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First confirmed report of a bacterial brood disease in stingless bees
Accepted Manuscript First confirmed report of a bacterial brood disease in stingless bees Jenny Lee Shanks, Anthony Mark Haigh, Markus Riegler, Robert Neil SpoonerHart PII: DOI: Reference:
S0022-2011(16)30189-6 http://dx.doi.org/10.1016/j.jip.2017.01.004 YJIPA 6906
To appear in:
Journal of Invertebrate Pathology
Received Date: Accepted Date:
6 November 2016 8 January 2017
Please cite this article as: Lee Shanks, J., Mark Haigh, A., Riegler, M., Neil Spooner-Hart, R., First confirmed report of a bacterial brood disease in stingless bees, Journal of Invertebrate Pathology (2017), doi: http://dx.doi.org/ 10.1016/j.jip.2017.01.004
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Short Communication
First confirmed report of a bacterial brood disease in stingless bees
Jenny Lee Shanksa,b, Anthony Mark Haigha, Markus Riegler c, Robert Neil Spooner-Hart1a,c
a
School of Science and Health, Western Sydney University, Locked Bag 1797, Penrith, NSW
2751, Australia b
c
Plant Health Australia, Phipps Close, Deakin, ACT 2600, Australia
Hawkesbury Institute for the Environment, Western Sydney University, Locked Bag 1797,
Penrith, NSW 2751, Australia
ABSTRACT
Susceptibility to brood pathogens in eusocial stingless bees (Meliponini), alternative pollinators to honey bees, is unknown. Brood losses in managed colonies of the Australian stingless bee, Tetragonula carbonaria, were studied over 20 months. We isolated a diseasecausing bacterium, Lysinibacillus sphaericus (Firmicutes, Bacillaceae), from worker and queen larvae, brood cell provisions and honey stores. Pathogenicity experiments confirmed this bacterium as the causal organism. It took 22 days from infection to first appearance of brood disease symptoms. This is the first confirmed record of a brood pathogen in stingless bees.
Keywords: Tetragonula carbonaria; honey bee; brood disease; Lysinibacillus sphaericus
1. Introduction Brood diseases are of concern for beekeeping because of the effects they have on hive population numbers. Control measures can include antibiotics; however, bacterial resistance is common. Apis mellifera can be infected with a number of diseases caused by pathogenic bacteria and fungi (Aronstein 2010, Genersch 2010, Genersch et al. 2010) whereas stingless bees appear to have few brood diseases. The only report is by Kerr (1948), who noted diseased pupae in the Brazilian stingless bees Melipona quadrifasciata and Melipona bicolor bicolor. Bacillus para-alvei was identified in 1957 as the possible causal organism (Nogueira-Neto 1997). However, at the time only microscopic investigations were performed without any description of pathogenicity, symptoms of the infection or molecular diagnostics. Apart from this, no other reports have been published. Prior to the current study, there were no recorded cases of brood disease in Australian stingless bees.
The Australian stingless bee, Tetragonula carbonaria, has been reported to be an effective crop pollinator (Dollin et al. 1997), due to its generalist foraging behaviour (Slaa et al. 2006). Tetragonula carbonaria is the most commonly kept (Halcroft et al. 2013) and widespread species of stingless bee, distributed from the warm tropical areas of coastal Queensland (QLD) (Cape York, 16º S, 145º E) to the temperate areas of southern New South Wales (NSW) (Bega, 36º 40.27’ S, 149º 50.34’ E), and has a flight temperature range between 18– 35ºC (Rayment 1935). We report the first detection and identification of a bacterial brood disease, discovered in a T. carbonaria hive showing brood losses from December 2012. Over a 20-month observational period starting from March 2013, a further seven colonies showed symptoms of infection.
2. Materials and Methods
Tetragonula carbonaria colonies were kept at the Hawkesbury campus apiary, Western Sydney University in Richmond (33º 36.42’ S, 150º 44.44’ E), NSW, Australia, within their natural range. They were originally sourced from south-east QLD (Australian Stingless Native Bees, Hatton Vale). Four colonies arrived at the apiary in March 2011, and appeared healthy. From then to the first symptoms of disease they experienced normal environmental conditions, with ambient temperatures ranging between 1.5 and 37ºC. However, in December 2012 (after 18 months), upon opening, one hive showed atypical spiral brood structures containing discoloured larvae. The hive was subsequently assessed for its internal colour, structural appearance of the brood, texture of storage pots, involucrum appearance and coverage, formation of brood nest, worker population, in-hive worker behaviour, and colony odour. All brood cells were removed from the colony, and any larvae showing discolouration or fluid appearance in opened cells were separately placed into 1.5 mL sterile Eppendorf tubes. Samples were processed under aseptic conditions, using procedures reported for diagnosis of Paenibacillus larvae-infected honey bee larvae (World Organisation for Animal Health 2013). Individual samples were plated onto Sheep Blood Agar (SBA) containing the antibiotic nalidixic acid (3 mg/ mL), and incubated for 24–48 h at 37ºC in 5% CO2 for further characterisation and pathogenicity testing. After incubation, discrete colonies were observed. Individual colonies were plated onto fresh SBA plates. The SBA cultures were subsequently subcultured and maintained on nutrient agar at 37ºC in 5% CO2. Samples were prepared for Gram staining and biochemical profiling via catalase testing (Reiner 2010, World Organisation for Animal Health 2013). The pathogenicity of the isolated bacterium was tested against healthy T. carbonaria brood. Two strong hives of equal strength with regard to honey and pollen provisions, workers and brood, determined by estimating the brood populations (Michener 1961, Roubik 1979), were selected. Before sunrise (i.e. prior to bee flight activity), one hive was split into two halves;
these two halves were used as the uninoculated control. Each of the two control halves were treated with a total of 105 mL of sterile water. Water was applied first using a 10 mL sterile plastic syringe to dispense the water directly into all opened brood cells and storage pots, after which any remaining water was uniformly applied using a 500 mL sterile plastic hand sprayer with fine spray nozzle over the entire brood chamber, storage pots and nest structures. A 3 mm thick acrylic lid was placed onto the open side and sealed with 48 mm wide masking tape. Both control box halves were placed in a temperature controlled room at 26ºC. The second hive was split and processed in the same manner as the control, but treated with a highly concentrated spore suspension (total 105 mL) containing approximately 400 million CFUs of the isolated bacterium. To reduce possible environmental contamination, the colonies were not allowed to forage; however, in-hive stores were plentiful. Hives were observed 48 hours after treatment, then weekly until first symptoms of disease. The inoculated lower hive half showed symptoms of disease after three weeks and was examined before sunrise. Samples of brood, callows (newly emerged workers), adult workers and honey were collected as described previously. The inoculated upper half was also removed and sampled similarly. Bacteria were isolated and cultured on SBA media as previously described. Furthermore, discrete, re-isolated colonies from these bacterial cultures were used for DNA extraction (Isolate II Genomic DNA Kit, Bioline, London, UK), following the manufacturer’s protocol; and then amplified using 16S rDNA primers (530F and 1495R) (Madrid et al 2001), with the following modifications: 95ºC, 5 min, 35 cycles of 94ºC, 1 min; 50ºC, 30 sec; 72ºC, 1.5 min, followed by a final extension step at 72ºC for 10 min. This was followed by molecular cloning using the pGEM®–T Easy Vector System I (Promega, Madison, Wisconsin, USA). Cleaned colony PCR products were sent to Macrogen (Geumcheon, South Korea) for sequencing. In addition, the bacterial samples were subjected to multi locus sequence typing (MLST) analysis of four loci (adk, glcK, glpF, glyA)
following the protocol outlined by Ge et al. (2011). The sequences of the MLST loci were aligned with sequences of eight previously characterised L. sphaericus isolates, four that are toxic (ST-2, ST-4, ST-5, ST-9) and four non-toxic (ST-3, ST-7, ST-8, ST-17) to mosquitos (Ge et al. 2011). Then, a neighbour-joining phylogenetic tree based on the Tamura 3parameter using a discrete Gamma distribution (T92+G) model was constructed for the 1,622 bp concatenated sequence of the four MLST loci of L. sphaericus. Samples of brood collected from the initial infected hive were also sent to the State Veterinary Diagnostic Laboratory of the Elizabeth Macarthur Agricultural Institute, NSW Department of Primary Industries, for isolation and identification of the putative pathogen.
3. Results The diseased hive identified in December 2012 had reduced forager activity on an ideal foraging summer day (warm and sunny). Most workers were observed to be moving very slowly or were motionless, not performing any tasks in the colony. The few workers that were performing normal nest behaviours were slower in performance and action than normal (Table 1). The brood structure was small, non-uniform, scattered, and with few brood cells (Figure 1). The hive materials were dark in colour and thick in structure. Developing larvae had changed in appearance, colour and texture (Table 1, Figure 1). Many infected larvae had been removed from their cells and were located singly or in small groups, mainly on the involucrum. A prominent rotting smell was detected in infected brood cells; cell provisions had a greenish-yellow colour (Figure 1). It appeared that the queen was absent as no newly laid eggs were observed. SBA plates were dominated by one bacterial species, which was both Gram and catalase positive. In the pathogenicity tests, the bacterium induced disease symptoms (i.e. the presence of brown larvae) at 22 days after inoculation. The re-isolated bacterium (ex larvae, brood provisions and honey stores) was confirmed as Lysinibacillus
sphaericus comb. nov. (Ahmed et al. 2007) formerly known as Bacillus sphaericus Meyer and Neide, 1904 (Firmicutes, Bacillaceae), by sequencing of the 16S rDNA (GenBank accession numbers: KR947300–KR947307). The16S rDNA sequences matched L. sphaericus and L. fusiformes isolates on GenBank. The State Veterinary Diagnostic Laboratory confirmed this also for samples from the initially infected hive. According to a phylogenetic sequence analysis for the four MLST loci adk (KT285615), glcK (KT285614), glpF (KT285616), and glyA (KT285613) the T. carbonaria isolate was similar to non-toxic ST-7 L. sphaericus NRS-1693 (Supplementary Figure S1), with a sequence divergence of about 1.6% (Supplementary Table S1).
4. Discussion This is the first account of a brood disease that was confirmed by pathogenicity testing in stingless bees. The pathogen L. sphaericus was isolated from T. carbonaria worker and queen larvae, cell provisions and honey stores and was shown to infect a new, healthy, colony. Lysinibacillus sphaericus occurs widely in the environment, including in soil (Massie et al. 1985), as an endophyte in plant material (Melnick et al. 2011), and has also been isolated from aquatic habitats and mosquito cadavers (Guerineau et al. 1991, Ludwig et al. 2009), under aerobic conditions (Hu et al. 2008). It has also been recognised as an emerging insect pathogen that is also used for mosquito population control (Berry 2011). However, the L. sphaericus strain detected in T. carbonaria honey stores is different to the strains used for mosquito control and is related to L. sphaericus NRS-1693, a non-toxic strain with similarities also to L. fusiformis (Ge et al. 2011, Xu et al. 2015). It is not clear how it causes pathology in T. carbonaria, and perhaps infections have developed when the colonies were weakened due to external factors, such as environmental stress. Tetragonula carbonaria
colony members do not progressively feed their young. If cells are provisioned with contaminated food sources, the young larvae feeding on the contaminated provisions will ingest the spores. When used to control mosquito populations, spores of several virulent strains of L. sphaericus are ingested by the larvae, germinate inside the host and cause disease (World Health Organisation 1985). Similarly, P. larvae spores are only infectious to 12–36 h-old A. mellifera larvae and not adults, after ingestion of contaminated food (Genersch 2010). A similar process may occur for the L. sphaericus strain infection of T. carbonaria, where it resulted in reduced colony populations and lack of maintenance of hive structures. Recently, a L. sphaericus strain with an identical 16S rDNA sequence (KR947308) has been isolated from a symptomatic Austroplebeia australis colony (M. Halcroft 2013 pers. comm., J. Shanks, personal observations), and these findings warrant further research into the prevalence of L. sphaericus infections across Meliponini.
5. Acknowledgements The authors would like to thank Dr Tim Heard, and Russell Zabel for the supply of stingless bee hives, Western Sydney University apiarist Michael Duncan for his assistance and input in the meliponary and apiary aspects. MLST sequences for L. sphaericus were kindly provided by Dr. Zhiming Yuan. The project was funded by Western Sydney University and the Hawkesbury Institute for the Environment.
6. References Ahmed I, Yokota A, Yamazoe A & Fujiwara T (2007). Proposal of Lysinibacillus boronitolerans gen. nov. sp. nov., and transfer of Bacillus fusiformis to Lysinibacillus fusiformis comb. nov. and Bacillus sphaericus to Lysinibacillus sphaericus comb.
nov. International Journal of Systematic and Evolutionary Microbiology, 57, 11171125. Aronstein KA & Murray KD (2010). Chalkbrood disease in honey bees. Journal of Invertebrate Pathology, 103, s20-s29. Berry C (2011). The bacterium, Lysinibacillus sphaericus, as an insect pathogen. Journal of Invertebrate Pathology, 109, 1-10. Dollin AE, Dollin LJ & Sakagami SF (1997). Australian stingless bee of the genus Trigona (Hymenoptera: Apidae). Invertebrate Taxonomy, 11, 861-896. Ge Y, Hu X, Zheng D, Wu Y & Yuan Z (2011). Allelic diversity and population structure of Bacillus sphaericus as revealed by multilocus sequence typing. Applied and Environmental Microbiology, 77, 5553-5556. Genersch E (2010). American Foulbrood in honeybees and its causative agent, Paenibacillus larvae. Journal of Invertebrate Pathology, 103, s10-s19. Genersch E, Evans JD & Fries I (2010). Honey bee disease overview. Journal of Invertebrate Pathology, 103, 52-54. Guerineau M, Alexander B & Priest FG (1991). Isolation and identification of Bacillus sphaericus strains pathogenic for mosquito larvae. Journal of Invertebrate Pathology, 57, 325-333. Halcroft MT, Spooner-Hart R, Haigh AM, Heard TA & Dollin A (2013). The Australian stingless bee industry: a follow-up survey, one decade on. Journal of Apicultural Research, 52, 1-7. Hu X, Fan W, Liu H, Zheng D, Li Q, Dong W, Yan J, Gao M, Berry C & Yuan Z (2008). Complete genome sequence of the mosquitocidal bacterium Bacillus sphaericus C341 and comparison with those of closely related Bacillus species. Journal of Bacteriology, 190, 2892-2902.
Kerr WE (1948). Estudos sôbre o gênero Melipona. Doctoral thesis, Universidade de São Paulo. Ludwig W, Schleifer KL & Whitman WB (2009). Class I. Bacilli class. nov.: Bacillus sphaericus. pp 117. In: Bergey's Manual of Systematic Bacteriology. Volume 3: The Firmicutes. De Vos P, Garrity GM, Jones D, Krieg NR, Ludwig W, Rainey FA, Schleifer KH & Whitman WB (eds.) 2nd ed. New York: Springer. Massie J, Roberts G & White PJ (1985). Selective isolation of Bacillus sphaericus from soil by use of acetate as the only major source of carbon. Applied and Environmental Microbiology, 49, 1478-1481. Melnick RL, Suárez C, Bailey BA & Backman PA (2011). Isolation of endophytic endsporeforming bacteria from Theobroma cacao as potential biological control agents of cacao disease. Biological Control, 57, 236-245. Michener CD (1961). Observations on the nests and behavior of Trigona in Australia and New Guinea (Hymenoptera, Apidae). American Museum Novitates, 2026, 1-46. Nogueira-Neto P (1997). Life and the creation of indigenous stingless bee. 447 pages. São Paulo, Parma Publishing Ltd. Rayment T (1935). A cluster of bees. 390 pages. Sydney, The Endeavour Press. Reiner K (2010). ASM microbe library: catalase test protocol [Online]. Washington, American Society for Microbiology. Available: http://www.microbelibrary.org/library/laboratory-test/3226-catalase-test-protocol (20/11/2015). Roubik DW (1979). Nest and colony characteristics of stingless bees from French Guiana (Hymenoptera: Apidae). Journal of the Kansas Entomological Society, 52, 443-470. Slaa EJ, Sanchez Chaves LA, Malagodi-Braga KS & Hofstede FE (2006). Stingless bees in applied pollination: practice and perspectives. Apidologie, 37, 293-315.
World Health Organization (1985). Informal consultation on the development of Bacillus sphaericus as a microbial larvicide. Program for Research and Training in Tropical Diseases. TDR/BCV/SPHAERICUS/85.3. 24 pages. Geneva, World Health Organization. Available: http://apps.who.int/iris/bitstream/10665/60326/1/TDR_BCV_SPHAERICUS_85.3.pd f (29/01/2016). World Organisation for Animal Health (2013). American foulbrood of honey bees. Manual of diagnostic tests and vaccines for terrestrial animals. Available: http://www.oie.int/fileadmin/Home/fr/Health_standards/tahm/2.02.02_AMERICAN_ FOULBROOD.pdf (29/01/2016). Xu K, Yuan Z, Rayner S & Hu X (2015). Genome comparison provides molecular insights into the phylogeny of the reassigned new genus Lysinibacillus. BMC Genomics 16, 140.
Figure 1. Signs and symptoms of a bacterial brood infection in a Tetragonula carbonaria colony. a) Typical healthy nest showing a central circular disc with good structural involucrum surrounding the brood chamber. b) Unhealthy nest, the brood cells are scattered, forming no clear disc structure and little structural involucrum is present. c) Brood with cell caps removed. Darker coloured cell caps containing greyish to brown discoloured larvae; light coloured cells containing healthy, white, larvae. d) Greenishyellow coloured, thick fluid cell provisions of an infected hive. e) to h) Unhealthy T. carbonaria larvae displaying varying colour symptoms, depending on stage of disease development. Larvae are occasionally deposited into small groups once they are removed from their brood cells by workers. All images, except a), were recorded from one hive on the same day.
Table 1. Comparison of healthy and unhealthy nest characteristics. Healthy
Colony strength Odour
Storage pots
Unhealthy
Large adult population, with
Adult population reduced, little
strong entrance activity of
entrance activity
foragers and cleaners Strong smell of plant resins
Pungent, decaying, rotten smell
Oval, bright orange-brown, thin,
Oval, darker brown, thick and
with smooth appearance. Glossy
tough appearance. Honey and
honey and fresh, moist pollen
pollen may be present but do not appear to be newly collected
Involucrum
Brood chamber
Orange, soft and malleable,
Dark brown, tough and thick
smooth lines and finishes, strong
network, dry and brittle in
network in nest and multiple (4+)
advanced stages; may cover the
thin layers covering brood
brood chamber but limited to 1–2
chamber
layers
Large, characteristic spiral
Variety of sizes (depending on
formation, with leading edge of
infection stage), may lack spiral
newly laid eggs, fresh healthy
formation, cells are scattered and
colour of newly made cerumen
may lack leading edge (absence of queen), lack of developing pupae, overall colour variable
Brood cell exterior Cell provisions
Oval, fully formed swollen caps,
Oval or irregular oval with
soft texture, bright yellow-orange
flattened caps, thick dark orange
colour Smooth, glossy, yellow to orange
Thick, dark yellow to green
Glossy, white, soft, solid mass
Half to full brown to black in colour, fluid-like or dry, may form short rope with matchstick
Larvae
test, may be dumped singly or in small groups on nest structures Worker behaviour
Actively moving (flying, walking,
Motionless, lethargic, will
preening), performing hive tasks,
walk/crawl out of nest, will not
will defend nest if opened
aggressively defend nest if opened
The article describes the first confirmed record of a brood disease in stingless bees (Meliponini). We anticipate that our manuscript will find high interest in the research community working on stingless bees. In particular we have been recently contacted by researchers in South America who also found similar symptoms of a potential bacterial brood disease in South American bees, and they are keen to already refer to our study.
S0022-2011(16)30189-6 http://dx.doi.org/10.1016/j.jip.2017.01.004 YJIPA 6906
To appear in:
Journal of Invertebrate Pathology
Received Date: Accepted Date:
6 November 2016 8 January 2017
Please cite this article as: Lee Shanks, J., Mark Haigh, A., Riegler, M., Neil Spooner-Hart, R., First confirmed report of a bacterial brood disease in stingless bees, Journal of Invertebrate Pathology (2017), doi: http://dx.doi.org/ 10.1016/j.jip.2017.01.004
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Short Communication
First confirmed report of a bacterial brood disease in stingless bees
Jenny Lee Shanksa,b, Anthony Mark Haigha, Markus Riegler c, Robert Neil Spooner-Hart1a,c
a
School of Science and Health, Western Sydney University, Locked Bag 1797, Penrith, NSW
2751, Australia b
c
Plant Health Australia, Phipps Close, Deakin, ACT 2600, Australia
Hawkesbury Institute for the Environment, Western Sydney University, Locked Bag 1797,
Penrith, NSW 2751, Australia
ABSTRACT
Susceptibility to brood pathogens in eusocial stingless bees (Meliponini), alternative pollinators to honey bees, is unknown. Brood losses in managed colonies of the Australian stingless bee, Tetragonula carbonaria, were studied over 20 months. We isolated a diseasecausing bacterium, Lysinibacillus sphaericus (Firmicutes, Bacillaceae), from worker and queen larvae, brood cell provisions and honey stores. Pathogenicity experiments confirmed this bacterium as the causal organism. It took 22 days from infection to first appearance of brood disease symptoms. This is the first confirmed record of a brood pathogen in stingless bees.
Keywords: Tetragonula carbonaria; honey bee; brood disease; Lysinibacillus sphaericus
1. Introduction Brood diseases are of concern for beekeeping because of the effects they have on hive population numbers. Control measures can include antibiotics; however, bacterial resistance is common. Apis mellifera can be infected with a number of diseases caused by pathogenic bacteria and fungi (Aronstein 2010, Genersch 2010, Genersch et al. 2010) whereas stingless bees appear to have few brood diseases. The only report is by Kerr (1948), who noted diseased pupae in the Brazilian stingless bees Melipona quadrifasciata and Melipona bicolor bicolor. Bacillus para-alvei was identified in 1957 as the possible causal organism (Nogueira-Neto 1997). However, at the time only microscopic investigations were performed without any description of pathogenicity, symptoms of the infection or molecular diagnostics. Apart from this, no other reports have been published. Prior to the current study, there were no recorded cases of brood disease in Australian stingless bees.
The Australian stingless bee, Tetragonula carbonaria, has been reported to be an effective crop pollinator (Dollin et al. 1997), due to its generalist foraging behaviour (Slaa et al. 2006). Tetragonula carbonaria is the most commonly kept (Halcroft et al. 2013) and widespread species of stingless bee, distributed from the warm tropical areas of coastal Queensland (QLD) (Cape York, 16º S, 145º E) to the temperate areas of southern New South Wales (NSW) (Bega, 36º 40.27’ S, 149º 50.34’ E), and has a flight temperature range between 18– 35ºC (Rayment 1935). We report the first detection and identification of a bacterial brood disease, discovered in a T. carbonaria hive showing brood losses from December 2012. Over a 20-month observational period starting from March 2013, a further seven colonies showed symptoms of infection.
2. Materials and Methods
Tetragonula carbonaria colonies were kept at the Hawkesbury campus apiary, Western Sydney University in Richmond (33º 36.42’ S, 150º 44.44’ E), NSW, Australia, within their natural range. They were originally sourced from south-east QLD (Australian Stingless Native Bees, Hatton Vale). Four colonies arrived at the apiary in March 2011, and appeared healthy. From then to the first symptoms of disease they experienced normal environmental conditions, with ambient temperatures ranging between 1.5 and 37ºC. However, in December 2012 (after 18 months), upon opening, one hive showed atypical spiral brood structures containing discoloured larvae. The hive was subsequently assessed for its internal colour, structural appearance of the brood, texture of storage pots, involucrum appearance and coverage, formation of brood nest, worker population, in-hive worker behaviour, and colony odour. All brood cells were removed from the colony, and any larvae showing discolouration or fluid appearance in opened cells were separately placed into 1.5 mL sterile Eppendorf tubes. Samples were processed under aseptic conditions, using procedures reported for diagnosis of Paenibacillus larvae-infected honey bee larvae (World Organisation for Animal Health 2013). Individual samples were plated onto Sheep Blood Agar (SBA) containing the antibiotic nalidixic acid (3 mg/ mL), and incubated for 24–48 h at 37ºC in 5% CO2 for further characterisation and pathogenicity testing. After incubation, discrete colonies were observed. Individual colonies were plated onto fresh SBA plates. The SBA cultures were subsequently subcultured and maintained on nutrient agar at 37ºC in 5% CO2. Samples were prepared for Gram staining and biochemical profiling via catalase testing (Reiner 2010, World Organisation for Animal Health 2013). The pathogenicity of the isolated bacterium was tested against healthy T. carbonaria brood. Two strong hives of equal strength with regard to honey and pollen provisions, workers and brood, determined by estimating the brood populations (Michener 1961, Roubik 1979), were selected. Before sunrise (i.e. prior to bee flight activity), one hive was split into two halves;
these two halves were used as the uninoculated control. Each of the two control halves were treated with a total of 105 mL of sterile water. Water was applied first using a 10 mL sterile plastic syringe to dispense the water directly into all opened brood cells and storage pots, after which any remaining water was uniformly applied using a 500 mL sterile plastic hand sprayer with fine spray nozzle over the entire brood chamber, storage pots and nest structures. A 3 mm thick acrylic lid was placed onto the open side and sealed with 48 mm wide masking tape. Both control box halves were placed in a temperature controlled room at 26ºC. The second hive was split and processed in the same manner as the control, but treated with a highly concentrated spore suspension (total 105 mL) containing approximately 400 million CFUs of the isolated bacterium. To reduce possible environmental contamination, the colonies were not allowed to forage; however, in-hive stores were plentiful. Hives were observed 48 hours after treatment, then weekly until first symptoms of disease. The inoculated lower hive half showed symptoms of disease after three weeks and was examined before sunrise. Samples of brood, callows (newly emerged workers), adult workers and honey were collected as described previously. The inoculated upper half was also removed and sampled similarly. Bacteria were isolated and cultured on SBA media as previously described. Furthermore, discrete, re-isolated colonies from these bacterial cultures were used for DNA extraction (Isolate II Genomic DNA Kit, Bioline, London, UK), following the manufacturer’s protocol; and then amplified using 16S rDNA primers (530F and 1495R) (Madrid et al 2001), with the following modifications: 95ºC, 5 min, 35 cycles of 94ºC, 1 min; 50ºC, 30 sec; 72ºC, 1.5 min, followed by a final extension step at 72ºC for 10 min. This was followed by molecular cloning using the pGEM®–T Easy Vector System I (Promega, Madison, Wisconsin, USA). Cleaned colony PCR products were sent to Macrogen (Geumcheon, South Korea) for sequencing. In addition, the bacterial samples were subjected to multi locus sequence typing (MLST) analysis of four loci (adk, glcK, glpF, glyA)
following the protocol outlined by Ge et al. (2011). The sequences of the MLST loci were aligned with sequences of eight previously characterised L. sphaericus isolates, four that are toxic (ST-2, ST-4, ST-5, ST-9) and four non-toxic (ST-3, ST-7, ST-8, ST-17) to mosquitos (Ge et al. 2011). Then, a neighbour-joining phylogenetic tree based on the Tamura 3parameter using a discrete Gamma distribution (T92+G) model was constructed for the 1,622 bp concatenated sequence of the four MLST loci of L. sphaericus. Samples of brood collected from the initial infected hive were also sent to the State Veterinary Diagnostic Laboratory of the Elizabeth Macarthur Agricultural Institute, NSW Department of Primary Industries, for isolation and identification of the putative pathogen.
3. Results The diseased hive identified in December 2012 had reduced forager activity on an ideal foraging summer day (warm and sunny). Most workers were observed to be moving very slowly or were motionless, not performing any tasks in the colony. The few workers that were performing normal nest behaviours were slower in performance and action than normal (Table 1). The brood structure was small, non-uniform, scattered, and with few brood cells (Figure 1). The hive materials were dark in colour and thick in structure. Developing larvae had changed in appearance, colour and texture (Table 1, Figure 1). Many infected larvae had been removed from their cells and were located singly or in small groups, mainly on the involucrum. A prominent rotting smell was detected in infected brood cells; cell provisions had a greenish-yellow colour (Figure 1). It appeared that the queen was absent as no newly laid eggs were observed. SBA plates were dominated by one bacterial species, which was both Gram and catalase positive. In the pathogenicity tests, the bacterium induced disease symptoms (i.e. the presence of brown larvae) at 22 days after inoculation. The re-isolated bacterium (ex larvae, brood provisions and honey stores) was confirmed as Lysinibacillus
sphaericus comb. nov. (Ahmed et al. 2007) formerly known as Bacillus sphaericus Meyer and Neide, 1904 (Firmicutes, Bacillaceae), by sequencing of the 16S rDNA (GenBank accession numbers: KR947300–KR947307). The16S rDNA sequences matched L. sphaericus and L. fusiformes isolates on GenBank. The State Veterinary Diagnostic Laboratory confirmed this also for samples from the initially infected hive. According to a phylogenetic sequence analysis for the four MLST loci adk (KT285615), glcK (KT285614), glpF (KT285616), and glyA (KT285613) the T. carbonaria isolate was similar to non-toxic ST-7 L. sphaericus NRS-1693 (Supplementary Figure S1), with a sequence divergence of about 1.6% (Supplementary Table S1).
4. Discussion This is the first account of a brood disease that was confirmed by pathogenicity testing in stingless bees. The pathogen L. sphaericus was isolated from T. carbonaria worker and queen larvae, cell provisions and honey stores and was shown to infect a new, healthy, colony. Lysinibacillus sphaericus occurs widely in the environment, including in soil (Massie et al. 1985), as an endophyte in plant material (Melnick et al. 2011), and has also been isolated from aquatic habitats and mosquito cadavers (Guerineau et al. 1991, Ludwig et al. 2009), under aerobic conditions (Hu et al. 2008). It has also been recognised as an emerging insect pathogen that is also used for mosquito population control (Berry 2011). However, the L. sphaericus strain detected in T. carbonaria honey stores is different to the strains used for mosquito control and is related to L. sphaericus NRS-1693, a non-toxic strain with similarities also to L. fusiformis (Ge et al. 2011, Xu et al. 2015). It is not clear how it causes pathology in T. carbonaria, and perhaps infections have developed when the colonies were weakened due to external factors, such as environmental stress. Tetragonula carbonaria
colony members do not progressively feed their young. If cells are provisioned with contaminated food sources, the young larvae feeding on the contaminated provisions will ingest the spores. When used to control mosquito populations, spores of several virulent strains of L. sphaericus are ingested by the larvae, germinate inside the host and cause disease (World Health Organisation 1985). Similarly, P. larvae spores are only infectious to 12–36 h-old A. mellifera larvae and not adults, after ingestion of contaminated food (Genersch 2010). A similar process may occur for the L. sphaericus strain infection of T. carbonaria, where it resulted in reduced colony populations and lack of maintenance of hive structures. Recently, a L. sphaericus strain with an identical 16S rDNA sequence (KR947308) has been isolated from a symptomatic Austroplebeia australis colony (M. Halcroft 2013 pers. comm., J. Shanks, personal observations), and these findings warrant further research into the prevalence of L. sphaericus infections across Meliponini.
5. Acknowledgements The authors would like to thank Dr Tim Heard, and Russell Zabel for the supply of stingless bee hives, Western Sydney University apiarist Michael Duncan for his assistance and input in the meliponary and apiary aspects. MLST sequences for L. sphaericus were kindly provided by Dr. Zhiming Yuan. The project was funded by Western Sydney University and the Hawkesbury Institute for the Environment.
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Figure 1. Signs and symptoms of a bacterial brood infection in a Tetragonula carbonaria colony. a) Typical healthy nest showing a central circular disc with good structural involucrum surrounding the brood chamber. b) Unhealthy nest, the brood cells are scattered, forming no clear disc structure and little structural involucrum is present. c) Brood with cell caps removed. Darker coloured cell caps containing greyish to brown discoloured larvae; light coloured cells containing healthy, white, larvae. d) Greenishyellow coloured, thick fluid cell provisions of an infected hive. e) to h) Unhealthy T. carbonaria larvae displaying varying colour symptoms, depending on stage of disease development. Larvae are occasionally deposited into small groups once they are removed from their brood cells by workers. All images, except a), were recorded from one hive on the same day.
Table 1. Comparison of healthy and unhealthy nest characteristics. Healthy
Colony strength Odour
Storage pots
Unhealthy
Large adult population, with
Adult population reduced, little
strong entrance activity of
entrance activity
foragers and cleaners Strong smell of plant resins
Pungent, decaying, rotten smell
Oval, bright orange-brown, thin,
Oval, darker brown, thick and
with smooth appearance. Glossy
tough appearance. Honey and
honey and fresh, moist pollen
pollen may be present but do not appear to be newly collected
Involucrum
Brood chamber
Orange, soft and malleable,
Dark brown, tough and thick
smooth lines and finishes, strong
network, dry and brittle in
network in nest and multiple (4+)
advanced stages; may cover the
thin layers covering brood
brood chamber but limited to 1–2
chamber
layers
Large, characteristic spiral
Variety of sizes (depending on
formation, with leading edge of
infection stage), may lack spiral
newly laid eggs, fresh healthy
formation, cells are scattered and
colour of newly made cerumen
may lack leading edge (absence of queen), lack of developing pupae, overall colour variable
Brood cell exterior Cell provisions
Oval, fully formed swollen caps,
Oval or irregular oval with
soft texture, bright yellow-orange
flattened caps, thick dark orange
colour Smooth, glossy, yellow to orange
Thick, dark yellow to green
Glossy, white, soft, solid mass
Half to full brown to black in colour, fluid-like or dry, may form short rope with matchstick
Larvae
test, may be dumped singly or in small groups on nest structures Worker behaviour
Actively moving (flying, walking,
Motionless, lethargic, will
preening), performing hive tasks,
walk/crawl out of nest, will not
will defend nest if opened
aggressively defend nest if opened
The article describes the first confirmed record of a brood disease in stingless bees (Meliponini). We anticipate that our manuscript will find high interest in the research community working on stingless bees. In particular we have been recently contacted by researchers in South America who also found similar symptoms of a potential bacterial brood disease in South American bees, and they are keen to already refer to our study.