasite with detrimental fitness effects Oliver Otti a,*, Paul Schmid-Hempel a,b a Experimental Ecology, Institute of Integrative Biology Zurich (IBZ), ETH Zurich, 8092 Zurich, Switzerland b Wissenschaftskolleg zu Berlin, Wallotstrasse 19, D-14193 Berlin, Germany Received 30 October 2006; accepted 27 March 2007 Available online 31 March 2007 Abstract Nosema bombi is an obligate intracellular parasite that infects different bumblebee species at a substantial, though variable, rate. To date its pathology and impact on host fitness are not well understood. We performed a laboratory experiment investigating the pathology and fitness effects of this parasite on the bumblebee Bombus terrestris. We experimentally infected one group of colonies with N. bombi spores at the start of the worker production, while a second uninfected group of colonies served as controls. During colony development we collected live workers for dissections to measure infection intensities. In parallel, we measured several life history traits, to investigate costs to the host. We succeeded in infecting 11 of 16 experimental colonies. When infection occurred at an early stage of colony devel- opment, virtually all individuals were infected, with spores being found in a number of tissues, and the functional fitness of males and young queens was reduced to zero. Further, the survival of workers from infected colonies and infected males were reduced. With such severe effects, N. bombi appears to decrease its opportunities for transmission to the next host generation. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Host–parasite interaction; Nosema bombi; Bombus terrestris; Pathology; Fitness effects; Microsporidia; Virulence; Pollinator parasites 1. Introduction could play a role in reducing populations, at least when they invade populations that are diminishing for other Bumblebees are important natural pollinators for a wide reasons. range of flowering plants (Goulson, 2003) and are of eco- Studies on other economically important insect species, nomic importance as they pollinate a substantial propor- e.g. the silkworm moth and the honeybee, have shown that tion of our crops. They are therefore commercially bred parasites can inflict substantial financial losses to managed in large numbers to be used in the pollination of a range populations. For example, in the 19th century, the micro- of greenhouse crops, such as tomato, capsicum and melon sporidian parasite N. bombycis, the agent of pebrine disease (Al-Attal et al., 2003; Banda and Paxton, 1991; de Ruijter, in the silkworm Bombyx mori (an insect important for the 1997). In the United States alone, the annual economic production of textiles), was spread throughout France and value of pollination services is estimated at 1.25 billion caused massive losses to the silk industry (Cadeddu, 2000). USD, of which bumblebees provide a large share (Ghazoul, Currently, the Varroa mite in honeybees poses financial 2005). These major ecological and economic roles have led problems to the apiculture industry as it impairs the honey to widespread concern over recent declines in pollinator production and kills off reproductive animals so that the populations, including bumblebees (Goulson, 2003; Wil- colonies can no longer be propagated (Sammataro et al., liams, 2003). The major causes of this decline are thought 2000). In the United States, the industry is under additional to relate to changes in the use of agricultural land (Goulson pressure due to the spread of the Africanized honeybee et al., 2005; Williams, 2005), but also the effects of parasites (Whitfield et al., 2006). There is, however, a concern that a parasite, the microsporidian Nosema bombi Fantham and Porter, 1914, could cause the decline of the native * Corresponding author. Fax: +41 44 632 1271. bumblebee community (Kearns and Thomson, 2001; Whit- E-mail address: [email protected] (O. Otti). tington and Winston, 2003), but too little is known to date 0022-2011/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2007.03.016 O. Otti, P. Schmid-Hempel / Journal of Invertebrate Pathology 96 (2007) 118–124 119 about the interactions of N. bombi with its bumblebee host, 2. Materials and methods e.g. impact on host fitness and the transmission dynamics (Fries et al., 2001), to make any informed estimate of the 2.1. Preparation of inoculum threat posed by this microsporidium. The microsporidians are related to fungi (Adl et al., To prepare the inoculum, abdomens of infected bumble- 2005; James et al., 2006), and all the members of this phy- bees from four different colonies (50 workers per colony) lum are obligatory intracellular parasites. The host range were homogenised in Krebs Ringer solution. The homoge- includes nearly all animal groups from protists to humans, nate was filtered through glass wool. The filtered spore solu- with insects as one of the major host groups (Cali and tion was centrifuged for 5 min at 3000g and 4 °Cand Takvorian, 1999). In general, microsporidians have two checked for N. bombi using a light microscope. Ammonium major life cycle stages, a spore stage and a vegetative stage. chloride, which inhibits the germination process, was added The infective or environmental stage is the spore, and in to the solution to minimize the loss of infective spores (Und- most cases is ingested by the host and first infects host cells een and Avery, 1988). Subsequently, the spores were washed in the gut lumen and the Malpighian tubules. During and centrifuged another two times. After washing, the con- reproduction and proliferation the parasite spreads within centration of spores was determined in a haemocytometer the host. Fresh spores are then released into the environ- (Neubauer improved). Aliquots were prepared (100 llwith ment via faeces or a decaying host (Cali and Takvorian, a concentration of 5.5 · 105 spores llÀ1)andkeptat 1999). The spore has a thick chitinous cell wall that pro- À80° C until needed for the experiment. tects it from harsh environmental conditions, i.e. tempera- ture and humidity (Li et al., 2003; Maddox and Solter, 2.2. Exposure to N. bombi (treatments) 1996; Malone et al., 2001). Bombus terrestris and other Bombus species are infected We selected 94 B. terrestris queens that originated from by N. bombi at substantial though variable rates (Fries first generation laboratory-reared colonies and were mated et al., 2001; Imhoof and Schmid-Hempel, 1999; Schmid- randomly with males from other laboratory colonies. After Hempel and Loosli, 1998; Shykoff and Schmid-Hempel, artificial hibernation the queens were allowed to oviposit in 1991; Tay et al., 2005). N. bombi primarily infects the Mal- the laboratory under standard conditions (Gerloff and Sch- pighian tubules, but also infects the thorax muscles, fat mid-Hempel, 2005). After the first egg batch was laid, the body tissue, nerve tissue, midgut and the muscle tissue queens were assigned at random to one of two treatments. surrounding the gut epithelium (Fries et al., 2001). In the Queens in the ‘‘Exposed’’ treatment received a pollen pellet Malpighian tubules spores and vegetative stages are found with 18 ll of spore solution (5.5 · 105 spores llÀ1, or a total in the regenerative cell layer of the gut epithelium near the of 9.9 · 106 spores per feeding) twice a week for 4 weeks. basement membrane (McIvor and Malone, 1995). In heavy Queens in the ‘‘Control’’ treatment were fed pollen pellets infections, the structure of the tissues is destroyed and with only the suspension solution. Because imagos cannot mature spores are released into the lumen. The queen is be reliably infected with N. bombi (Otti, unpublished data) the primary source of N. bombi infection for the next our method mimics a natural exposure of larvae to the para- generation, and infection has seemingly little impact on sitevia contact withthe nursing queen. To reduce the potential her nest initiation and egg-laying capacity (Fisher and for inflated re-infection rates due to the housing conditions of Pomeroy, 1989b). colonies in the laboratory, dead workers were removed every Data on the effect of N. bombi on the fitness of its second day. Separate feeding and defecating boxes with cat lit- bumblebee host is scarce and contradicting. Some authors ter substrate were exchanged weekly, and sugar water feeders have reported chronic rather than fatal effects (Fisher and were cleaned and filled with fresh sugar water every week. Pomeroy, 1989a; Imhoof and Schmid-Hempel, 1999), while During the treatment period 18 queens died (11 controls, 7 others found more severe effects (DeJonghe, 1986; Fantham exposed), 43 queens either stopped egg-laying or their brood and Porter, 1914; Macfarlane et al., 1995; Schmid-Hempel did not develop (20 controls, 23 exposed); from 30 broods at and Loosli, 1998). Most of these reports are based on either least one worker emerged (14 controls, 16 exposed). Three accidental field observations or unintended (natural occur- queens were excluded from the experiment as they died due ring) infections in laboratory experiments. Data from to a malfunction of the feeding equipment. As soon as the field-collected live bumblebees that are dissected and colony size reached 10 workers we culled each week 20% of assessed for parasite infestation might give a biased picture the workers to mimic a natural mortality rate. The culled of the overall effect. For example, parasites inflicting heavy workers were used to check for infection status, and these mortality may go undetected, and prevalence and effects of revealed that 11 of the 16 exposed colonies had at least one disease underestimated. We, therefore, conducted an exper- infected individual (infection success 68.75%). iment under standardised laboratory conditions to investi- gate the impact and pathology of N. bombi in its 2.3. Colony level measures bumblebee host B.