Greener Journal of Biological Sciences

Vol. 9(2), pp. 29-34, 2019

ISSN: 2276-7762

Copyright ©2019, the copyright of this article is retained by the author(s)

DOI Link: http://doi.org/10.15580/GJBS.2019.2.090119163  

https://gjournals.org/GJBS

 

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Detection of Aethina tumida Murray 1867 (Coleoptera: Nitidulidae) and the yeast Kodamaea ohmeri in Africanized honey bees from southeastern of Brazil

 

 

1 Edlene Ribeiro Prudêncio; 2 Geraldo dos Santos Oliveira; 3Maria Cristina Affonso Lorenzon*; 3 Alexandre dos Santos Nogueira; 1 Rosa Helena Luchese

 

 

1Food Microbiology Laboratory, Departmentof Food Technology, Federal Rural University of Rio de Janeiro, 23897-970 Seropédica, Rio de Janeiro, Brazil.

2 Fernandes Figueira Institute – Fiocruz; Av. Rui Barbosa 716, 22.250-020, Rio de Janeiro, RJ, Brazil.

3 Animal Production Department, Universidade Federal Rural do Rio de Janeiro, CEP 23897-000, Seropédica, Rio de Janeiro, Brazil.

 

 

ARTICLE INFO

ABSTRACT

 

Article No.: 090119163

Type: Research

DOI: 10.15580/GJBS.2019.2.090119163

 

 

The small hive beetle (SHB), is a significant pest of honeybee (Apis mellifera L.) hives in various regions throughout the world, including the southeastern Brazil. Samples of honeycombs and pollen, bees and SHB were collected from two apiaries located nearby Rio de Janeiro city, southeastern Brazil. Samples were homogenized and plated in Sabouraud agar. White, moist, creamy colonies were counted and identified phenotyphically as Kodamaea ohmeri. At the time the sampling was done, the yeast contamination was low in all parts of the hives but, yet above the Brazilian legal standards for honey, which established a maximum of 102 CFU g-1 of mold and yeasts.

 

Submitted: 01/09/2019

Accepted:  03/09/2019

Published: 26/09/2019

 

*Corresponding Author

Maria CA Lorenzon

E-mail: affonsoneta@ gmail. com

Phone: +55213787 3975

 

Keywords: honeybee; symbiosis; small beetle; beekeeping; Microbiology

 

 

 

Abbreviations used: SHB, small hive beetle; AHB, Africanized honey bee; CFU, colony forming unity; LAB, lactic acid bacteria.

 


 

 

INTRODUCTION

 

The first report of Aethina tumida (small hive beetle) in South America occurred in 2015, specifically in the state of São Paulo, Brazil (Al Toufailia et al., 2017; Cordeiro et al., 2019; Teixeira et al., 2016).One year later, a new invasion of this small beetle was registered in the neighboring state, Rio de Janeiro; its presence was documented in regions nearby the city of Rio de Janeiro (Lorenzon, 2017).

 

In the last three decades, this small beetle has drawn attention worldwide after a series of successful invasions into contrastingly different habitats (Lounsberry et al., 2010).What caused the entry of this animal in Brazil is still unknown, however the direction point out to the global travels and transportation of goods that enhance the chances in favor to biological invasions (Cassey et al., 2005; Cuthbertson et al., 2013; Cuthbertson and Brown, 2009).

 

As happened in other countries, the range of the spread of SHB(small honey beetle) in some Brazilian regions was surprising, which has mobilized intensive efforts to control the source of the invasions and, how to mitigate this feral beetle’s damage and managed honey bees (Al Toufailia et al., 2017; Mutinelli, 2011; Teixeira et al., 2016).

 

The factors that explain efficiency in long-range transportation from these beetles are due to their natural way of life, to voracity as a predator and a scavenger, to the wide flight (6 and 13 km from their nest), the use of counter resistant tactics, trophallactic mimicry etc.., under honeybee hives (Cuthbertson et al., 2013). Nowadays, Brazilian beekeepers were able to identify SHB immediately, this beetle easily found in the feral swarms of Africanized honey bees, which people removed and depend on them to supply its apiaries (Lorenzon et al., 2019). But moving honey bees like that, become beekeepers man-mediated migration to SHB.

 

SHB in its native range, feeds on a mixture of pollen, honey and bee brood (Neumann and Elzen, 2004).  From a mixture of pollen and honey, some volatiles are released which attracted SHB (Torto et al., 2007). These volatiles are due to fermentation by microorganisms including the yeast Kodamea ohmeri (Etchells and Bell) (Ascomycota: Saccharomycotina), isolated from the beetle feeding on pollen (Benda et al., 2008; Teal, 2006). Thus there is a relationship between the yeast K. ohmeri and A. tumida. Benda et al., (2008)detected yeasts of two strains de K. ohmerii samples obtained from hives in Florida (USA) and Kenya affected by Aethina. They found volatile profiles which were attractive to SHB and contained compounds also found in honey bee alarm pheromone.

 

Although a global potential threat for apiculture and wild swarms, the knowledge about the small hive beetle in Brazil is still limited, creating demands for more researches. In our short communication, we search for yeasts and molds involved in symbiotic relationship with SHB.

 

 

MATERIALS AND METHODS

 

Samples from apiaries

 

Sampling was done in May 2019 using six AHB (Africanized honeybee hives), which were located in two apiaries at county of Seropédica, state of Rio de Janeiro, southeastern Brazil (22°44'29''S, 43°42'19''W). The AHB hives were strong, with one to two supers, in a typical period of mass-flowering. Samples were obtained from the upper sides of the nest, taking: a) pieces of honeycombs and pollen (10g), b) bees from the honey combs (10g), d) SHB (10 specimens). These samples were stored into sterile containers, under refrigeration and sent to the laboratory at the same day, where they were stored at 5°C overnight before plating.

 

Yeast isolation

 

Honey, pollen and bees were homogenized in 90 mL of peptone water; small hive beetles (10 specimens) were homogenized in 10 mL at the same substrate, further all samples were diluted and surface plated on Sabouraud dextrose agar (Merck, Germany). Plates were incubated at 30°C for 72 hours for colony counting. At least five colonies of the predominant yeast of each sample were identified by morphological and biochemical tests

 

Microscopic Examination and Biochemical Identification

 

The isolated yeast colonies were re-isolated in special media, potato dextrose agar (Difco) and Lacrimel, (Sidrim and Moreira, 1999).They were also seeded in rice medium (Difco) for germ tube testing and chlamydoconides formation. In addition to the morphological tests, yeast isolates were identified through biochemical tests with the aid of equipment VITEC (BioMérieux, France).

                                         

 

RESULTS AND DISCUSSION

 

Yeast counts and Identification

 

            Plating of homogenized bodies of beetles, bees and honey and pollen combs resulted in smooth cream-colored yeast colonies.Yeast colony counts of all three samples ranged from 1.5 x 102 to 5.0 x 102 CFU g-1. No difference was observed between counts of different samples. At the time the sampling was done, the yeast contamination was low in all parts of the hives, probably due to the fast hygienic behaviour and defence mechanism of AHB (Guerra et al., 2000; Spivak, 1996). According to beekeepers from the same study site, they had a few losses caused by Aethina (personal observation). However, the values did not meet the legal standards (Brasil, 2000), which establish a maximum of102 CFU g-1 of mold and yeasts. Spiewok et al. (2007) observed that apiaries of one region are not equally infested and attributed to different environmental factors such as soil moisture or local beekeeping facilities.

 

The great majority of the yeast colonies in Sabouraud agar were white, moist, creamy (Figure 1). The yeast cells were ovoid to cylindrical (Figure 2). The 72h old culture showed 76% cell size variation, with dimensions ranging from 8.5 - 3.4x 6.4-2.6 µm (Figures 3). Neither isolates formed germ tubes but formed pseudomycelial cells as they aged. The isolates grew well at 37°C but showed only small growth at 42°C. The identity of the isolates using VITEC gave a level of confidence ranging from 88 to 93% probability for Kodamaea ohmeri.The same yeast was recovered not only in honey and combs, but also in bees and SHB.The relationship between K. ohmeri and its host is important for the development of management strategies for this important pest (Amos et al., 2018)

 

K. ohmeri, previously known as Yamadazymaohmeri, is ateleomorphic state of Candida guilliermondii or Trichosporonarenicola (Mycobank, 2019) and is also found in fermented foods like Asian indigenous fermented food (Aidoo et al., 2006) but is also recognized as a cause of life-threatening human infections being an opportunistic fungal pathogen (Shang et al., 2010).

 

According to Hayes et al. (2015) the attractiveness for the beetle of the fermenting hive products (‘slime’) increases as fermentation progressed, and volatile profiles become more complex; these volatile substances are produced by K. ohmeri fermentation. Fermenting hive products remain extremely attractive for more than 30 days, allowing the SHB to proliferate. Actually, the fermentation of hive products, caused by the yeast, can be a destructive and so economically significant part of A. tumida’s impact on commercial honeybee products in various regions throughout the world (Amos et al., 2019). In Brazil, dealing with AHB in a tropical area, the swarm absconding is typical, and can occur within seven days when it is due to the loss of its products (food and brood), that happens with Brazilian Sac Brood disease, which results in rapid fungal hive infection (Deveza et al., 2015). Thus, AHB can probably control the progress of fermentation from K. ohmeri. However, if the yeasts disseminate all over the hive, certainly the AHB swarm will abscond, and after that, the SHB will destroy the hive immediately. In this sense, this incidence widens to the margin of losses of hives from beekeeping in the state of Rio de Janeiro, which is already revealed to a high degree (>30%) (Lorenzon et al., 2012)

 

            Conklin (2012) examines the factors that affect the growth of K.ohmeri in the bee hive, including water activity, nutrient availability, the presence of beetles and bees. K. ohmeri is capable of growing on bee bread from bee hives without beetles present, and that water activity is important to the growth of K. ohmeri on bee bread and pollen. Beetle frass, which contains a high quantity of K. ohmeri, may be the source of yeast inoculum. According to (McGlashan, 2011) K. ohmeri, like most yeast associated with insects, is thought to inhabit its host’s intestines and probably be transmitted vertically via egg coating or mucilage, although horizontal transmission may also occur during contact between adults (as mating). Brogan et al. (2018) related the presence of K. ohmeri in each life stage of SHB (larva, pupa and adult).

 

K. ohmeri, like most yeast associated with insects and reducing the presence of this yeast would represent a strategy to control SHB. According to Olofsson et al.(2014) there is a symbiotic interaction between lactic acid bacteria and honey bees around the world. Lamei et al.(2019)showed that honey bee-specific lactic acid bacteria (hbs–LAB) had a major inhibitory effect on Paenibacillus larvae, a serious bacterial disease of honey bees.


 

 

Figure 1. Kodamae ohmeri colonies after 72h growing at 30°C on Sabouraud dextrose agar.

 

 

Figure 2.  Wet mount prepared (400x magnification) from a 72h old culture of Kodamae ohmeri grown on Sabouraud dextrose agar

 

Figure 3. Variation in cell size of Kodamae ohmeri with an average size of 5.7 x 4.3 µm and median size of 6.0 x 4.3 µm.

 

 


 

CONCLUSIONS

 

This is the first report in Brazil, presenting Kodamaea ohmeri in bee products, in AHB hives affected by SHB. Initially, for Brazilian beekeeping the impact of SHB on the parasitized AHB colonies may even be minimal. As rapid occupation throughout the world are strong signs for Brazilian beekeepers to worry about. Thus, it is crucial to prevent by starting in the knowledge of this small beetle in tropical regions. We believe that the reduction of K.ohmeri hive contamination may be related to the presence of competing microorganisms. In future work we intend to evaluate the presence of lactic acid bacteria in the hives and their relationship with the reduction of bee diseases and honey production.

 

Competing interests

 

We presented the need and the relevance of the knowledge about the intrinsic relationship between the small beetle Aethina and Africanized honeybees to warn about this parasite. Thus, our team will be assisting producers and scientists who must achieve more information of this new pest of honeybee in Brazil.

 
Authors' contributions
 

M Lorenzon, A Nogueira –Organized the sampling, reviewed and wrote part of the manuscript.

R.H.Luchese - Assistedin microbiological analysis and wrote and revised part of the manuscript 
E.R.Prudêncio- Performed the microbiological assay and the formatting of all paper and references
G.Oliveira - He was responsible for yeast identification.

 

 

Acknowledgements

 

The authors are thankful to beekeepers for helping with insect sampling. We are also grateful to Dr. Roberto Laureano, Department of Food Technology, Federal Rural University of Rio de Janeiro, for performing yeast cell size measurement.

 

 

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Cite this Article: Prudêncio ER; Oliveira GS; Lorenzon MCA; Nogueira AS; Luchese RH (2019). Detection of Aethina tumida Murray 1867 (Coleoptera: Nitidulidae) and the yeast Kodamaea ohmeri in Africanized honey bees from southeastern of Brazil. Greener Journal of Biological Sciences, 9(2): 29-34, https://doi.org/10.15580/GJBS.2019.2.090119163.