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.
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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