Journal of Phytopathology and Pest Management 5(3): 67-76, 2018
pISSN:2356-8577 eISSN: 2356-6507
Journal homepage: http://ppmj.net/
∗
Corresponding author:
A.S.M.H. ELRoby,
E-mail: ahmed.hussien1@mu.edu.eg
67
Copyright © 2018
Biological activity of certain natural products
against
Varroa destructor
(Acari:Varroidae)
and their selectivity against
Apis mellifera
(Hymenoptera: Apidae)
A. S. M. H. ELRoby
*
, M. G. Darwish
Plant Protection Department, Faculty of Agriculture, Minia University, Minia, Egypt
Abstract
Keywords: Varroa destructor, biological activity, natural products, Apis mellifera.
68
Introduction
Honey bee colonies are subject to
infestation by insects, mites and diseases.
The ectoparasitic mite,
Varroa destructor
(Anderson and Trueman) is considered
one of the most serious pests of beehives.
Parasitism can result in a loss of up to
25% of adult weight, severe deformations
of the wing and reduced longevity of
worker and drone honey bees (Hussein et
al., 2016). Colonies infested with this
mite have significantly reduced worker
bee populations and eventually die if left
without controlling (Al-Waili et al., 2012;
Ariana et al., 2002). The widespread use
of synthetic acaricides has led to the
accumulation of residues in beeswax,
propolis and to a much lesser degree, in
honey (Eshbah et al., 2018; Calatayud-
Vernich et al., 2017; Nai et al., 2017;
Bargańska et al., 2016; Calatayud-
Vernich et al., 2016; Chiesa et al., 2016;
Porrini et al., 2016; Sattler et al., 2016;
Hussein & Mostafa, 2009; Hussein et al.,
2002). The development of acaricide
resistance in
V. destructor
populations
and the spectra of the contamination of
hive products provide considerable need
to develop new treatment strategies that
minimize the potential for the rapid
development of resistance and the
accumulation of residues. Natural
products have potential as elements for
varroa mite control because some of them
are selective and have little or no harmful
effects on non-target species. Many
natural products known to possess
various bio efficacies such as ovicidal,
anti-feeding and bioactivities against
various pests without any adverse effects
on non-target species. and they are found
to be highly effective against insecticide
resistant pests (Benelli et al., 2018;
Eshbah et al., 2018; Calo et al., 2015).
Thus, the present studies were carried out
to evaluate the efficacy of certain natural
products on the parasitic mite
V.
destructor
(adult and brood infestation)
and their selectivity on
Apis mellifera
to
find out one of the natural products
maximizing Varroa control and
minimizing the site effect on the
honeybee colonies and environment, with
minimal costs.
Materials and methods
Experimental honeybee colonies:
Honeybee colonies (63 colonies) of the
first hybrid carnelian bees,
Apis mellifera
Carnica
were selected among the
colonies of a private apiary at Shosha
village 7, Minia Governorate. The
experimental colonies were divided into
7 groups, each having three sub groups
each sub group contains three colonies as
replicates. Average strength of the tested
colonies was 8 combs covered with bees
housed in Langstroth hive and the
experiment was repeated twice during
season 2018 and the averages of the
treatments were calculated.
Preparations products and application
of the tested plant
: Six plant natural
products were evaluated against varroa
mite infested the honeybee colonies
represented in Table 1. Three features
(extract, powder and original raw
materials (used as fumigation) of the
tested plant products were prepared as
follows.
Extracts:
The used part of the tested
plant products (Table.1) were washed
more once and left to dry under room
temperature (30
o
C±2). They were
grounded in an electric mill and sieved
through 0.5 mm sieve. Samples
powdered plant material (1000 g) except
69
Garlic were blended in 70% aqueous
ethanol and kept in dark container for 24
hours. The mixture of the dried plant
material and the solvent was stirred for
30 minutes using a magnetic stirrer. The
cured extracts were filtered firstly over
clean cotton followed by a filter paper
with anhydrous sodium sulfate. Then it as
evaporated under reduced pressure
using
a rotary evaporator. The farmed
concentrated extract was stored in glass
containers and maintained in the
refrigerator for the experimental tests.
Preparation of garlic extract:
The
Garlic extract prepared according to
Vijayalakshmi et al. (1999) method; in
brief 5 ml of (vegetable oil) was added to
85 grams of chopped Garlic and allowed
to stand for 24 hours, then 950 ml of
water was added and they mixed with 10
ml of soap. The mixture was filtered and
stored in covered bottle until treatment.
The extracts were used as 20 ml /colony
from the previous extract.
Powders:
The dried portions of the
tested plant products (Table 1) were
grinded and each properly mixed with
powdered sugar (1 part of the tested
product to 4 parts of powdered sugar to
constitute 6 powdered products, in
addition to powdered sugar which used
as control treatment. The powder
preparations were used through dusting
often achieved before sunset. Each
prepared powdered were applied as 40
gm/colony.
Table 1: English and scientific name of the source of the natural products.
English name
Scientific name
Family
The part used
Pomegranate
Punica granatum
Punicaceae
Peel
Garlic
Allium sativum
Liliaceae
Bulb
Marjoram
Majorana hortensis
Lamiaceae
Whole plant
Harmal
Peganum Harmala
Zyghophyllaceae
Seeds
Black cumin
Nigella sativa
Ranunculaceae
Seeds
Ambrosia
Ambrosia martitima
Compositeae
Leaves and flowers
The original raw materials
(Fumigation):
The dried parts of the
tested natural plant products were used
for producing a smoke through burning in
smoker which used in smoking on the
experimental colonies. Smoking of the
experimental bees was done through
fumigation. The colonies were divided
into seven groups for obtained the effect
of plant material treated with the plant
products as smoke material for 60
seconds every week for one month the
Varroa fallen on the sticky bottom board
of hives (covered with a plastic sheet
coated with raw Vaseline) were counted.
Evaluation the tested materials:
Level
of infestation on live bees was
determined through randomly collection
of about 50 bees /colony in a gar partially
filled with water and few drops of
detergent (Liquid soap). The collected
bees were shaken for 30 seconds, then
bee were filtered through muslin (8 to 12
mesh/ inch) to remove the bees, and the
passed adults of mite were then counted
(Komeili, 1988). This procedure was
done before the application and after 24,
48 hrs; 7, and 14 days of the application
and the treatment was applied new. Also,
infestation of brood was determined at
the same time of examination of the adult
bees. One hundred of sealed brood
worker per colony were randomly
70
chosen. The cell capping were removed
by forceps and the pupae were picked up
to examine for mites presence
(Marcangel et al., 1992). Counting the
number of dead mites was done through
providing the bottom brood of each brood
chamber with sticky white paper smeared
with thin layer of Vaseline. Evaluation
the efficacy of the tested natural plant
products in controlling Varroa mite, with
calculation reduction percentage of mite
infestation by applying Henderson and
Telton (1955) equation.
Where: T
a
is % infestation of mite after
treatment; T
b
is % infestation of mite
before treatment; C
b
is % infestation of
mite before treatment for the control. C
a
is % infestation of mite after treatment
for the control.
Assessment of the side effects of
applied natural plant products on daily
rate egg lying of the queens:
The combs
of the treated colonies were inspected at
4 days intervals for successive two days
period. Daily egg laying rate was
calculated by subtracting total number of
eggs of the first day out of the total
number of eggs of consequent day.
Counting of the egg cells based on the
method reported by (Kefuss, 1978).
Assessment of the side effects of
applied natural plant products on
larvae viability:
Determination of
pupation percentages was based on
calculation the percent of open brood
cells larva reached to sealed brood stage
(Pupa stage). Pupation percentages was
determined twice for each treatment
period. Pupation percentages was
determined through marking an area of
brood cells (100 cells) of each
experimental hive using transparent sheet
(Hassan & Aly, 1998). The marked area
was examined again after 5 days for
measurement the number of cells reached
to pupa stage and the larva viability
percentage was estimated by applying
the following equation
Assessment of the side effects of
applied natural plant products on
worker longevity:
Second day after the
treatment, the half of comb cages were
used for caging on a compact area (4 x 6
inch for each colony) of a sealed brood
comb of the treated colonies, twelve days
later, the caged area was inspected and
the combs with their loads of the half
comb cages having emerged bees were
got out from their colonies. The drawn
combs were brushed for removing the
un-caged bees; one hundred of emerged
bees were individually cached gently by
forces through a hole of their cage and
marked on their thorax with a pot of
quick- drying paint having a particular
color. All marked bees along with their
combs were subsequently reintroduced to
their respective colony by the swarm
introduction method (Grout, 1960). The
tested colonies were inspected in the
dusk at 4 days intervals until the
disappearance of the marked workers.
The longevity was calculated as the
midpoint between the rest day the bee
was observed and the next observation
date (Terada et al., 1975).
Assessment of the side effects of
applied natural plant products on
estimation of honey production:
Honey
71
yield in experimental colonies were
estimated by measuring open and sealed
honey areas in June and September and
transformed into weight using the
following formula according to (Shawer
et al., 1986).
10.64 =amount in grams of honey in one
square inch, based on averages calculated
from unsealed and sealed honey from
combs of different thickness.
Statistical analysis:
The collected data
were subjected to one way analysis of
variance according to method of Mead et
al. (1993). The statistical analyses were
conducted using the SPSS 16 and F- test.
Differences among means were
determined by Duncan's multiple range
tests.
Results and Discussion
Spraying application of the extracts of
tested natural products:
Results in
Table 2 showed that treatments the
infested colonies with dosage (20
ml/colony) of extracts of the tested
natural plant products resulted in
reduction of Varroa infestation by
various levels. The highest average
reduction percentage (91.88%) of adult
bee infestation was recorded with
spraying the extract of Harmal followed
with Pomegrante (91.06%) followed by
Black-cumin (89.11%) by the rate of 20
ml /colony and Garlic (88.82%) with
significant differences among them as
shown in Table 2. At the same time,
treatment of the infested colonies with
these natural products showed much
variation in brood infestation. The
average number of dead Varroa mite
fallen was obtained when the colonies
sprayed twice with the extract of Garlic
(91.15%) followed by Black-cumin
(86.57%) by the rate of 20 ml /colony.
The differences among the reduction
percentages of mite infestation resulted
through treatment of these natural
products from statistical view point were
significant. Regarding the average %
reduction on infestation after 24 hrs the
treatment with different tested
substances, the highest reduction % in
the number mites was recorded for
application of 20 ml/ colony of Black-
cumin (78.84%) followed by Garlic
(70.99%). Marjoram preparation gave the
lowest one (59.17%) (Table 2). The
statistical analysis of the obtained data
showed significant differences among
means of reduction percentages of mite
infestation in the two treatments as
shown in Table 2.
Table 2: Effect of spraying of extracts plant products on their infestation level of the parasitic
mite, Varroa destructor after 14 days post treatments (General Average).
Treatments
1
st
spray
Reduction of infestation (%)
2
nd
spray
Reduction of infestation (%)
Average of
Reduction of infestation (%)
Adult
brood
fallen varroa mite
Adult
brood
fallen varroa mite
Adult bees
Brood
Fallen mite
Pomegranate
93.27
77.08
36.67
90.48
62.50
101.50
91.88
69.79
59.34
Garlic
94.62
77.08
40.67
91.67
86.67
104.50
93.15
81.88
64.50
Marjoram
96.01
69.44
32.83
93.33
91.67
117.50
94.67
80.56
69.00
Harmal
96.01
88.54
36.00
93.33
84.62
115.50
94.67
86.58
69.50
Black- cumin
95.51
79.63
31.67
93.33
80.00
120.30
94.42
79.82
71.40
Ambrosia
94.95
69.44
24.50
85.19
83.33
107.80
90.07
76.39
63.99
LSD 0. 05
3.12
7.82
12.41
ns
10.61
17.30
-
-
72
Dusting application of different
powder concentrations of tested
natural products:
Reduction
percentages of Varroa mite infestation on
adult bees in Table 3. It was clear that
treatment with Marjoram and Harmal
(94.67%) followed by the Black-cumin
(94.42%) followed Pomegranate powder
by the rate of 40 gm/ colony resulted in
decreasing the infestation by 91.88 %. by
the rate of 40 g / colony. Also, mite
infestation in the brood of the treated
colonies suppressed by 86.58%, 81.88 %
and 80.56 % for treatment with Harmal,
Garlic, and Marjoram, respectively. The
differences among general reduction
percentages of mite infestation resulted
through treatment of these plant products
were significant (0.05%) as shown in
Table 3. With looking to the number of
dead Varroa mite fallen after 24 hrs of
the treatment with different tested
substances, the highest reduction % was
71.4% that recorded for application of 40
gm /colony of the Black- cumin
preparation, while the lowest one was
associated using Pomegranate
preparation (59.34%). Significant
differences among means of reduction
percentages of mite infestation resulted
by application of different substances as
dust formulation.
Table 3: Effect of dusting powder of certain plant products on the infestation level of the parasitic mite
(Varroa destructor) during the two spray and their average.
Treatments
1
st
spray
Reduction of infestation (%)
2
nd
spray
Reduction of infestation (%)
Average of
Reduction of infestation (%)
Adult
brood
fallen varroa mite
Adult
brood
fallen varroa mite
Adult bees
Brood
Fallen mite
Pomegranate
88.46
84.72
36.67
93.65
80.00
89.25
91.06
82.36
62.96
Garlic
85.04
95.63
40.67
92.59
86.67
101.30
88.82
91.15
70.99
Marjoram
89.23
79.63
32.83
88.89
c
73.33
85.50
89.06
76.48
59.17
Harmal
93.27
92.36
36.00
90.48
80.00
102.00
91.88
86.18
69.00
Black- cumin
86.54
89.81
31.67
91.67
83.33
126.00
89.11
86.57
78.84
Ambrosia
85.64
69.44
24.50
87.30
73.33
96.00
86.47
71.39
60.25
LSD 0. 05
3.62
3.39
8.94
4.87
2.89
11.89
-
-
-
Treatment of fumigation of certain
plant products during first season:
Concerning the general reduction
percentages of mite population data
presented in Table 4 showed that the
efficiency of the tested material for
reducing Varroa population on adult bees
could be managed in the following
descending order Pomegranate > Black
cumin> Garlic > Marjouram and Harmal
> Ambrosia. The least effect was
observed in treatment with Ambrosia.
The average percentages of reduction
were 94.6, 91.1, 89.83, 89.80, 87.9 and
83.5 % treatment by the rates of 40
gm/colony for the above-mentioned
substances, respectively. General
reduction of mite infestation in brood of
the treated colonies showed the highest
reduction percentage (86.10%) was
obtained when the colonies treated with
Garlic (86.10%), followed by
Pomegranate (85.70%) by the rate of 40
gm/ colony, Black- cumin (85.10%) and
Ambrosia (73.60%). Regarding the
number of dead Varroa mite fallen
(average of post-treatment with different
tested substances, the highest reduction%
(56%) when the colonies treated with
Marjoram plant fumigation. While the
lowest one (45.6 %) was associated using
dosage 40 gm / colony of the Harmal
73
preparation. Statistical analysis indicated
that there were significant differences
among means of reduction percentages of
mite infestation resulted by application
of different substances in the two
treatments.
Table 4: Effect of fumigation with certain plant products on their infestation level of the parasitic mite,
Varroa destructor during two sprays with three concentration 40 gm/colony after 14 days post treatment
and their average.
Treatments
1
st
spray
Reduction of infestation (%)
2
nd
spray
Reduction of infestation (%)
Average of
Reduction of infestation (%)
Adult
brood
fallen varroa mite
Adult
brood
fallen varroa mite
Adult bees
Brood
Fallen mite
Pomegranate
97.00
91.27
18.5
93.65
92.10
80.00
94.6
85.6
50.40
Garlic
82.20
84.70
15.5
92.59
97.50
87.50
89.83
86.1
46.70
Marjoram
92.30
89.80
17.5
88.89
87.30
75.00
89.80
82.4
56.00
Harmal
89.23
88.54
17.00
90.48
86.60
73.30
87.90
80.9
45.60
Black- cumin
94.80
92.40
18.00
91.67
87.30
77.80
91.10
85.1
54.00
Ambrosia
89.20
84.70
14.00
87.30
77.80
62.50
83.50
73.6
46.80
LSD 0. 05
8.16
4.32
3.20
1.98
9.86
4.79
-
-
-
Selectivity of treatments with different
method of application of natural
products on bees:
As shown in Figure 1,
treatment colonies with 20 ml of extracts
of certain plant products showed various
effects on daily rate of egg. The average
two sprays showed that the daily rates of
egg laying were 655.00, 634.00 and
630.00 eggs /day for the queens of the
colonies treated with Harmal, Garlic and
Pomegranate respectively. In comparison
to (287.50 eggs/ day) for the queens of
untreated colonies. Larval viability the
highest percentage (91.00%) was
recorded with those colonies treated with
Black cumin, with no different significant
effect between treatments but there a
significant effect between treatments and
control. Larvae viability percentage of
the brood of untreated colonies was
(87.50%). Worker longevity revealed that
the workers of the treated colonies lived
various periods longevity of the workers
belong to those colonies treated with the
Harmal, Garlic, Pomegranate were 43, 37
and 34.90 days respectively in
comparison to 21.13 days for the workers
of the control colonies. The honey
production of the experimental bees, data
showed that the highest rate (10.34 kg/
colony) of the honey was produced by
the colonies treated with the Garlic
followed by those colonies treated by the
Harmal (9.75 kg/ colony). Treatment
with Pomegranate fumigation gave the
highest efficacy against Varroa mite
followed by, Garlic and then Marjoram.
Spraying with extract of Black-cumin
showed the highest efficacy against
Varroa mite followed by Garlic and then
Harmal. By dusting application powder
of Pomegranate recorded the highest
efficacy against Varroa mite followed by
Garlic then Marjoram. Investigation on
mode of action of essential oils or natural
products chemical is important for mite
control because it may give useful
information on the most appropriate
formulation and delivery means. Volatile
compounds of many plant extracts and
essential oils are composed alkanes,
alcohols, aldehydes and terpenoids,
particularly mono-terpenoids, and exhibit
fumigant activity (Al-Waili et al. 2012,
Ariana et al. 2002) indicated that
aromatic plants and their essential oils
74
have been used as antimicrobial,
acaricidal and insecticidal agents and to
repel insect and mites or protect stored
products. These constitute effective
alternatives to synthetic pesticides
without producing adverse effects or the
environment (Isman 2006, Isman &
Machial 2006)
.
Figure 1: Effect of spraying, dusting and fumigation of certain plant products on daily rate of egg laid, larvae
viability %, worker longevity and productivity of honey bees for season of 2018 (P: Pomegrnate- GA: Garlic –
M.: Marjorum, H: Harmal, B.C.: Black cumin, A.: Ambrosia, C.: Control).
Moreover, the interest is natural products
such as essential oils has regained
momentum during the last decade
primary due to their fumigant and
contact acricidal activities and the less
stringent regulatory approval
mechanisms for their exploration due to
long history of use (Isman, 2006). In
general new alternatives for the control
of
V. destractor
are necessary because of
the rapid and widespread development of
pyrethroid and organophosphate resistant
mite population and because of the
potential for contamination of hive
products by these chemicals (Hussein &
Mostafa, 2009). Natural products
especially contain essentials, and
especially components of essential oils,
may serve as alternatives or as adjuvants
to traditional treatment measures. From
our results we can concluded that
treatment the Varroa infested colonies
with the different features of the plant
products indicated that fumigations was
more efficient than spraying while
treatment with dusting of the powder of
this materials recorded the lowest
efficacy and all treatments were good
selective against bees. Future research
can assist in this effect by focusing on
the characterization of the dose response
relationships between components and
mite/ bee toxicity and effects on mite
behaviors.
References
Al-Waili N, Salom K, Al-Ghamdi A, Ansari,
MJ, 2012. Antibiotic, pesticide, and
microbial contaminants of honey:
Human health hazards. The Scientific
World Journal 2012: 930849.
Ariana A, Ebadi, R, Tahmasebi G. 2002.
Column1
daily rate of egg laying
Larvae viability %
Worker longevity (day )
Honey production (kg / colony )
10
210
410
610
810
spray
p
Ga
M
H
B.C
A
C
Dusting
P
Ga
M.
H
B.C.
A
C
Fumigation
P
Ga
M.
H.
B.C.
A.
C
daily rate of egg laying
Larvae viability %
Worker longevity (day )
Honey production (kg / colony )
75
Laboratory evaluation of some plant
essences to control Varroa destructor
(Acari: Varroidae). Experimental &
Applied Acarology 27: 319.
Bargańska Z, Ślebioda M, Namieśnik J, 2016.
Honey bees and their products:
Bioindicators of environmental
contamination. Critical Reviews in
Environmental Science and Technology
46: 235–248.
Benelli G, Pavela R, Giordani C, Casettari L,
Curzi G, Cappellacci L, Petrelli R,
Maggi F, 2018. Acute and sub-lethal
toxicity of eight essential oils of
commercial interest against the filariasis
mosquito Culex quinquefasciatus and the
housefly Musca domestica. Industrial
Crops and Products 112: 668–680.
Calatayud-Vernich P, Calatayud F, Simó E,
Picó Y, 2017. Occurrence of pesticide
residues in Spanish beeswax. Science of
the Total Environment 605: 745–754.
Calatayud-Vernich P, Calatayud F, Simó, E,
Suarez-Varela MM, Picó Y, 2016.
Influence of pesticide use in fruit
orchards during blooming on honeybee
mortality in 4 experimental apiaries.
Science of the Total Environment 541:
33–41.
Calo JR, Crandall PG, O'Bryan CA, Ricke
SC, 2015. Essential oils as antimicrobials
in food systems–A review. Food Control
54: 111–119.
Chiesa LM, Labella GF, Giorgi A, Panseri S,
Pavlovic R, Bonacci S, Arioli F, 2016.
The occurrence of pesticides and
persistent organic pollutants in Italian
organic honeys from different productive
areas in relation to potential
environmental pollution. Chemosphere
154: 482–490.
Eshbah H, Mohamed, A, Hassan A,
Mahmoud M, Shaban M 2018.
Efficiency of feeding honey bee
colonies, Apis mellifera L., with mixture
of natural products and sugar syrup on
brood and adult population. Scientia 21:
14–18.
Grout RA, 1960. The hive and the honeybee.
Hamilton IL, 3
rd
revision. Dadant and
Sons, USA, pp. 740.
Hassan A, Aly AM, 1998. Comparative
studies of the efficiency of some
chemicals used for controlling wax
moths and their effects on honey bees
behavior towards the treated combs, Ain
Shams University, Cairo, Egypt, pp.
244–255.
Hussein S, Hafez H, Kchrobok C, Thiemann
W, 2002. Contamination of different
sources of water in Minia, Egypt by
selected chlorinated insecticides,
Proceeding of Minia 1
st
Conference of
Agriculture and environmental Sciences,
pp. 25–28.
Hussein SM, Mostafa DM, 2009. Resduies of
certain varrocides in the honeybee
products p. abstract, KSU, Abha Saudia
arab.(abstract)
Hussein SM, Moustafa DM, Hassan AR,
2016. The efficacy of certain essential
oils and their mixtures on the parasitic
mite varroa destructor. 13
th
AAA
Conference, Jada, Saudia Arabia, pp. 65.
Isman MB, 2006. Botanical insecticides,
deterrents, and repellents in modern
agriculture and an increasingly regulated
world. Annual Review of Entomology
51: 45–66.
Isman MB, Machial CM, 2006. Pesticides
based on plant essential oils: from
traditional practice to commercializ-
ation. In: Rai, M., Carpinella, M.C.
76
(Eds.), Naturally Occurring Bioactive
Compounds. Advances in
Phytomedicine, Elsevier, 3: 29–44.
Kefuss JA, 1978. Influence of photoperiod on
the behavior and brood rearing activity
of honeybees in a flight room. Journal of
Apicultural Research 17(3): 137–151.
Komeili A, 1988. The impact of the Varroa
mite on Iranian commercial beekeeping.
American Bee Journal 128(6): 423–424.
Marcangel JM, Eguaras JM, Fernandez NA,
1992. Reproduction of Varroa jacobsoni
(Acardi: Mesostigmata: Varroidae in
temperate dimates of Argentina.
Apidologie 23: 57–60.
Mead R, Curnow RN, Harted AM, 1993.
Statistical methods in agriculture and
experimental Biology, Chapman and
Hall, London, England, pp.498.
Nai YS, Chen TY, Chen YC, Chen CT, Chen,
BY and Chen, YW, 2017. Revealing
Pesticide Residues under High Pesticide
Stress in Taiwan's Agricultural
Environment Probed by Fresh Honey
Bee (Hymenoptera: Apidae) Pollen.
Journal of Economic Entomology
110(5): 1947–1958.
Porrini C, Mutinelli F, Bortolotti L, Granato
A, Laurenson L, Roberts K, Gallina A,
Silvester N, Medrzycki P, Renzi T,
Sgolastra F, Lodesani M, 2016. The
status of honey bee health in Italy:
Results from the nationwide bee
monitoring network. PLoS ONE 11(5):
e0155411.
Sattler JAG, De-Melo AAM, do Nascimento
KS, de Melo ILP, Mancini-Filho J,
Sattler A, de Almeida-Muradian LB,
2016. Essential minerals and inorganic
contaminants (Barium, cadmium,
lithium, lead and vanadium) in dried bee
pollen produced in Rio Grande do Sul
State, Brazil. Food Science and
Technology 36(3): 505–509.
Shawer MB, Shenishen Z, El- Dakhakhni
NM, 1986. Effect of colony strength
activity and productivity of honeybee
colonies. Bulletin of the Entomological
Society of Egypt 66: 239–249.
Terada YC, Garofalo A and Sakagami A,
1975. Age survival curve for workers of
two susocial bees (Apis mellifera) and
plebeian droyana) in a subtropical
climate, with notes on worker plythism
in P. draryana. Journal of Apicultural
Research 14(3): 161–170.
Vijayalakshmi K, Subhashini B, Koul S
1999. Plants in pest control: Garlic and
onion. Chennai, India, pp. 1–23.
