Infestation of commercial bumblebee ( Bombus impatiens ) ﬁ eld colonies by small hive beetles ( Aethina tumida )

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Introduction

The small hive beetle (SHB), Aethina tumida Murray (Coleoptera: Nitidulidae), is a parasite of honeybee, Apis mel- lifera L. (Hymenoptera: Apidae), colonies native to sub-Saharan Africa ( Hepburn & Radloff, 1998 ). In the last decade, it became an invasive species in North America, Egypt, Australia ( Elzen et al. , 1999; Neumann & Elzen, 2004 ), and Europe ( Ritter, 2004 ). In its native range, SHB is considered to be only a minor pest of honeybee colonies ( Lundie, 1940; Hepburn & Radloff, 1998 ). However, in its new ranges the beetle can have a consid-

erable impact on local honeybee populations ( Hood, 2004;

Neumann & Elzen, 2004 ).

Bumblebees, Bombus Latreille, and honeybees are close phy- logenetic relatives, both belonging to the taxon Apidae ( Michener, 2000 ). While the colonies of cavity-nesting Apis species, e.g. Apis mellifera , can usually be found above ground (e.g. in hollow trees), the nests of most Bombus species are lo- cated underground (e.g. in abandoned small mammal nests).

Moreover, Bombus colonies typically consist of some hundred workers and are much smaller than honeybee colonies, which consist of several thousand individuals ( Michener, 1974 ).

Nevertheless, honeybees and bumblebees share essential fea- tures such as the storage of pollen and nectar and the usage of wax for nest construction. In the endemic range of SHB, Bombus species do not naturally occur but they are native to North America and Europe ( Michener, 2000 ). Because of the similarities Correspondence: Sebastian Spiewok, Martin-Luther-Universität

Halle-Wittenberg, Institut für Molekulare Ökologie, Hoher Weg 4, D-06099 Halle (Saale), Germany. E-mail: s.spiewok@web.de

Infestation of commercial bumblebee ( Bombus impatiens ) fi eld colonies by small hive

beetles ( Aethina tumida )

S E B A S T I A N S P I E W O K

¹

and P E T E R N E U M A N N

^{2 , 3 , 4}

¹ Institut für Zoologie, Martin-Luther-Universität Halle-Wittenberg, Halle (Saale), Germany, ² Swiss Bee Research Centre, Agroscope Liebefeld-Posieux, Research Station, Bern, Switzerland, ³ Department of Zoology and Entomology,

Rhodes University, Grahamstown, South Africa, and ⁴ Eastern Bee Research Institute of Yunnan Agricultural University, Kunming, China

Abstract .

1. The small hive beetle, Aethina tumida , is a parasite of honeybee (

Apis mellifera ) colonies native to sub-Saharan Africa and has become an invasive species. In

North America the beetle is now sympatric with bumblebees,

Bombus , not occurring

in its native range. Laboratory studies have shown that small hive beetles can reproduce in bumblebee colonies but it was not known whether infestations occur in the field.

2. For the first time, infestation of bumblebee colonies by small hive beetles was investigated in the field. Commercial

Bombus impatiens colonies ( n =

10) were installed in proximity to infested apiaries. Within 8 weeks, all colonies that were alive in the 5-week observation period (

n =

9) became naturally infested with adult small hive beetles and successful small hive beetle reproduction occurred in five colonies.

3. In four-square choice tests, the beetles were attracted to both adult bumblebee workers and pollen from bumblebee nests, suggesting that these odours may serve as cues for host finding.

4. The data indicate that bumblebee colonies may serve as alternative hosts for small hive beetles in the field. To foster the conservation of these essential native pollinators, investigations on the actual impact of small hive beetles on wild bumblebee populations are suggested.

Key words .

Aethina tumida , Apis , Bombus , bumblebee , honeybee , host fi nding , host

switch , small hive beetle .

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between Apis and Bombus a host switch of SHB to bumblebees might be possible. Indeed, laboratory studies demonstrated that SHB are able to reproduce successfully inside artificially infested colonies of B. impatiens Cresson ( Stanghellini et al. , 2000; Ambrose et al. , 2000 ) and that SHB larvae caused considerable damage. However, crucial for an infestation of bumblebee colonies is host finding in the field, which has not been investigated previously. Since pollen and honey as well as adult workers from honeybee colonies are attractive to SHB ( Suazo et al. , 2003 ), the stores and workers of bumblebee nests may me- diate host location; but whether cues derived from bumblebee colonies are attractive to SHB is unknown. If SHB are attracted to cues derived from Bombus colonies, host finding should also occur in the field, thereby enabling a host switch of SHB from honeybees to bumblebees. In this study, the possibility of natural infestation of bumblebee colonies by SHB is investigated.

Furthermore, olfactory orientation of SHB towards bumblebee workers and pollen collected from bumblebee colonies was studied to identify possible cues for the host finding of SHB.

Materials and methods Field colonies

Experiments on the natural infestation of bumblebee nests by SHB in the field were conducted with commercial colonies ( B.

impatiens Cresson) from Koppert Biological Systems (Romulus, Michigan) in Umatilla, Florida, from June to August 2004. The colonies were placed inside carefully cleaned (mechanically and with soap), empty Langstroth hive boxes (bottom board, deep and lid ) to protect them from heavy rain. Since SHB popula- tion density may vary at different sites, thereby influencing the probability of colony infestation, two circles (diameter: 10 m) were arranged consisting of five colonies each. Group 1 was set up in a garden of a housing estate » 500 m away from a pri- vate apiary (20 honeybee colonies). Group 2 was placed a dis- tance of » 100 m from a honey house next to an adjacent apiary (32 honeybee colonies). To protect the bumblebee colonies against ants, the boxes were placed on bricks inside aluminium pans filled with water and washing liquid. Both circles were shaded by surrounding trees. The colonies were allowed to es- tablish for 5 weeks without any disturbance. For the following 5 weeks they were carefully screened for the occurrence and number of adult and larval SHB, other nest parasites, and bumblebee workers. While the number of bumblebees was estimated, the number of parasites was assessed by individually collecting them with aspirators and reintroducing them after- wards into the nest. Dying colonies were removed from the boxes and the empty boxes were subsequently screened during the following weeks to control for possible attraction of SHB to these boxes alone. Additionally, five empty Langstroth boxes (group 3) were set up as another control in an apiary, which was heavily infested with SHB (26 honeybee colonies). Finally, five Langstroth boxes containing only honeycombs (group 4), with a screen at the entrance allowing SHB but not bees to enter the hive box, were set up as a further control in another heavily infested apiary (22 honeybee colonies).

The number of adult and larval SHB and larvae of the greater wax moth, Galleria mellonella L. (Lepidoptera: Pyralidae), found within the boxes at the three locations were analysed for differences using a Kruskal – Wallis test and Mann – Whitney U - tests. Differences in the number of SHB at the beginning and at the end of the observation period (5th and 10th week) in group 2 were analysed with a Wilcoxon matched-pairs test. Since only two colonies of group 1 survived the observation period, the numbers of SHB of this group were not compared. The numbers of bumblebee workers and SHB larvae in the colonies were analysed for the eighth week using a Spearman rank correlation.

Olfactory orientation

Olfactory orientation of adult SHB towards bumblebee and honeybee workers and pollen freshly obtained from a bumblebee nest was evaluated using a four-square choice test. The tested SHB originated from the laboratory rearing at the USDA in Gainesville, Florida. Six live bumblebee workers or 4 g freshly collected pollen served as odour sources. To compare between the reaction of SHB towards bumblebees and honeybees an additional test was conducted with six honeybee workers caught from the entrance of an Apis mellifera ligustica colony. The bees were placed inside one of the squares of the test arena and were provided with honey served in the lid of a 1.5-ml Eppendorf vial. The other three squares contained only the honey-filled lids. During the trial with pollen, the control squares remained empty. The responses of 20 male and 20 female SHB to honeybees and pollen or of 30 males and 30 females to bumblebees were tested. Time spent in the four different squares was recorded for 3 min using the program The Observer Ver.

2.0 (Noldus, Wageningen, the Netherlands). To minimise posi- tional bias, the arena was rotated 90° after 10 or 15 SHB had been tested. The experiments were conducted at 29 ± 1 °C and 60% relative humidity. A red light bulb (25 W) illuminated the arena, which was placed 65 cm above its centre.

The proportional time p spent in each square was arcsin Ö p transformed and analysed for differences with a Friedman anova followed by Wilcoxon – Wilcox tests. The responses to the different odour sources were analysed for males and females using a Kruskal – Wallis test followed by Mann – Whitney U -tests (Bonferroni – Fisher adjusted level of significance: P < 0.0167).

Differences in the responsiveness to the odour sources between the sexes were tested for statistical significance using a Wilcoxon matched-pairs test. All tests except the Wilcoxon – Wilcox one, which was calculated following Köhler et al. (2002) , were car- ried out using the program Statistica ^® .

Results Field colonies

The detailed observations are shown in Table 1. Two weeks after the establishment of the experimental groups, one colony of group 1 was killed by Florida carpenter ants ( Camponotus flori- danus ). Two more colonies of this group died 6 weeks later after

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queen loss. All colonies that were alive in the 5-week observation period were naturally infested by adult SHB. Because no SHB were found in any of the control boxes, significantly more beetles were found in the bumblebee colonies than in the controls (groups 3 and 4, Table 2). Up to 15 adult SHB were found in one colony, but the numbers were highly variable between screenings and not significantly different between the test groups 1 and 2 ( Table 2 ). The number of adult SHB [median (first quartile; third quartile )] decreased significantly from the 5th to the 10th week [ group 1 : 5th week: 5 (0; 11) SHB/colony, 10th week:

1 ; group 2: 5th week: 9 (4; 11), 10th week: 1 (0; 1); Wilcoxon matched-pairs test: T = 0, P = 0.043]. A total of 51 SHB larvae was found in five colonies during the whole observation time ( Table 1 ). The larvae showed a range of developmental stages including those close to the wandering phase, when the larvae are leaving the nest to pupate in the soil ( Lundie, 1940 ). The number of SHB larvae was negatively correlated to the number of bumblebee workers in the colonies (8th week: Spearman rank correlation: r _s = – 0.80, t = – 3.54, d.f. = 7, P < 0.01).

Furthermore, all colonies of group 2 and one of group 1 were infested with larvae of the greater wax moth. The combs of two colonies were almost completely destroyed by the moth larvae at the end of the observation period. At the eighth week, significantly more wax moth larvae were found in the colonies of group 2 compared with group 1 (Mann – Whitney U -test: U = 2, d.f. = 1, P = 0.046).

Olfactory orientation

Proportionally, male and female SHB spent significantly more time in the square with the respective odour source (bumblebees, honeybees or pollen) than in the other three squares ( Fig. 1;

Table 3), while there were no significant differences between the proportions of time spent in the latter ones ( Fig. 1 ). Male beetles responded more strongly to bumblebees than females (Wilcoxon matched-pairs test: T = 126, d.f. = 1, P = 0.028), but no significant differences were detected between the sexes in their responses to honeybees ( T = 76, d.f. = 1, P = 0.279) and pollen ( T = 96, d.f. = 1, P = 0.498). In contrast to males, which showed no significant differences in their responsiveness to the different odours (Kruskal – Wallis test: H = 5.59, d.f. = 2, P = 0.061), females responded significantly more strongly to honeybees than to bumblebees ( H = 8.56, d.f. = 2, P = 0.014; Mann – Whitney U -test: U = 162.5, d.f. = 1, P = 0.007). However, no significant

Table 1. Observations on the infestation of Bombus impatiens colonies 5 – 10 weeks after exposure. The numbers of SHB adults and larvae, and of

greater wax moth larvae as well as the estimated numbers of bumblebee workers are shown (SHB, adult small hive beetles; SHBL, small hive beetle larvae; WML, wax moth larvae ).

Weeks after exposure

Group 1 ^a (garden) Group 2 (honey house) Groups 3 and

4 (controls) Boxes 1 – 10 Colony 1 Colony 2 Colony 3 Colony 4 Colony 6 Colony 7 Colony 8 Colony 9 Colony 10

5 Bees 12 25 40 80 100 40 100 50 70 0

SHB 0 0 14 10 9 3 15 11 4 0

SHBL 0 0 0 0 0 0 0 0 0 0

WML 0 0 0 0 0 0 0 0 0 0

6 Bees 12 23 40 70 80 30 100 70 100 0

SHB 1 1 2 6 6 4 9 7 5 0

SHBL 0 0 0 6 0 0 0 0 0 0

WML 0 0 0 0 0 0 1 0 0 0

8 Bees 0 5 50 90 120 30 90 50 110 0

SHB 1 0 2 3 0 3 14 1 5 0

SHBL 14 2 7 0 0 6 0 0 0 0

WML 0 0 0 13 4 55 73 15 7 0

10 Bees – – 10 65 100 14 33 46 80 0

SHB – – 0 2 0 2 0 1 1 0

SHBL – – 6 10 0 0 0 0 0 0

WML – – 0 0 0 89 106 7 0 0

a Colony 5 of group 1 died before the start of the observations.

Table 2. Differences in the number of adult SHB between the two test

groups and the controls. H -, Z -, and P -values of the Kruskal – Wallis tests or Mann – Whitney U -tests are shown.

Test H d.f. Z P

5th week 7.19 2 0.028

Group 1 – Control 1 1.68 0.090

Group 1 – Group 2 1 – 0.74 0.461

6th week 10.58 2 0.005

Group 1 – Group 2 1 – 1.52 0.128

8th week 7.32 2 0.026

Group 1 – Group 2 1 – 0.45 0.651

10th week –

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differences were found between the female responses to honeybees and pollen ( U = 125, d.f. = 1, P = 0.027) as well as to adult bumblebees and pollen ( U = 272, d.f. = 1, P = 0.411).

Discussion

The observations clearly show that SHB can naturally infest commercial B. impatiens colonies in the field. In fact, at both locations all colonies surviving until the end of the observation period were infested with up to 15 adult SHB. Moreover, a total number of 51 larvae was found in five colonies, showing that successful reproduction of SHB in bumblebee colonies is not confined to the laboratory ( Ambrose et al. , 2000 ), but may also occur under field conditions. These figures represent minimum numbers of both larval and adult SHB, because it was impossible to completely screen the boxes without destroying the colony due to the nest architecture. The laboratory choice test demonstrated the attractiveness of bumblebee workers and pollen obtained from bumblebee nests to SHB. The lack of SHB in the control boxes is in line with previous studies ( Elzen et al. , 1999 ), showing that free-flying SHB cannot be trapped in the field even with bee products unless adult bees are present.

The data give strong support to laboratory studies ( Stanghellini et al. , 2000 ) that SHB are capable of reproducing successfully in association with bumblebee colonies, because SHB larvae of different developmental stages were found in five colonies. This successful reproduction was not destructive to the field colonies, similar to the low level cryptic reproduction of SHB within honeybee colonies ( Spiewok & Neumann, 2006 ). Since the number of SHB larvae was negatively correlated to the number of bumblebee workers, small colonies may be less efficient in preventing SHB reproduction than larger ones. Alternatively, but not mutually exclusively, SHB may more readily lay eggs in smaller bumblebee colonies. There was no evidence that adult or larval SHB caused the decline of one of the dying colonies.

In the laboratory experiments of Stanghellini et al. (2000) , SHB were able to produce more offspring (> 1000 larvae per colony) and to cause considerable damage in terms of lower numbers of live workers and comb damage. This high reproductive success occurred although the colonies consisted of 100 – 200 bumblebee workers and infestation levels were 20 adult SHB per colony. It is unclear to what extent the laboratory conditions or the introduction of a higher number of SHB favoured this development. Indeed, in the study of Stanghellini et al. (2000) , fairly large numbers of adult SHB were introduced all at the same time as compared with the assumed smaller number of SHB entering the field colonies in the present study over an extended period of time. Nevertheless, Stanghellini et al. (2000) demonstrated that SHB are in principle also capable of mass reproducing in

Table 3. Responses of male and female SHB to the respective odour

sources in the four-square choice test. The results of the Friedman anova are shown.

Odour source SHB sex ␹²3-value P-value

Bumblebees Males 36.29 <0.00001

Females 24.36 <0.00002

Honeybees Males 24.90 <0.00002

Females 34.50 <0.00001

Fresh pollen Males 18.33 <0.00038

Females 19.06 <0.00027

Response (% of time)

0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100

1 2 3 4 Squares

Pollen Honeybees Bumblebees A

A

A a

a a

B

B B

B

B B B

b b b

b b

b

b b b

Fig. 1. Proportional time spent in the four different squares of the choice test by male (fi lled boxes) and female (open boxes) SHB. Square 1 contained the respective odour source, while squares 2, 3, and 4 remained empty. Values are medians with fi rst and third quartiles (boxes) as well as standard deviation (bars). Signifi cant differences between times spent in the four squares are indicated for males by different upper case letters and for females by different lower case letters (Wilcoxon – Wilcox test for P < 0.0125).

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bumblebee colonies and thereby damaging them. No aggression of B. impatiens either towards adult or larval SHB was observed in this laboratory study, which may suggest that bumblebees lack behavioural resistance against SHB ( Stanghellini et al. , 2000 ). However, in this study SHB reproduced successfully only in a few colonies, although all were infested by adult beetles. Thus, it is likely that the bumblebee workers were some- how able to prevent SHB mass reproduction. Indeed, defensive behaviour towards beetle larvae similar to the jettisoning behaviour of honeybees towards SHB ( Lundie, 1940; Schmolke, 1974; Neumann & Härtel, 2004 ) has been described for bumblebees ( Michener, 1974 ).

While all colonies next to the honey house (group 1) were infested with greater wax moth larvae, only one colony in the garden (group 2) housed moth larvae. This suggests that scavengers/parasites infesting both bumblebee and honeybee colonies might be more frequent around large commercial honey houses. Infestations with wax moths led to a steep decline of bumblebee colonies, which can also happen with weak honeybee colonies ( Williams, 1997 ). Therefore, bumblebee colonies may be exposed to a higher threat by parasites usually associated with honeybees in the close vicinity of honey houses.

However, there was no significant difference in the numbers of adult SHB between the two locations, presumably because both apiaries were heavily infested with adult SHB. Scavengers and parasites of Apidae colonies like SHB, which seek actively for host colonies, seem to express rather low host specificity, e.g.

the nitidulid beetle Epuraea depressa Illiger is reported from the nests of different bumblebee species ( Scott, 1920; Cumber, 1949 ). Similarly, the greater wax moth G. mellonella , which is usually associated with colonies of different Apis species ( Williams, 1997 ), was also found in nests of meliponids ( Nogueira-Neto, 1953 ) and bumblebees ( Oertel, 1963 ; this study). Moreover, the lesser wax moth Achroia grisella Fabricius, another pest of honeybee colonies ( Williams, 1997 ), is also reported from stingless bees ( Cepeda-Aponte et al. , 2002 ). A further example is the bumblebee wax moth Aphomia sociella L., which infests various Bombus species ( Free &

Butler, 1959; Pouvreau, 1967 ) but is also rarely found in honeybee colonies ( Toumanoff, 1939 ). In light of these previous re- ports it is not surprising that SHB do not only infest honeybee but also bumblebee colonies in the field. Indeed, several trans- mission events of parasites from invaders to native species are already known ( Prenter et al. , 2004 ). The close taxonomic rela- tionship between Bombus and Apis ( Michener, 2000 ) appears to facilitate such a host switch. As a consequence of the general low host specificity of those mobile colony parasites, SHB can be expected to infest not only B. impatiens but also nests of other bumblebee species.

The four-square choice test showed that SHB were attracted to adult bumblebee workers and to freshly collected pollen from bumblebee nests, suggesting that those may constitute cues for host finding of SHB. Since SHB are also attracted to pollen and other volatiles obtained from honeybee colonies ( Suazo et al. , 2003; Torto et al. , 2005 ), the positive response to this protein source is not unexpected. Similarly, the nitidulid pollen beetle Meligethes aeneus Fabricius is also attracted by pollen ( Cook et al. , 2002 ). A surprising result was the high responsiveness

to bumblebee workers, especially by male SHB, whose response is similar to that to honeybee workers. Thus, SHB may react to the same odour components or to a broader spectrum of similar odour cues emitted by both adult bumblebee and honeybee workers. The so far analysed volatiles of honeybees that elicit an orientation response of SHB ( Torto et al. , 2005 ) are either com- mon in a variety of insects or have not yet been identified from bumblebees . Similar to Suazo et al. (2003) , significant differ- ences in the responsiveness of the two sexes were also found, but in their experiments, females were more responsive than males. Sex-specific host-finding behaviour of adult SHB, e.g.

males seem to infest honeybee colonies before females ( Elzen et al. , 1999 ), may contribute to this, but the underlying reasons for the different responsiveness of male and female SHB remain unclear and require further investigation. Experimental nest boxes above ground might be more easily detectable for host- seeking SHB than natural underground bumblebee nests, caus- ing higher infestation rates in this study. Nevertheless, some bumblebee species nest above the ground surface ( Michener, 1974 ) and might be as easily infested by SHB as the commercial colonies in this study. Bumblebee colonies may be more endangered in close proximity to apiaries. However, SHB can emerge when honeybee colonies are no longer in close proximity, e.g.

after migratory beekeepers have left. Moreover, to the authors’

knowledge no field data are available for the flight range of host- finding SHB. Since all colonies were infested by SHB, it was not possible to evaluate their impact on colony growth. Furthermore, a comparison of the present observations on colony development with the literature is not possible as, to the authors’ knowledge, no respective data exist for B. impatiens in the field, only for laboratory studies ( Cnaani et al. , 2002 ). However, even if SHB infestations do not necessarily result in the death of a colony, they could still be damaging, e.g. by reducing the number of successfully reared sexuals. Therefore, a more detailed study on the impact of SHB infestation on bumblebee colonies in the field is desirable.

While commercial honeybee colonies are under the surveil- lance of beekeepers, who have chemical control methods against the SHB at their disposal ( Baxter et al. , 1999; Elzen et al. , 1999 ), this is clearly not the case for wild bee colonies. The presented data suggest the strong possibility that SHB may have an impact on wild bumblebee populations at least near apiaries, which cannot be protected like managed honeybee colonies.

However, not a single study has yet addressed this issue. It has to be stressed that killing bumblebee colonies to get rid of SHB breeding opportunities is not an appropriate solution for apicul- turists. On the contrary, it is essential to protect these important pollinators ( Crane & Walker, 1984; Corbet et al. , 1991; Free, 1993; Osborne & Williams, 1996; Carreck & Williams, 1998;

Stanghellini et al. , 1997, 1998 ), especially since many bumble- bee species are already endangered in both North America and Europe ( Williams, 1986; Banaszak, 1996; Buchmann &

Nabhan, 1996 ). An additional stress caused by a newly introduced pest might be critical for some bumblebee species.

Further comparative studies on a variety of bumblebee species and on a range of colony sizes would help to understand the impact of the SHB on these essential native pollinators in different habitats.

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Acknowledgements

We thank John Wolf from Koppert for the kind gift of bumblebee colonies. We also thank Baldwyn Torto (USDA, Gainesville), who provided adult SHB from the laboratory rearing and David Westervelt (FDA) for logistical support. We also thank Johannes Steidle (University of Hohenheim) for the four-square arena and two anonymous referees for their constructive comments as well as Sarah Radloff for proofreading. Financial support was granted by an Emmy Noether fellowship of the DFG to P.N.

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Accepted 1 June 2006

First published online 28 November 2006