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Bacterial diversity in worker adults of Apis mellifera capensis and Apis mellifera scutellata (Insecta: Hymenoptera) assessed using 16S rRNA sequences
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INVERTEBRATE PATHOLOGY Journal of Invertebrate Pathology 84 (2003) 96–103 www.elsevier.com/locate/yjipa
Bacterial diversity in worker adults of Apis mellifera capensis and Apis mellifera scutellata (Insecta: Hymenoptera) assessed using 16S rRNA sequences Ayyamperumal Jeyaprakash,a,* Marjorie A. Hoy,a and Michael H. Allsoppb b
a Department of Entomology and Nematology, University of Florida, Gainesville, FL 32611, USA Plant Protection Research Institute, Agricultural Research Council, Private Bag X5017, Stellenbosch 7599, South Africa
Received 13 June 2003; accepted 28 August 2003
Abstract High-fidelity PCR of 16S rRNA sequences was used to identify bacteria associated with worker adults of the honeybee subspecies Apis mellifera capensis and Apis mellifera scutellata. An expected 1.5-kb DNA band, representing almost the entire length of the 16S rRNA gene, was amplified from both subspecies and cloned. Ten unique sequences were obtained: one sequence each clustered with Bifidobacterium (Gram-positive eubacteria), Lactobacillus (Gram-positive eubacteria), and Gluconacetobacter (Gram-negative a-proteobacteria); two sequences each clustered with Simonsiella (b-proteobacteria) and Serratia (c-proteobacteria); and three sequences each clustered with Bartonella (a-proteobacteria). Although the sequences relating to these six bacterial genera initially were obtained from either A. m. capensis or A. m. scutellata or both, newly designed honeybee-specific 16S rRNA primers subsequently amplified all sequences from all individual workers of both subspecies. Attempts to amplify these sequences from eggs have failed. However, the wsp primers designed to amplify Wolbachia DNA from arthropods, including these bees, consistently produced a 0.6kb DNA band from individual eggs, indicating that amplifiable bacterial DNA was present. Hence, the 10 bacteria could have been acquired orally from workers or from other substrates. This screening of 16S rRNA sequences from A. m. capensis and A. m. scutellata found sequences related to Lactobacillus and Bifidobacterium which previously had been identified from other honeybee subspecies, as well as sequences related to Bartonella, Gluconacetobacter, Simonsiella/Neisseria, and Serratia, which have not been identified previously from honeybees. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Apis mellifera capensis; Apis mellifera scutellata; Bacterial diversity; 16S rRNA gene
1. Introduction Arthropods are associated with a wide variety of bacteria, including beneficial endosymbionts, occasional disease-causing entomopathogens, and harmless transient cuticle contaminants acquired from the environment (Boucias and Pendland, 1998; Bourtzis and Miller, 2003; Wernegreen, 2002). Endosymbiotic bacteria may be extracellular or intracellular and can be found free in the digestive system, hemocoel, reproductive tract, or localized in specialized cells. Buchnera in aphids, for example, are tightly held in 60–90 bacteriocytes in a
* Corresponding author. Fax: 1-352-392-0190. E-mail address: [email protected]fl.edu (A. Jeyaprakash).
0022-2011/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2003.08.007
specialized structure (bacteriome) and maintain a mutualistic relationship with their aphid hosts by producing tryptophan to supplement the nutrient-deficient plant sap diet (Baumann et al., 1995; Baumann and Moran, 1997). In contrast, Wolbachia are present in the reproductive tissues and implicated in a variety of anomalies such as reproductive incompatibilities (Laven, 1951), thelytoky or parthenogenesis induction in parasitoid wasps (Stouthamer et al., 1990), male-killing in coccinellid beetles (Hurst et al., 1999) and feminization of genetic males in isopods (Rousset et al., 1992). The Western honeybee, Apis mellifera, is a semi-domesticated insect reared in apiaries to produce honey for human consumption and for the pollination of commercial crops. Two subspecies, Apis mellifera capensis and Apis mellifera scutellata, exist in Southern Africa
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(Hepburn and Crewe, 1991). A. m. capensis workers are known to be able to invade A. m. scutellata colonies and lay eggs. A. m. scutellata workers are unable to detect or eliminate A. m. capensis eggs (Martin et al., 2002), allowing capensis bees to eventually eliminate the scutellata colony. The unique parasitic behavior of A. m. capensis is not observed in any other bee subspecies and the reason(s) for the A. m. capensis phenotype is unknown, but could involve microorganisms (Hoy et al., 2003). The European honeybee, Apis mellifera mellifera, has been reported to contain multiple gut symbionts, including about 1% yeast-like microbes, 29% Gram-positive bacteria such as Bacillus, Lactobacillus, Bifidobacterium, Corynebacterium, Streptococcus, and Clostridium, and 70% Gram-negative or Gram-variable bacteria such as Achromobacter, Citrobacter, Enterobacter, Erwinia, Escherichia coli, Flavobacterium, Klebsiella, Proteus, and Pseudomonas (see Gilliam, 1997; Snowdon and Cliver, 1996 for reviews). However, 16S rRNA sequences have not been obtained from these bacteria. 16S rRNA sequences have, however, been obtained from the bacteria Melissococcus plutonius (formerly Melissococcus pluton) and Paenibacillus larvae var. larvae, the cause of European and American foulbrood diseases, respectively, in honeybee larvae (Alippi et al., 2002; Govan et al., 1998). In this study, microbial DNA from the abdomens of an A. m. capensis and an A. m. scutellata worker was amplified using High-fidelity PCR (Barnes, 1994) with eubacterial 16S rRNA primers (27f and 1495r) (Weisburg et al., 1991). The sequences and relationships of the bacteria present in adult workers of these subspecies is provided, and their potential role as endosymbiont or entomopathogen is discussed.
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magnitude more sensitive than Standard PCR (Hoy et al., 2001, 2003; Jeyaprakash and Hoy, 2000). Hence, High-fidelity PCR was used to screen the honeybee DNA. High-fidelity PCR was performed using 1 ll genomic DNA preparation in a 50-ll reaction volume containing 50 mM Tris (pH 9.2), 16 mM ammonium sulfate, 1.75 mM MgCl2 , 350 lM dATP, dGTP, dCTP, dTTP, 800 pM of eubacterial primers (27f, 50 -GAGAG TTTGATCCTGGCTCAG-30 and 1495r, 50 -CTACG GCTACCTTGTTACGA-30 ) (Weisburg et al., 1991), 0.2 U Tgo and 5 U of Taq DNA polymerases (Roche Molecular Biochemicals, Indianapolis, IN) (Barnes, 1994). The primers and dNTPs were mixed in a 25-ll volume and combined with another 25-ll volume containing the buffer, genomic DNA and DNA polymerases in a thin-walled 0.5-ml centrifuge tube and kept on ice prior to amplification. The reaction mix was then overlaid with 100 ll of mineral oil and amplified using three linked profiles; (i) one cycle consisting of 94 °C denaturation for 2 min, (ii) 10 cycles consisting of 94 °C denaturation for 10 s, 55 °C annealing for 30 s and 68 °C extension for 2 min, and (iii) 25 cycles consisting of 94 °C denaturation for 10 s, 55 °C annealing for 30 s and 68 °C extension for 2 min with 20 s added for every consecutive cycle; extension time for Cycle 1 is 2 min, Cycle 2 is 2 min and 20 s, Cycle 3 is 2 min and 40 s, etc., for 25 cycles. The quality of bacterial DNA present was determined by amplifying a 0.6-kb Wolbachia bacterial DNA sequence from both adults and eggs using the wsp primers 81F (50 -TGGTCCAATAAGTGATGAAGAAAC-30 ) and 691R (50 -AAAAATTAAACGCTACTCCA-30 ) (Braig et al., 1998). The conditions used for Wolbachia wsp DNA amplification were similar to 16S rRNA amplification, but the extension time was 1 min (Hoy et al., 2003).
2. Materials and methods 2.3. Cloning and RFLP analysis 2.1. DNA extraction Workers and eggs of the honeybee subspecies A. m. capensis and A. m. scutellata were collected from different locations in Southern Africa, shipped in 95% ethanol and stored at )70 °C. The abdomen was cut off from workers using a new single-edged razor blade for each bee and the internal tissues were removed for extraction of genomic DNA. Total genomic DNA was extracted from the abdomen of an individual worker or a batch of 20 pooled eggs using PUREGENE reagents following the procedure suggested by the manufacturer (Gentra Systems, Minneapolis, MN) and resuspended in 100 ll sterilized water. 2.2. High-fidelity PCR The High-fidelity PCR method makes surveys of bacteria associated with insects five to six orders of
High-fidelity PCR products were cleaned using QIAquick Columns (QIAGEN, Valencia, CA) and cloned into pCR2.1 TOPO following the manufacturerÕs suggested protocol (Invitrogen, Carlsbad, CA). Individual E. coli transformants were selected at random, plasmid DNA extracted using QIAGEN Plasmid Mini Columns (QIAGEN, Valentia, CA), digested with RsaI and the DNA fragments separated on a 2% agarose TBE gel. Clones displaying similar RFLP banding patterns were identified for further sequence analysis. 2.4. Phylogenetic analysis Sequencing was performed at the University of Florida Interdisciplinary Core Facility. The sequences obtained were searched for close similarities in GenBank using BLAST. A CLUSTAL W alignment was made using the closely related DNA sequences retrieved from
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Table 1 Honeybee bacterial 16S rRNA-specific forward primers and the expected size of the DNA band amplified when used in combination with the eubacterial reverse primer (1495r) Name 197-F 207-F 203/208/210-F 205-F 198-F 199-F 204/206-F
Sequence 0
Expected size of a DNA band (bp) 0
5 -TGCTCAAGCAGTAAAAGGCGGC-3 50 -GGATCCGCCAGGCTTGCTTG-30 50 -GAAGATAATGACGGTAACCGG-30 50 -TACTTTCGGTAGGGACGATG-30 50 -GCATCGGAACGTACCGAGTAA-30 50 -GGTAACGAGGAAGGTGGTG-30 50 -GTCGATTTGGAGTTTGTTGCC-30
BLAST searches. Phylogenetic trees were constructed using PAUP 4.0b10. Genetic distance between any two bacterial sequences was measured on a scale of 0–1 using the Distance and Neighbor-joining Kimura 2-parameter in PAUP 4.0 and then converted into a percentage to calculate sequence divergence between the bacteria. 2.5. Diagnostic High-fidelity PCR 16S rRNA sequences were aligned and honeybeespecific forward primers were designed (Table 1) and used in combination with the eubacterial reverse primer (r1345) to amplify genomic DNA extracted from each of five individual A. m. capensis and A. m. scutellata abdomens or 20 pooled eggs of each subspecies using the same High-fidelity PCR conditions. Clean laboratory practices, plugged pipet tips, and fresh reagents were used to avoid contamination. DNAaseAWAY solution (Fisher Scientific, Pittsburgh, PA) was used to clean bench surfaces and pipets.
1331 1429 1045 1057 1392 1073 693
an individual bee worker or that Taq DNA polymerase introduced DNA mismatches during amplification (Kunkel and Eckert, 1989). Others have found similar sequence variations in gut symbionts; for example, 98 spirochaetal 16S rRNA sequences were analyzed from the gut of the termite Reticulotermes flavipes and 21 unique strains of Treponema were recognized (Lilburn et al., 1999). Because we used the High-fidelity PCR procedure, Taq-introduced errors should have been removed from the amplified DNA fragments (Barnes, 1994). Therefore, nucleotide differences observed could be real and indicate the probable presence of multiple bacterial strains. Two clones (pAJ196 and pAJ197) representing the eighth banding pattern showed 100% sequence match. 3.2. Phylogenetic analyses of 16S rRNA sequences identify bacteria belonging to six genera from honeybees Complete sequences from a random clone representing each banding pattern were used for further analysis. Thus, three sequences were obtained from A. m. scu-
3. Results and discussion 3.1. 16S rRNA sequence amplification from honeybees using High-fidelity PCR The eubacterial 16S rRNA primers (27f and 1495r) produced an expected 1.5-kb DNA band from both subspecies and these were cloned. No amplification was ever obtained in the no-DNA controls, as expected. A total of 30 E. coli transformants was selected at random from each of the two honeybee subspecies and typed using an RFLP strategy (Fukatsu and Nikoh, 1998). RsaI-digested plasmid DNA produced three unique patterns from A. m. scutellata and eight from A. m. capensis; one banding pattern was shared by both for a total of 10 (Fig. 1). Two independent clones were sequenced from the transformants representing each of 8 banding patterns, but only one clone was available for 2 of the 10 banding patterns. Sequences from two clones from 7 of the 8 banding patterns displayed 4–7 bp differences, which could indicate that different bacterial strains co-exist in
Fig. 1. RFLP banding patterns of RsaI-digested plasmid DNA in 2% agarose TBE gel from 10 different E. coli transformants generated by cloning the 16S rRNA PCR products obtained from A. m. capensis and A. m. scutellata. Lanes: 1, DNA marker VI; 2, pAJ196; 3, pAJ198; 4, pAJ199; 5, pAJ203; 6, pAJ204; 7, pAJ205; 8, pAJ206; 9, pAJ207; 10, pAJ208; and 11, pAJ210. pAJ196–pAJ199 were cloned from A. m. scutellata and pAJ203–pAJ210 were from A. m. capensis.
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tellata (pAJ197, pAJ198, and pAJ199) and seven sequences from A. m. capensis (pAJ203–pAJ208 and pAJ210) for BLAST searches. All 10 sequences were unique and not represented in GenBank. Sequences from Lactobacillus crispatus (AF257097), Simonsiella muelleri (AF328148), Neisseria meningitidis (AF337937), Bartonella henselae (AJ223780), Serratia marcescens (AB061685), DpLE (U20279), Bifidobacterium indicum (D86188), and Gluconacetobacter sacchari (AF127412) were found to be related to one or more of the sequences from these honeybees. Nine other sequences, Escherichia coli (AE000474), Buchnera aphidicola (M27039), Bacillus subtilis (Z99107), M. plutonius (X75752), Rickettsia japonica (L36213), Wolbachia pipientis (U23709), and an endosymbiont of the rice weevil Sitophilus oryzae (AF005235) and parasitoid wasp Encarsia pergandiella (AF319783), were selected as outgroups. A CLUSTAL W alignment was made using
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these 26 bacterial 16S rRNA sequences. Phylogenetic trees were constructed using Maximum Parsimony, Neighbor-joining, and Maximum Likelihood methods. A phylogenetic tree constructed using the Distance and Neighbor-joining Kimura 2-parameter was found to display bootstrap values >50 for all branches (Fig. 2). A 1532-bp sequence obtained from A. m. scutellata (pAJ197) clustered with Lactobacillus crispatus (6.9% sequence divergence) (Fig. 2), a Gram-positive eubacterium. Stackebrandt and Goebel (1994) determined that bacterial strains displaying 3% divergence, or greater, in 16S rRNA sequences are nearly always from different species. Hence, it appears that this 16S rRNA sequence (AY370183) may be an unidentified honeybee Lactobacillus species. Two cultures of pleomorphic bacteria isolated from A. m. mellifera worker bees by Gilliam (1997) appeared to be members of the genus Lactobacillus based on microscopic observations and
Fig. 2. A Neighbor-joining tree generated for bacterial 16S rRNA sequences using a CLUSTAL W alignment with the optimality criteria set for Distance and Kimura 2-parameter in PAUP 4.0b10. Bootstrap values detected for 100 replicates are shown before the nodes. The bacterial 16S rRNA sequences obtained from A. m. capensis and A. m. scutellata are shown in bold.
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their ability to grow in different synthetic media. Recently, a partial 16S rRNA sequence related to Lactobacillus has been cloned during a survey for gut bacteria in the wasp Vespula germanica (Reeson et al., 2003). Lactobacillus bacteria are normal inhabitants of the gastrointestinal tract of man and animals and are known to be beneficials, involved in immunomodulation, interference with enteric pathogens and maintenance of a healthy intestinal microflora (Mitsuoka, 1992). They are facultatively anaerobic, non-motile, and non-spore forming, rod-shaped bacteria that adhere to the intestinal cell wall. They are known to produce D -lactic acid as a metabolic end product from hexose sugars via the Embden–Meyerhof pathway (Axelsson, 1998). A 1491-bp sequence obtained from A. m. capensis (pAJ207) (AY370184) was found to cluster with B. indicum (1.9% sequence divergence) (Fig. 2), a Gram-positive eubacterium. B. asteroides previously was identified from the A. m. mellifera midgut and rectum by microscopic observation (Rada et al., 1997). A 16S rRNA sequence of B. asteroides has been obtained from the human gut (M58730). However, the 16S rRNA sequence from A. m. capensis displayed 6.2% sequence divergence from the B. asteroides sequence isolated from the human gut, indicating that a bacterium more closely related to B. indicum appears to reside in A. m. capensis. Bifidobacteria are anaerobic branched or pleomorphic rod-shaped bacteria that commonly reside in the intestinal tract of humans and animals, adhering to the cell wall (Biavati et al., 2000). Bifidobacteria can be distinguished from other genera by the presence of an unique Ôbifidus pathwayÕ that is involved in fermenting glucose to produce acetate and lactate (Scardovi, 1986). Three sequences obtained from A. m. capensis [pAJ203 (1454 bp) (AY370185), pAJ208 (1453 bp) (AY370186), and pAJ210 (1454 bp) (AY370187)] exhibited a close relationship with each other; sequence divergences of 0.9 and 3.1% were obtained when the sequence that cloned into pAJ203 was compared to the sequences from pAJ208 and pAJ210, respectively. All three sequences clustered with a B. henselae sequence (Fig. 2), an a-proteobacterium and the casual agent of Bartonellosis in humans (Piemont and Heller, 1998). The three sequences displayed 3.6, 2.8, and 5% sequence divergences from B. henselae, respectively. A variety of arthropods are known to transmit Bartonella species; Bartonella quintana is vectored by human lice (Pediculus humanus and Pediculus capitis) and B. bacilliformis is carried by sandflies (Lutzomyia, Brumptomyia, and Warileya) (Marquardt et al., 2000). Recently, several 16S rRNA sequences closely related to human- and animal-pathogenic Bartonella spp., including B. henselae, B. quintana, and Bartonella vinsonii, were detected from ticks (Chang et al., 2001; Sanogo et al., 2003) and fleas (Parola et al., 2002). Detection of three Bartonella-related strains from these honeybees was novel and unexpected.
A 1455-bp sequence obtained from A. m. capensis (pAJ205) (AY370188) was found to cluster with a sequence from the a-proteobacterium G. sacchari, which is an intracellular gut symbiont located in the caecal bacteriocytes of pink sugarcane mealybug (Saccharococcus sacchari) (Fig. 2) (Franke et al., 2000). A divergence of 7.1% was detected between the 16S rRNA sequences obtained from A. m. capensis and S. sacchari, indicating that different bacterial species reside in these insects. G. sacchari cells are ellipsoidal to rod-shaped, and are present both as endophytes in sugarcane leaves as well as gut symbionts in the mealybug (Franke et al., 1999). Gluconacetobacter bacteria are known to live in a sugarrich environment, producing acetic acid from ethanol. Acetic acid-producing bacteria (called Gluconobacters) were reported earlier from A. m. mellifera (Lambert et al., 1981), but their 16S rRNA sequences have never been determined. The 16S rRNA sequence obtained from A. m. capensis (pAJ205) appears to be related to Gluconacetobacter and could be from a honeybee gut symbiont. Two sequences obtained from A. m. scutellata, pAJ198 (1505 bp) (AY370189) and pAJ199 (1503 bp) (AY370190), displayed 8.1% sequence divergence from each other and clustered with both S. muelleri and N. meningitidis sequences (Fig. 2), which are extracellular b-proteobacteria and human oral commensals, as well as facultative human pathogens (Hedlund and Staley, 2002; Taha et al., 2002). The 16S rRNA sequence from A. m. scutellata that cloned into pAJ198 displayed 5.6 and 7.3% sequence divergences from S. muelleri and N. meningitidis, respectively. An additional 16S rRNA sequence from A. m. scutellata that cloned into pAJ199 displayed 10.2 and 11.6% sequence divergences, respectively, from the same bacterial species. Bacteria related to Simonsiella and Neisseria have not been reported from arthropods, but an unidentified Neisseria species was detected in honey (Snowdon and Cliver, 1996). These sequences appear to be from two undescribed b-proteobacterial species because the sequence divergence detected was high (8.1%). Two sequences obtained from A. m. capensis [pAJ204 (1505 bp) (AY370191) and pAJ206 (1506 bp) (AY370192)] were similar (0.6% sequence divergence), differing by 6 substitutions and 3 insertions. Both sequences are related to sequences from an entomopathogen from Drosophila paulistorum (known by the common name DpLE) (Miller et al., 1995), and to Serratia marcescens, which is an opportunistic entomopathogen (Boucias and Pendland, 1998). The two sequences from A. m. capensis displayed 10 and 10.4% sequence divergences, respectively, from DpLE, while the same sequences from A. m. capensis displayed 10.5 and 10.6% sequence divergences, respectively, from S. marcescens. All four sequences (pAJ204, pAJ206, DpLE, and S. marcescens) clustered with c-proteobacteria, including Buchnera aphidicola,
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Escherichia coli, a primary endosymbiont from S. oryzae, and R. japonica (Fig. 2). Serratia are rod-shaped bacteria and attach to the foregut and midgut wall. S. marcescens is isolated frequently from insectaryreared insects kept under crowded unnatural conditions (King et al., 1975; Steinhaus, 1959). The sequences from A. m. capensis appear to be from two strains (0.6% sequence divergence) of an undescribed c-proteobacterial species related to S. marcescens.
among the bacteria from the two honeybee subspecies. Further assays might uncover more bacterial strains or species from these honeybees. The new honeybeespecific PCR primers (Table 1) provide a tool for researchers to investigate the role of these microbes in the biology and ecology of A. m. capensis and A. m. scutellata.
3.3. Bacterial genera detected from either A. m. capensis or A. m. scutellata are present in both subspecies
To resolve whether the bacteria are maternally transmitted to honeybee progeny or acquired from contaminated workers through feeding, genomic DNA was extracted from 20 pooled A. m. scutellata and A. m. capensis eggs and screened using the 7 honeybee-specific primer pairs (Table 1). No PCR products were obtained, indicating either that all the bacteria were acquired after the honeybees hatched or that the number of bacteria in the eggs was below detectable level. However, Wolbachia wsp primers readily amplified a 0.6-kb DNA band from these honeybee egg DNA preparations (data not shown here), indicating that microbial DNA can be amplified from eggs.
Because 16S rRNA sequences related to two genera were detected from A. m. scutellata (Lactobacillus and Simonsiella) and sequences related to five genera were detected from A. m. capensis (Bifidobacterium, Gluconacetobacter, Bartonella, Serratia, and Simonsiella) with only one sequence being common to both subspecies (Simonsiella), it was not clear whether A. m. scutellata and A. m. capensis have a different bacterial fauna or whether the eubacterial 16S rRNA primers annealed at random to some bacterial 16S rRNA sequences early in the High-fidelity PCR cycles and amplified only a subset of all the sequences present. In order to resolve this issue, honeybee bacteria-specific primers were designed. Sequences related to Lactobacillus (pAJ197), Bifidobacterium (pAJ207) and Gluconacetobacter (pAJ205), Bartonella (pAJ203, pAJ208 and pAJ210) and Serratia (pAJ204 and pAJ206) were used to design a specific primer for each (Table 1). Two sequences related to Simonsiella (pAJ198 and pAJ199) displayed sufficient sequence divergence (8.1%) that two specific primers could be designed (Table 1). Thus, seven different honeybee bacteria-specific forward primers were designed to detect 16S rRNA sequences related to six bacterial genera in A. m. capensis and/or A. m. scutellata. DNA was extracted from each of five individual honeybee abdomens belonging to workers of both A. m. scutellata and A. m. capensis and screened using Highfidelity PCR and the honeybee-specific forward primers (Table 1) in combination with the eubacterial reverse primer (1495r). All five A. m. scutellata and the five A. m. capensis bees tested produced the expected size DNA bands (Table 1) for all seven primer pairs tested (data not shown here). Hence, the initial limited number of cloned 16S rRNA sequences from A. m. scutellata (pAJ197–pAJ199) and A. m. capensis (pAJ203–pAJ208 and pAJ210) were likely due to the primers binding to some bacterial sequences by chance during the early High-fidelity PCR cycles and preferentially amplifying only some sequences. It appears that all honeybees contained all bacterial sequences identified so far. However, because these PCR products were not sequenced, we do not know if there were strain differences
3.4. Specific primers fail to amplify 16S rRNA from eggs
4. Conclusions Our screening of 16S rRNA sequences from A. m. capensis and A. m. scutellata found sequences related to two genera (Lactobacillus and Bifidobacterium) previously identified from honeybees, as well as sequences related to bacterial genera (Bartonella, Gluconacetobacter, Simonsiella, and Serratia) not previously identified from honeybees (Fig. 2). Honeybees are known to have complex interactions with their diet and with diverse microorganisms (Snowdon and Cliver, 1996). Bacteria belonging to Lactobacillus, Corynebacterium, and Bifidobacterium have been detected from A. m. mellifera, primarily by conventional culture or microscopic observations (Gilliam, 1997). Well over 6000 bacterial strains have been cultured from honeybees and frozen (Gilliam, 1997). Although it has been known for a long time that the honeybee gut has a rich microbial fauna (White, 1921), DNA sequence analyses of these microbes have been limited. A single Wolbachia wsp sequence (w Cap-B1) has been detected from both A. m. capensis and A. m. scutellata but is most likely associated with the reproductive tissues (Hoy et al., 2003) and not from gut microflora. The 16S rRNA sequences of M. plutonius and P. l. var. larvae, which causes European and American foulbrood diseases in honeybee larvae, have also been characterized (Alippi et al., 2002; Govan et al., 1998). None of the 10 16S rRNA sequences obtained in this study are related to either Wolbachia or M. plutonius or P. l. larvae.
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One objective of this study was to identify a bacterium uniquely associated with the A. m. capensis phenotype, but none has been detected so far. Despite this, if additional surveys are conducted, the possibility of finding additional bacterial species associated with these honeybees still remains, because well over 6000 bacterial strains are known to be associated with honeybees (Gilliam, 1997). Acknowledgments This work was supported in part by the Davies, Fisher, and Eckes Endowment in Biological Control to Marjorie A. Hoy at the University of Florida. We thank Carol Lauzon for comments on an earlier draft of the manuscript. This is Florida Agricultural Experiment Station journal publication R-09157.
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A. Jeyaprakash et al. / Journal of Invertebrate Pathology 84 (2003) 96–103 Reeson, A.F., Jankovic, T., Kasper, M.L., Rogers, S., Austin, A.D., 2003. Application of 16S rDNA-DGGE to examine the microbial ecology associated with a social wasp Vespula germanica. Insect Mol. Biol. 12, 85–91. Rousset, F., Bouchon, D., Pintureau, B., Juchault, P., Solignac, M., 1992. Wolbachia endosymbionts responsible for various alterations of sexuality in arthropods. Proc. R. Soc. Lond. B 250, 91–98. Sanogo, Y.O., Zeaiter, Z., Caruso, G., Merola, F., Shpynov, S., Brouqui, P., Raoult, D., 2003. Bartonella henselae in Ixodes ricinus ticks (Acari: Ixodida) removed from humans, Belluno Province, Italy. Emerg. Infect. Dis. 9, 329–332. Scardovi, V., 1986. Genus Bifidobacterium. In: Sneath, P.H.A., Mair, N.S., Sharpe, M.E., Holt, J.G. (Eds.), BergeyÕs Manual of Systematic Bacteriology, vol. 2. Williams and Watkins, Baltimore, MD, pp. 1418–1434. Snowdon, J.A., Cliver, D.O., 1996. Microorganisms in honey. Int. J. Food Microbiol. 31, 1–26. Stackebrandt, E., Goebel, B.M., 1994. Taxonomic note: a place for DNA:DNA reassociation and 16S rRNA sequence analysis in the
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INVERTEBRATE PATHOLOGY Journal of Invertebrate Pathology 84 (2003) 96–103 www.elsevier.com/locate/yjipa
Bacterial diversity in worker adults of Apis mellifera capensis and Apis mellifera scutellata (Insecta: Hymenoptera) assessed using 16S rRNA sequences Ayyamperumal Jeyaprakash,a,* Marjorie A. Hoy,a and Michael H. Allsoppb b
a Department of Entomology and Nematology, University of Florida, Gainesville, FL 32611, USA Plant Protection Research Institute, Agricultural Research Council, Private Bag X5017, Stellenbosch 7599, South Africa
Received 13 June 2003; accepted 28 August 2003
Abstract High-fidelity PCR of 16S rRNA sequences was used to identify bacteria associated with worker adults of the honeybee subspecies Apis mellifera capensis and Apis mellifera scutellata. An expected 1.5-kb DNA band, representing almost the entire length of the 16S rRNA gene, was amplified from both subspecies and cloned. Ten unique sequences were obtained: one sequence each clustered with Bifidobacterium (Gram-positive eubacteria), Lactobacillus (Gram-positive eubacteria), and Gluconacetobacter (Gram-negative a-proteobacteria); two sequences each clustered with Simonsiella (b-proteobacteria) and Serratia (c-proteobacteria); and three sequences each clustered with Bartonella (a-proteobacteria). Although the sequences relating to these six bacterial genera initially were obtained from either A. m. capensis or A. m. scutellata or both, newly designed honeybee-specific 16S rRNA primers subsequently amplified all sequences from all individual workers of both subspecies. Attempts to amplify these sequences from eggs have failed. However, the wsp primers designed to amplify Wolbachia DNA from arthropods, including these bees, consistently produced a 0.6kb DNA band from individual eggs, indicating that amplifiable bacterial DNA was present. Hence, the 10 bacteria could have been acquired orally from workers or from other substrates. This screening of 16S rRNA sequences from A. m. capensis and A. m. scutellata found sequences related to Lactobacillus and Bifidobacterium which previously had been identified from other honeybee subspecies, as well as sequences related to Bartonella, Gluconacetobacter, Simonsiella/Neisseria, and Serratia, which have not been identified previously from honeybees. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Apis mellifera capensis; Apis mellifera scutellata; Bacterial diversity; 16S rRNA gene
1. Introduction Arthropods are associated with a wide variety of bacteria, including beneficial endosymbionts, occasional disease-causing entomopathogens, and harmless transient cuticle contaminants acquired from the environment (Boucias and Pendland, 1998; Bourtzis and Miller, 2003; Wernegreen, 2002). Endosymbiotic bacteria may be extracellular or intracellular and can be found free in the digestive system, hemocoel, reproductive tract, or localized in specialized cells. Buchnera in aphids, for example, are tightly held in 60–90 bacteriocytes in a
* Corresponding author. Fax: 1-352-392-0190. E-mail address: [email protected]fl.edu (A. Jeyaprakash).
0022-2011/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2003.08.007
specialized structure (bacteriome) and maintain a mutualistic relationship with their aphid hosts by producing tryptophan to supplement the nutrient-deficient plant sap diet (Baumann et al., 1995; Baumann and Moran, 1997). In contrast, Wolbachia are present in the reproductive tissues and implicated in a variety of anomalies such as reproductive incompatibilities (Laven, 1951), thelytoky or parthenogenesis induction in parasitoid wasps (Stouthamer et al., 1990), male-killing in coccinellid beetles (Hurst et al., 1999) and feminization of genetic males in isopods (Rousset et al., 1992). The Western honeybee, Apis mellifera, is a semi-domesticated insect reared in apiaries to produce honey for human consumption and for the pollination of commercial crops. Two subspecies, Apis mellifera capensis and Apis mellifera scutellata, exist in Southern Africa
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(Hepburn and Crewe, 1991). A. m. capensis workers are known to be able to invade A. m. scutellata colonies and lay eggs. A. m. scutellata workers are unable to detect or eliminate A. m. capensis eggs (Martin et al., 2002), allowing capensis bees to eventually eliminate the scutellata colony. The unique parasitic behavior of A. m. capensis is not observed in any other bee subspecies and the reason(s) for the A. m. capensis phenotype is unknown, but could involve microorganisms (Hoy et al., 2003). The European honeybee, Apis mellifera mellifera, has been reported to contain multiple gut symbionts, including about 1% yeast-like microbes, 29% Gram-positive bacteria such as Bacillus, Lactobacillus, Bifidobacterium, Corynebacterium, Streptococcus, and Clostridium, and 70% Gram-negative or Gram-variable bacteria such as Achromobacter, Citrobacter, Enterobacter, Erwinia, Escherichia coli, Flavobacterium, Klebsiella, Proteus, and Pseudomonas (see Gilliam, 1997; Snowdon and Cliver, 1996 for reviews). However, 16S rRNA sequences have not been obtained from these bacteria. 16S rRNA sequences have, however, been obtained from the bacteria Melissococcus plutonius (formerly Melissococcus pluton) and Paenibacillus larvae var. larvae, the cause of European and American foulbrood diseases, respectively, in honeybee larvae (Alippi et al., 2002; Govan et al., 1998). In this study, microbial DNA from the abdomens of an A. m. capensis and an A. m. scutellata worker was amplified using High-fidelity PCR (Barnes, 1994) with eubacterial 16S rRNA primers (27f and 1495r) (Weisburg et al., 1991). The sequences and relationships of the bacteria present in adult workers of these subspecies is provided, and their potential role as endosymbiont or entomopathogen is discussed.
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magnitude more sensitive than Standard PCR (Hoy et al., 2001, 2003; Jeyaprakash and Hoy, 2000). Hence, High-fidelity PCR was used to screen the honeybee DNA. High-fidelity PCR was performed using 1 ll genomic DNA preparation in a 50-ll reaction volume containing 50 mM Tris (pH 9.2), 16 mM ammonium sulfate, 1.75 mM MgCl2 , 350 lM dATP, dGTP, dCTP, dTTP, 800 pM of eubacterial primers (27f, 50 -GAGAG TTTGATCCTGGCTCAG-30 and 1495r, 50 -CTACG GCTACCTTGTTACGA-30 ) (Weisburg et al., 1991), 0.2 U Tgo and 5 U of Taq DNA polymerases (Roche Molecular Biochemicals, Indianapolis, IN) (Barnes, 1994). The primers and dNTPs were mixed in a 25-ll volume and combined with another 25-ll volume containing the buffer, genomic DNA and DNA polymerases in a thin-walled 0.5-ml centrifuge tube and kept on ice prior to amplification. The reaction mix was then overlaid with 100 ll of mineral oil and amplified using three linked profiles; (i) one cycle consisting of 94 °C denaturation for 2 min, (ii) 10 cycles consisting of 94 °C denaturation for 10 s, 55 °C annealing for 30 s and 68 °C extension for 2 min, and (iii) 25 cycles consisting of 94 °C denaturation for 10 s, 55 °C annealing for 30 s and 68 °C extension for 2 min with 20 s added for every consecutive cycle; extension time for Cycle 1 is 2 min, Cycle 2 is 2 min and 20 s, Cycle 3 is 2 min and 40 s, etc., for 25 cycles. The quality of bacterial DNA present was determined by amplifying a 0.6-kb Wolbachia bacterial DNA sequence from both adults and eggs using the wsp primers 81F (50 -TGGTCCAATAAGTGATGAAGAAAC-30 ) and 691R (50 -AAAAATTAAACGCTACTCCA-30 ) (Braig et al., 1998). The conditions used for Wolbachia wsp DNA amplification were similar to 16S rRNA amplification, but the extension time was 1 min (Hoy et al., 2003).
2. Materials and methods 2.3. Cloning and RFLP analysis 2.1. DNA extraction Workers and eggs of the honeybee subspecies A. m. capensis and A. m. scutellata were collected from different locations in Southern Africa, shipped in 95% ethanol and stored at )70 °C. The abdomen was cut off from workers using a new single-edged razor blade for each bee and the internal tissues were removed for extraction of genomic DNA. Total genomic DNA was extracted from the abdomen of an individual worker or a batch of 20 pooled eggs using PUREGENE reagents following the procedure suggested by the manufacturer (Gentra Systems, Minneapolis, MN) and resuspended in 100 ll sterilized water. 2.2. High-fidelity PCR The High-fidelity PCR method makes surveys of bacteria associated with insects five to six orders of
High-fidelity PCR products were cleaned using QIAquick Columns (QIAGEN, Valencia, CA) and cloned into pCR2.1 TOPO following the manufacturerÕs suggested protocol (Invitrogen, Carlsbad, CA). Individual E. coli transformants were selected at random, plasmid DNA extracted using QIAGEN Plasmid Mini Columns (QIAGEN, Valentia, CA), digested with RsaI and the DNA fragments separated on a 2% agarose TBE gel. Clones displaying similar RFLP banding patterns were identified for further sequence analysis. 2.4. Phylogenetic analysis Sequencing was performed at the University of Florida Interdisciplinary Core Facility. The sequences obtained were searched for close similarities in GenBank using BLAST. A CLUSTAL W alignment was made using the closely related DNA sequences retrieved from
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Table 1 Honeybee bacterial 16S rRNA-specific forward primers and the expected size of the DNA band amplified when used in combination with the eubacterial reverse primer (1495r) Name 197-F 207-F 203/208/210-F 205-F 198-F 199-F 204/206-F
Sequence 0
Expected size of a DNA band (bp) 0
5 -TGCTCAAGCAGTAAAAGGCGGC-3 50 -GGATCCGCCAGGCTTGCTTG-30 50 -GAAGATAATGACGGTAACCGG-30 50 -TACTTTCGGTAGGGACGATG-30 50 -GCATCGGAACGTACCGAGTAA-30 50 -GGTAACGAGGAAGGTGGTG-30 50 -GTCGATTTGGAGTTTGTTGCC-30
BLAST searches. Phylogenetic trees were constructed using PAUP 4.0b10. Genetic distance between any two bacterial sequences was measured on a scale of 0–1 using the Distance and Neighbor-joining Kimura 2-parameter in PAUP 4.0 and then converted into a percentage to calculate sequence divergence between the bacteria. 2.5. Diagnostic High-fidelity PCR 16S rRNA sequences were aligned and honeybeespecific forward primers were designed (Table 1) and used in combination with the eubacterial reverse primer (r1345) to amplify genomic DNA extracted from each of five individual A. m. capensis and A. m. scutellata abdomens or 20 pooled eggs of each subspecies using the same High-fidelity PCR conditions. Clean laboratory practices, plugged pipet tips, and fresh reagents were used to avoid contamination. DNAaseAWAY solution (Fisher Scientific, Pittsburgh, PA) was used to clean bench surfaces and pipets.
1331 1429 1045 1057 1392 1073 693
an individual bee worker or that Taq DNA polymerase introduced DNA mismatches during amplification (Kunkel and Eckert, 1989). Others have found similar sequence variations in gut symbionts; for example, 98 spirochaetal 16S rRNA sequences were analyzed from the gut of the termite Reticulotermes flavipes and 21 unique strains of Treponema were recognized (Lilburn et al., 1999). Because we used the High-fidelity PCR procedure, Taq-introduced errors should have been removed from the amplified DNA fragments (Barnes, 1994). Therefore, nucleotide differences observed could be real and indicate the probable presence of multiple bacterial strains. Two clones (pAJ196 and pAJ197) representing the eighth banding pattern showed 100% sequence match. 3.2. Phylogenetic analyses of 16S rRNA sequences identify bacteria belonging to six genera from honeybees Complete sequences from a random clone representing each banding pattern were used for further analysis. Thus, three sequences were obtained from A. m. scu-
3. Results and discussion 3.1. 16S rRNA sequence amplification from honeybees using High-fidelity PCR The eubacterial 16S rRNA primers (27f and 1495r) produced an expected 1.5-kb DNA band from both subspecies and these were cloned. No amplification was ever obtained in the no-DNA controls, as expected. A total of 30 E. coli transformants was selected at random from each of the two honeybee subspecies and typed using an RFLP strategy (Fukatsu and Nikoh, 1998). RsaI-digested plasmid DNA produced three unique patterns from A. m. scutellata and eight from A. m. capensis; one banding pattern was shared by both for a total of 10 (Fig. 1). Two independent clones were sequenced from the transformants representing each of 8 banding patterns, but only one clone was available for 2 of the 10 banding patterns. Sequences from two clones from 7 of the 8 banding patterns displayed 4–7 bp differences, which could indicate that different bacterial strains co-exist in
Fig. 1. RFLP banding patterns of RsaI-digested plasmid DNA in 2% agarose TBE gel from 10 different E. coli transformants generated by cloning the 16S rRNA PCR products obtained from A. m. capensis and A. m. scutellata. Lanes: 1, DNA marker VI; 2, pAJ196; 3, pAJ198; 4, pAJ199; 5, pAJ203; 6, pAJ204; 7, pAJ205; 8, pAJ206; 9, pAJ207; 10, pAJ208; and 11, pAJ210. pAJ196–pAJ199 were cloned from A. m. scutellata and pAJ203–pAJ210 were from A. m. capensis.
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tellata (pAJ197, pAJ198, and pAJ199) and seven sequences from A. m. capensis (pAJ203–pAJ208 and pAJ210) for BLAST searches. All 10 sequences were unique and not represented in GenBank. Sequences from Lactobacillus crispatus (AF257097), Simonsiella muelleri (AF328148), Neisseria meningitidis (AF337937), Bartonella henselae (AJ223780), Serratia marcescens (AB061685), DpLE (U20279), Bifidobacterium indicum (D86188), and Gluconacetobacter sacchari (AF127412) were found to be related to one or more of the sequences from these honeybees. Nine other sequences, Escherichia coli (AE000474), Buchnera aphidicola (M27039), Bacillus subtilis (Z99107), M. plutonius (X75752), Rickettsia japonica (L36213), Wolbachia pipientis (U23709), and an endosymbiont of the rice weevil Sitophilus oryzae (AF005235) and parasitoid wasp Encarsia pergandiella (AF319783), were selected as outgroups. A CLUSTAL W alignment was made using
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these 26 bacterial 16S rRNA sequences. Phylogenetic trees were constructed using Maximum Parsimony, Neighbor-joining, and Maximum Likelihood methods. A phylogenetic tree constructed using the Distance and Neighbor-joining Kimura 2-parameter was found to display bootstrap values >50 for all branches (Fig. 2). A 1532-bp sequence obtained from A. m. scutellata (pAJ197) clustered with Lactobacillus crispatus (6.9% sequence divergence) (Fig. 2), a Gram-positive eubacterium. Stackebrandt and Goebel (1994) determined that bacterial strains displaying 3% divergence, or greater, in 16S rRNA sequences are nearly always from different species. Hence, it appears that this 16S rRNA sequence (AY370183) may be an unidentified honeybee Lactobacillus species. Two cultures of pleomorphic bacteria isolated from A. m. mellifera worker bees by Gilliam (1997) appeared to be members of the genus Lactobacillus based on microscopic observations and
Fig. 2. A Neighbor-joining tree generated for bacterial 16S rRNA sequences using a CLUSTAL W alignment with the optimality criteria set for Distance and Kimura 2-parameter in PAUP 4.0b10. Bootstrap values detected for 100 replicates are shown before the nodes. The bacterial 16S rRNA sequences obtained from A. m. capensis and A. m. scutellata are shown in bold.
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their ability to grow in different synthetic media. Recently, a partial 16S rRNA sequence related to Lactobacillus has been cloned during a survey for gut bacteria in the wasp Vespula germanica (Reeson et al., 2003). Lactobacillus bacteria are normal inhabitants of the gastrointestinal tract of man and animals and are known to be beneficials, involved in immunomodulation, interference with enteric pathogens and maintenance of a healthy intestinal microflora (Mitsuoka, 1992). They are facultatively anaerobic, non-motile, and non-spore forming, rod-shaped bacteria that adhere to the intestinal cell wall. They are known to produce D -lactic acid as a metabolic end product from hexose sugars via the Embden–Meyerhof pathway (Axelsson, 1998). A 1491-bp sequence obtained from A. m. capensis (pAJ207) (AY370184) was found to cluster with B. indicum (1.9% sequence divergence) (Fig. 2), a Gram-positive eubacterium. B. asteroides previously was identified from the A. m. mellifera midgut and rectum by microscopic observation (Rada et al., 1997). A 16S rRNA sequence of B. asteroides has been obtained from the human gut (M58730). However, the 16S rRNA sequence from A. m. capensis displayed 6.2% sequence divergence from the B. asteroides sequence isolated from the human gut, indicating that a bacterium more closely related to B. indicum appears to reside in A. m. capensis. Bifidobacteria are anaerobic branched or pleomorphic rod-shaped bacteria that commonly reside in the intestinal tract of humans and animals, adhering to the cell wall (Biavati et al., 2000). Bifidobacteria can be distinguished from other genera by the presence of an unique Ôbifidus pathwayÕ that is involved in fermenting glucose to produce acetate and lactate (Scardovi, 1986). Three sequences obtained from A. m. capensis [pAJ203 (1454 bp) (AY370185), pAJ208 (1453 bp) (AY370186), and pAJ210 (1454 bp) (AY370187)] exhibited a close relationship with each other; sequence divergences of 0.9 and 3.1% were obtained when the sequence that cloned into pAJ203 was compared to the sequences from pAJ208 and pAJ210, respectively. All three sequences clustered with a B. henselae sequence (Fig. 2), an a-proteobacterium and the casual agent of Bartonellosis in humans (Piemont and Heller, 1998). The three sequences displayed 3.6, 2.8, and 5% sequence divergences from B. henselae, respectively. A variety of arthropods are known to transmit Bartonella species; Bartonella quintana is vectored by human lice (Pediculus humanus and Pediculus capitis) and B. bacilliformis is carried by sandflies (Lutzomyia, Brumptomyia, and Warileya) (Marquardt et al., 2000). Recently, several 16S rRNA sequences closely related to human- and animal-pathogenic Bartonella spp., including B. henselae, B. quintana, and Bartonella vinsonii, were detected from ticks (Chang et al., 2001; Sanogo et al., 2003) and fleas (Parola et al., 2002). Detection of three Bartonella-related strains from these honeybees was novel and unexpected.
A 1455-bp sequence obtained from A. m. capensis (pAJ205) (AY370188) was found to cluster with a sequence from the a-proteobacterium G. sacchari, which is an intracellular gut symbiont located in the caecal bacteriocytes of pink sugarcane mealybug (Saccharococcus sacchari) (Fig. 2) (Franke et al., 2000). A divergence of 7.1% was detected between the 16S rRNA sequences obtained from A. m. capensis and S. sacchari, indicating that different bacterial species reside in these insects. G. sacchari cells are ellipsoidal to rod-shaped, and are present both as endophytes in sugarcane leaves as well as gut symbionts in the mealybug (Franke et al., 1999). Gluconacetobacter bacteria are known to live in a sugarrich environment, producing acetic acid from ethanol. Acetic acid-producing bacteria (called Gluconobacters) were reported earlier from A. m. mellifera (Lambert et al., 1981), but their 16S rRNA sequences have never been determined. The 16S rRNA sequence obtained from A. m. capensis (pAJ205) appears to be related to Gluconacetobacter and could be from a honeybee gut symbiont. Two sequences obtained from A. m. scutellata, pAJ198 (1505 bp) (AY370189) and pAJ199 (1503 bp) (AY370190), displayed 8.1% sequence divergence from each other and clustered with both S. muelleri and N. meningitidis sequences (Fig. 2), which are extracellular b-proteobacteria and human oral commensals, as well as facultative human pathogens (Hedlund and Staley, 2002; Taha et al., 2002). The 16S rRNA sequence from A. m. scutellata that cloned into pAJ198 displayed 5.6 and 7.3% sequence divergences from S. muelleri and N. meningitidis, respectively. An additional 16S rRNA sequence from A. m. scutellata that cloned into pAJ199 displayed 10.2 and 11.6% sequence divergences, respectively, from the same bacterial species. Bacteria related to Simonsiella and Neisseria have not been reported from arthropods, but an unidentified Neisseria species was detected in honey (Snowdon and Cliver, 1996). These sequences appear to be from two undescribed b-proteobacterial species because the sequence divergence detected was high (8.1%). Two sequences obtained from A. m. capensis [pAJ204 (1505 bp) (AY370191) and pAJ206 (1506 bp) (AY370192)] were similar (0.6% sequence divergence), differing by 6 substitutions and 3 insertions. Both sequences are related to sequences from an entomopathogen from Drosophila paulistorum (known by the common name DpLE) (Miller et al., 1995), and to Serratia marcescens, which is an opportunistic entomopathogen (Boucias and Pendland, 1998). The two sequences from A. m. capensis displayed 10 and 10.4% sequence divergences, respectively, from DpLE, while the same sequences from A. m. capensis displayed 10.5 and 10.6% sequence divergences, respectively, from S. marcescens. All four sequences (pAJ204, pAJ206, DpLE, and S. marcescens) clustered with c-proteobacteria, including Buchnera aphidicola,
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Escherichia coli, a primary endosymbiont from S. oryzae, and R. japonica (Fig. 2). Serratia are rod-shaped bacteria and attach to the foregut and midgut wall. S. marcescens is isolated frequently from insectaryreared insects kept under crowded unnatural conditions (King et al., 1975; Steinhaus, 1959). The sequences from A. m. capensis appear to be from two strains (0.6% sequence divergence) of an undescribed c-proteobacterial species related to S. marcescens.
among the bacteria from the two honeybee subspecies. Further assays might uncover more bacterial strains or species from these honeybees. The new honeybeespecific PCR primers (Table 1) provide a tool for researchers to investigate the role of these microbes in the biology and ecology of A. m. capensis and A. m. scutellata.
3.3. Bacterial genera detected from either A. m. capensis or A. m. scutellata are present in both subspecies
To resolve whether the bacteria are maternally transmitted to honeybee progeny or acquired from contaminated workers through feeding, genomic DNA was extracted from 20 pooled A. m. scutellata and A. m. capensis eggs and screened using the 7 honeybee-specific primer pairs (Table 1). No PCR products were obtained, indicating either that all the bacteria were acquired after the honeybees hatched or that the number of bacteria in the eggs was below detectable level. However, Wolbachia wsp primers readily amplified a 0.6-kb DNA band from these honeybee egg DNA preparations (data not shown here), indicating that microbial DNA can be amplified from eggs.
Because 16S rRNA sequences related to two genera were detected from A. m. scutellata (Lactobacillus and Simonsiella) and sequences related to five genera were detected from A. m. capensis (Bifidobacterium, Gluconacetobacter, Bartonella, Serratia, and Simonsiella) with only one sequence being common to both subspecies (Simonsiella), it was not clear whether A. m. scutellata and A. m. capensis have a different bacterial fauna or whether the eubacterial 16S rRNA primers annealed at random to some bacterial 16S rRNA sequences early in the High-fidelity PCR cycles and amplified only a subset of all the sequences present. In order to resolve this issue, honeybee bacteria-specific primers were designed. Sequences related to Lactobacillus (pAJ197), Bifidobacterium (pAJ207) and Gluconacetobacter (pAJ205), Bartonella (pAJ203, pAJ208 and pAJ210) and Serratia (pAJ204 and pAJ206) were used to design a specific primer for each (Table 1). Two sequences related to Simonsiella (pAJ198 and pAJ199) displayed sufficient sequence divergence (8.1%) that two specific primers could be designed (Table 1). Thus, seven different honeybee bacteria-specific forward primers were designed to detect 16S rRNA sequences related to six bacterial genera in A. m. capensis and/or A. m. scutellata. DNA was extracted from each of five individual honeybee abdomens belonging to workers of both A. m. scutellata and A. m. capensis and screened using Highfidelity PCR and the honeybee-specific forward primers (Table 1) in combination with the eubacterial reverse primer (1495r). All five A. m. scutellata and the five A. m. capensis bees tested produced the expected size DNA bands (Table 1) for all seven primer pairs tested (data not shown here). Hence, the initial limited number of cloned 16S rRNA sequences from A. m. scutellata (pAJ197–pAJ199) and A. m. capensis (pAJ203–pAJ208 and pAJ210) were likely due to the primers binding to some bacterial sequences by chance during the early High-fidelity PCR cycles and preferentially amplifying only some sequences. It appears that all honeybees contained all bacterial sequences identified so far. However, because these PCR products were not sequenced, we do not know if there were strain differences
3.4. Specific primers fail to amplify 16S rRNA from eggs
4. Conclusions Our screening of 16S rRNA sequences from A. m. capensis and A. m. scutellata found sequences related to two genera (Lactobacillus and Bifidobacterium) previously identified from honeybees, as well as sequences related to bacterial genera (Bartonella, Gluconacetobacter, Simonsiella, and Serratia) not previously identified from honeybees (Fig. 2). Honeybees are known to have complex interactions with their diet and with diverse microorganisms (Snowdon and Cliver, 1996). Bacteria belonging to Lactobacillus, Corynebacterium, and Bifidobacterium have been detected from A. m. mellifera, primarily by conventional culture or microscopic observations (Gilliam, 1997). Well over 6000 bacterial strains have been cultured from honeybees and frozen (Gilliam, 1997). Although it has been known for a long time that the honeybee gut has a rich microbial fauna (White, 1921), DNA sequence analyses of these microbes have been limited. A single Wolbachia wsp sequence (w Cap-B1) has been detected from both A. m. capensis and A. m. scutellata but is most likely associated with the reproductive tissues (Hoy et al., 2003) and not from gut microflora. The 16S rRNA sequences of M. plutonius and P. l. var. larvae, which causes European and American foulbrood diseases in honeybee larvae, have also been characterized (Alippi et al., 2002; Govan et al., 1998). None of the 10 16S rRNA sequences obtained in this study are related to either Wolbachia or M. plutonius or P. l. larvae.
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One objective of this study was to identify a bacterium uniquely associated with the A. m. capensis phenotype, but none has been detected so far. Despite this, if additional surveys are conducted, the possibility of finding additional bacterial species associated with these honeybees still remains, because well over 6000 bacterial strains are known to be associated with honeybees (Gilliam, 1997). Acknowledgments This work was supported in part by the Davies, Fisher, and Eckes Endowment in Biological Control to Marjorie A. Hoy at the University of Florida. We thank Carol Lauzon for comments on an earlier draft of the manuscript. This is Florida Agricultural Experiment Station journal publication R-09157.
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