Variasi Densitas Dan Ultramorphometrik Dari Sensilla Antena Antara Apis Andreniformis Dan Apis Cerana Pada Ketiga Kasta Lebah Madu.
VARIATIONS IN THE DENSITY AND ULTRAMORPHOMETRIC
OF ANTENNAL SENSILLA BETWEEN Apis andreniformis AND
Apis cerana IN THE THREE CASTES OF HONEY BEES
CUT FERAWATI
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2015
STATEMENT LETTER
I hereby declare that this thesis entitled “Variations in the Density and
Ultramorphometric of Antennal Sensilla between Apis andreniformis and Apis
cerana in the Three Castes of Honey Bees” is the original result of my own
research under supervision of an advisory commitee and has never been submitted
in any form to any other institution before. All information from other authors
cited here are mentioned in the text and listed in the reference list at the end of the
thesis.
Bogor, October 2015
Cut Ferawati
Student ID G352130141
SUMMARY
CUT FERAWATI. Variations in the Density and Ultramorphometric of
Antennal Sensilla between Apis andreniformis and Apis cerana in the Three
Castes of Honey Bees. Supervised by RIKA RAFFIUDIN and BERRY
JULIANDI.
Honey bees live in two different types of nest, the single comb open air
nests of Apis andreniformis, A. florea and A. dorsata and multiple comb cavity
nests of A. cerana, A. koschevnikovi, A. nuluensis, A. nigrocincta, and Athra nests.
Honey bee that consist of three castes (queen, drone and worker caste) perform
complex communications. The communication mostly mediate by antennal
sensilla that receive odor or pheromone. However, the variations of the density,
distribution, and ultramorphometric of antennal sensilla of the open and cavity
nesting types is poorly understood and the available data is derived from the
worker caste.
The objectives of this research were to analyse: (1) the variations of the
type and density of antennal sensilla within the three castes of the open nesting A.
andreniformis and cavity nesting A. cerana, (2) the sensilla distribution between
the anterior and posterior sides of antenna and between lower and upper end of
each flagellar antenna within the three castes of A. andreniformis and A. cerana,
and (3) ultramorphometric of the length, width, and area of trichodea and
placodea sensilla in anterior and posterior sides of flagella within the three castes
in both species.
The honey bee samples of A. andreniformis and A. cerana were obtained
from Padang Pariaman (West Sumatera) and Bogor (West Java), Indonesia,
respectively. Antennal sensilla of honey bees were analysed by using scanning
electron microscope. The densities, distributions and ultramorphometric of
antennal sensilla were examined using the ImageJ program. Furthermore, the
ultramorphometric sensilla were analyzed with ANOVA and t-test.
This study observed six antennal sensilla types in A. andreniformis and A.
cerana. Those are trichodea (variants A, B, C, and D), bassiconica, placodea,
campaniform, ampullacea, and coeloconica. The last two types of sensilla are the
new report in the open nesting A. andreniformis.
Densities of sensilla in the cavity nesting honey bee A. cerana are higher
compare to the open air nesting A. andreniformis. This phenomena might be a
structure adaption due to the bees use antennal sensilla for communication inside
of dark cavity hive.
The density of sensilla is varied among the three castes of open nesting A.
andreniformis and cavity nesting A. cerana. The density of bassiconica in worker
caste is higher than in the other castes in both species. The density of placodea is
the highest in the drone caste of both species. Sensilla density of trichodea C,
trichodea D, and bassiconica are more abundant in worker flagella among other
castes in both species, while placodea, ampulacea, coeloconica sensilla are more
abundant in drone among other castes. Interestingly, this study did not find
coeloconica in the queen of A. cerana. Density of trichodea A is the highest in A.
cerana queen and trichodea B are more higher on in A. cerana worker.
Furthermore, bassiconica were not found in A. cerana drone.
This research also the first report of the variations of antennal sensilla
distribution found between the anterior and posterior sides, as well as between the
upper and the lower end of flagellar antenna. The number of sensilla on the
anterior side is twice compared to the posterior side of flagellar antenna in A.
andreniformis and A. cerana. This result suggests that anterior areas are more
prominent to receive odor comes from the frontal area of the bee head. This study
also observed that the sensilla distributions are more prominent on the upper end
of flagella compare to the lower end. This result might be due to the upper end of
antenna is directly contact to the substrate, thus serve as the chemo- and
mechanoreception.
Beside the variations of the density and distribution of antennal sensilla
types, almost all of ultramorphometrics of trichodea and placodea sensilla are
differ within the three castes of open nesting A. andreniformis and cavity nesting
A. cerana. Ultramorphometric measurements of A. andreniformis drone placodea
are the highest compare to other caste. This suggest that they need highly efficient
to detect the queen pheromones. The ultramorphometric sensilla on anterior and
posterior sides are also different, that might imply the functional of sensilla as an
effective ogan to capture the odor molecules exposure to the olfactory sensory
from the outside environment. Therefore, the diversity in the density and
distribution as well as the ultramorphometric of antennal sensilla might give an
indication of the adaption of honey bees to different nesting environments.
Key words: Olfactory sensory, antennal sensilla, open-nesting honey bee, cavitynesting honey bee, Apis andreniformis, Apis cerana
RINGKASAN
CUT FERAWATI. Variasi Densitas dan Ultramorphometrik dari Sensilla
Antena antara Apis andreniformis dan Apis cerana pada Ketiga Kasta Lebah
Madu. Dibimbing oleh RIKA RAFFIUDIN dan BERRY JULIANDI.
Lebah madu hidup pada dua tipe sarang yang berbeda, yaitu sarang
terbuka dengan satu sisir seperti sarang Apis andreniformis, A. florea and A.
dorsata dan sarang tertutup dengan beberapa sisir seperti A. cerana, A.
koschevnikovi, A. nuluensis, A. nigrocincta, dan A. mellifera. Lebah madu terdiri
atas tiga kasta (ratu, jantan, dan pekerja) yang melakukan komunikasi kompleks.
Komunikasi pada umumnya menggunakan sensila pada antenna untuk menerima
bau atau feromon. Namun, masih sedikit pengetahuan tentang variasi densitas,
distribusi, dan ultramorfometrik sensilla antena pada tipe sarang terbuka dan
tertutup dan data yang tersedia hanya kasta pekerja saja.
Tujuan penelitian ini adalah untuk menganalisis: (1) variasi tipe dan
densitas sensila antena pada ketiga kasta dari lebah sarang terbuka A.
andreniformis dan lebah sarang tertutup A. cerana, (2) distribusi sensila antara sisi
anterior dan posterior serta antara pangkal dan ujung dari masing-masing flagela
antena pada ketiga kasta A. andreniformis dan A. cerana, dan (3)
ultramorfometrik panjang, lebar dan area dari sensila trichodea dan placodea pada
sisi anterior and posterior flagela pada ketiga kasta dari kedua spesies.
Sampel lebah madu A. andreniformis dan A. cerana diperoleh dari Padang
Pariaman (Sumatera Barat) dan dari Bogor (Jawa Barat), Indonesia, secara
berurutan. Analisis Sensila antena lebah madu menggunakan scanning electron
microscope. Pengujian densitas, distribusi dan ultramorfometrik sensila antena
diuji menggunakan program ImageJ. Selanjutnya, data ultramorfometrik sensila
dianalisis menggunakan ANOVA dan uji t.
Pada penelitian ini ditemukan enam tipe sensila pada A. andreniformis dan
A. cerana, yaitu sensila trichodea (variasi A, B, C, dan D), bassiconica, placodea,
campaniform, ampullacea, dan coeloconica. Dua tipe sensilla terakhir merupakan
laporan baru pada lebah sarang terbuka A. andreniformis.
Total densitas sensila lebah sarang tertutup A. cerana memiliki yang lebih
banyak dibandingkan lebah sarang terbuka A. andreniformis. Fenomena ini
kemungkinan karena adanya adaptasi struktur karena lebah menggunakan sensilla
untuk berkomunikasi di dalam sarang yang gelap.
Densitas sensila bervariasi diantara ketiga kasta pada lebah sarang terbuka
A. andreniformis dan lebah sarang tertutup A. cerana. Densitas sensila bassiconica
pada kasta pekerja lebih tinggi dibandingkan kasta lainnya pada kedua spesies.
Densitas placodea paling tinggi ditemukan pada kasta jantan pada kedua spesies.
Densitas sensila trichodea C, trichodea D, dan bassiconica lebih banyak pada
flagela kasta pekerja dibandingkan kasta lainnya pada kedua spesies, sementara
itu sensila placodea, ampulacea, coeloconica lebih banyak pada kasta jantan
dibandingkan kasta lainnya. Menariknya, pada penelitian ini tidak ditemukan
sensila coeloconica pada kasta ratu A. cerana. Densitas trichodea A paling tinggi
ditemukan pada kasta ratu A. cerana dan trichodea B lebih banyak pada kasta
pekerja A. cerana. Selanjutnya, bassiconica tidak ditemukan pada kasta jantan A.
cerana.
Penelitian ini juga pertama kali mencatat adanya variasi distribusi sensila
antara sisi anterior dan posterior antena, sama halnya dengan sensila antara ujung
dan pangkal flagela antena. Densitas sensila pda sisi anterior dua kali lipat lebih
banyak dibandingkan sisi posterior dari flagela antena pada A. andreniformis dan
A. cerana. Hasil ini diperkirakan karena area anterior lebih dominan untuk
menerima bau dari area depan kepala lebah. Penelitian ini juga menemukan
bahwa distribusi sensila lebih dominan pada bagian ujung flagela dibandingkan
pangkal. Hasil tersebut diperkirakan karena bagian ujung antena kontak langsung
dengan substrat, dalam mengindera chemo dan mechanoreception.
Selain variasi densitas dan distribusi tipe sensila antena, hampir semua
ultramorfometrik sensila trichodea dan placodea berbeda pada ketiga kasta dari
lebah sarang terbuka A. andreniformis dan lebah sarang tertutup A. cerana. Hasil
pengukuran ultramorfometrik placodea pada kasta jantan A. andreniformis lebih
panjang dibandingkan kasta lainnya. Hal ini diperkirakan karena jantan
membutuhkan sensila tersebut agar lebih efisien dalam mendeteksi feromon ratu.
Ultramorfometrik sensila pada anterior dan posterior berbeda, diperkirakan fungsi
sensila agar efektif menangkap molekul bau dari lingkungan luar oleh sensorik
olfaktori. Oleh karena itu, keragaman densitas dan distribusi maupun
ultramorfometrik sensila antena mungkin mengindikasikan bahwa adaptasi lebah
madu terhadap perbedaan lingkungan di sekitar sarang.
Kata kunci: Sensorik olfaktori, sensilla antena, lebah madu sarang terbuka, lebah
madu sarang tertutup, Apis andreniformis, Apis cerana
Copyright © 2015 Bogor Agricultural University
Copyright are Protected By Law
It is prohibited to cite all or part of this thesis without referring to and
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research, scientific writing, report writing, critical writing or reviewing scientific
problem. Citation does not infict the name and honour of Bogor Agricultural
University
It is prohibited to publish or to reproduce all or part of thesis without the
written permission from Bogor Agricultural University
VARIATIONS IN THE DENSITY AND ULTRAMORPHOMETRIC
OF ANTENNAL SENSILLA BETWEEN Apis andreniformis AND
Apis cerana IN THE THREE CASTES OF HONEY BEES
CUT FERAWATI
Thesis
As a partial fulfilment of the requirements for a Master Degree
in Animal Biosciences Master Program of Graduate School of
Bogor Agricultural University
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2015
Non-Commitee Examiner: Dr. Ir. Sih Kahono, M.Sc
PREFACE
Praise and grateful to Allah the All Mighty, for the completion of this
Master Thesis entitled Variations in the Density and Ultramorphometric of
Antennal Sensilla between Apis andreniformis and Apis cerana in the Three
Castes of Honey Bees. The study was conducted from June 2014 to June 2015.
This research was supported by Master Program Scholarship (BPPDN) from the
Directorate of General Higher Education of Indonesia (DIKTI), grant no.
1094/E4.4/2013.
I thank profusely to Dr. Ir. Rika Raffiudin, M.Si and Dr. Berry Juliandi,
S.Si, M.Si as the supervising commitee, which have supervised and put so much
trust to me during my master study and the completion of the thesis. I sincerely
thank to Dr. Ir. Sih Kahono, M.Sc as the thesis defense examiner and to Dr. Ir.
R.R. Dyah Perwitasari, M.Sc as the Head of Animal Bioscience Postgraduate
Master Program, Department of Biology, for insightful comments and correction
of the thesis. My gratitude also to honey bee researcher Prof. Siti Salmah, Drs.
Mochammad Chandra Widjaja, MM and Dr. Jasmi, for the knowledge and
experience that you shared, that had raised my interest on honey bees.
I address my thankfulness also to staff Scanning Electron Microscope
Laboratory, Division of Zoology, Research Center for Biology of Indonesian
Institute of Sciences (LIPI) for their magnification technical assistance. I specially
thank to staff of National Beekeeping Center (PUSBAHNAS), sampling team in
Padang Pariaman (West Sumatra), and sampling team in Bogor (West Java) for
the invaluable assistance during field data collection.
I also would like to thank to all the lectures in Animal Bioscience Master
Program, who at the end shaping my knowledge up through their high dedication
into the research and teaching. The endless gratitude is addressed to my parents,
brothers and sister who have supported me by their own way during the study.
The least, but very important, I would like to thank to all my friends; class of BSH
2013, BSH students, Desmina Kristiani Hutabarat, Febri Ayu, and Rosi Fitri
Ramadani.
Hopefully this scientific work will be useful.
Bogor, October 2015
Cut Ferawati
CONTENT LIST
LIST OF TABLE
xv
LIST OF FIGURES
xv
LIST OF APPENDICES
xv
1 INTRODUCTION
Background
Objectives
1
1
2
2 LITERATURE REVIEW
Honey Bees Evolution
Honey Bees Castes
Distribution of Apis andreniformis and Apis cerana
Nest Types
Sex Determination and Sexual Dimorphism
Structure and Functions of Sensilla
3
3
4
4
5
5
7
3 METHODS
Study Site and Time
Samples Collection
Antenna Preparation and SEM Analysis
Data Analysis
8
8
8
9
9
4 RESULTS AND DISCUSSION
Results
Antennal structure and sensilla types of A. andreniformis and A.
cerana, and in the three castes
Densities of total antennal sensilla in bees exhibiting different type
of nest in the three castes of A. andreniformis and A. cerana
Different distributions of antennal sensilla between flagellar sides,
and between the upper and lower ends of flagella
Ultramorphometric variations of trichodea and placodea sensilla of
three castes in two sides of antenna in A. andreniformis and A.
cerana
Discussion
Density and distribution variations of sensilla types in open
and cavity nesting bees suggests an environmental adaptation
Antennal sensilla density varies among castes
Ultramorphometric sensilla are different among the three castes and
flagellar sides
10
10
10
12
16
17
19
19
20
21
5 CONCLUSSION
22
REFERENCES
22
APPENDICES
25
BIOGRAPHY
39
LIST OF TABLES
1 The three castes of A. andreniformis and A. cerana
2 Ultramorphometric of trichodea and placodea sensilla of the three
castes of A. andreniformis and A. cerana
3 Comparison ultramorphometric of trichodea and placodea sensilla on
anterior and posterior sides of the three castes of A. andreniformis and
A. cerana
8
17
18
LIST OF FIGURE
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
The type of honey bee nest
Bayesian consesus tree derived of Apis from the cox2 sequence
Polyagethism of A. cerana workers
Geographical distribution area of A. andreniformis and A. cerana
Sex determination of queen eggs
Reproduction system structures of three castes of honey bees
Schematic of sensilla anatomy
Morphology of honey bee antenna
Ultramorphometric measurement of honey bee sensilla
Structure of A. cerana worker antenna
Antennal sensilla types in several flagella of A. andreniformis and A.
cerana
Total densities of antennal sensilla of A. andreniformis and A. cerana
Sensilla distribution on the fl8 anterior side the three castes of A.
andreniformis and A. cerana
Density of antennal sensilla in A. andreniformis and A. cerana of queen,
drone, and worker
Density of antennal sensilla on the anterior and posterior side
1
3
4
5
6
6
7
9
10
11
11
12
13
16
16
LIST OF APPENDICES
1
2
3
4
5
6
Antennal sensilla of an A. andreniformis queen flagella
Antennal sensilla of an A. andreniformis drone flagella
Antennal sensilla of an A. andreniformis worker flagella
Antennal sensilla of an A. cerana queen flagella
Antennal sensilla of an A. cerana drone flagella
Antennal sensilla of an A. cerana worker flagella
25
27
30
32
34
37
1
1 INTRODUCTION
Background
Honey bees (Hymenoptera: Apidae) live in two different types of nest, the
single comb open air nests of Apis andreniformis (Figure 1a), A. florea and A.
dorsata and multiple comb cavity nests of A. cerana (Figure 1b), A. koschevnikovi,
A. nuluensis, A. nigrocincta, and A. mellifera nests (Smith 1991; Oldroyd and
Wongsiri 2006; Raffiudin and Crozier 2007). Honey bee in both nest types have
different dance behaviors, which are A. andreniformis perform a horizontal dance
compare to vertical dance of A. cerana (Winston 1991; Dyer 2002). The different
dance behaviors might adapt different nest environment (Raffiudin and Crozier
2007; Hepburn and Radloff 2011).
Social life is one of the characteristics of honey bee. A colony of honey
bee consist of three castes based on their tasks (Ruttner 1988). However, the
division of honey bee caste can also based on the reproductive ability. The
reproductive caste consist of queen and drones, whereas the non-reproductive
caste all workers (Winston 1991). Each caste of honey bees have spesific function
or task in the colony. The role of queen honey bee has the role in produce eggs
and control the colony using queen pheromone. Drones have the role to mate the
queen.
The inside role of workers in the colony are cleaning the cell, tending the
brood, receiving nectar, capping the cell, attending the queen, building the comb,
food handling, and cleaning debris. The outside role of worker are ventilation,
guarding, and foraging (Winston 1991). Therefore, honey bees need the spesific
communication to carry out vary kind activity between or within castes.
Those communications use odor or pheromones and are received by the
antennal sensilla. Antennal sensilla of the queen receive odor or pheromones that
mediate communications with the workers in the hive and with the drones during
mating flight. Drones use these sensilla to detect queen and workers pheromones
a
b
Figure 1 The type of honey bee nest. (a) open nesting A. andreniformis and (b)
cavity nesting A. cerana (Figure b from Suwannapong et al. 2011).
2
(Winston 1991). Workers use olfactory sensilla to detect odors inside the colony
such as brood pheromone, queen pheromone, and outside the colony for detected
floral odors (Chapman 1998). Besides chemoreceptor, the antennal sensilla
facilitates also as mechanoreceptor, hygroreceptor, thermoreceptor, and gustatory
receptor (Chapman 1998).
Four antennal sensilla types of open nesting A. andreniformis workers are
trichodea, bassiconica, placodea, and campaniform and 81.6% of the total sensilla
is trichodea (Suwannapong et al. 2012). In cavity nesting A. mellifera there are
five types of sensilla, those are trichodea, bassiconica, placodea, ampulacea, and
coeloconica (Dostal 1958). Placodea sensilla in the drones are higher than in the
workers in A. mellifera (Esslen and Kaissling 1976). The total number of hair-like
sensilla (trichodea) per antenna in workers is higher than the queens and drones in
(Fang et al. 2012).
Further examinations showed that the anterior side of A. mellifera worker
antennae has higher number of antennal sensilla compare to the posterior side
(Esslen and Kaissling 1976). The ultramorphometric analysis of antennal sensilla
are different between A. florea and A. mellifera worker. The average length of
bassiconica, area of placodea, and diameter of coeloconica of open nesting A.
florea worker are higher than cavity nesting A. mellifera African race worker (Al
Ghamdi 2006). This suggest that to the length of antennal sensilla is adapt to
maximize the effective to capture the odor molecules from the outside
environment (Wyatt 2003).
There is no information on the variations in antennal sensilla among
queen and drone castes of the open nesting A. andreniformis. This study
questioned other types of sensilla besides the four known types of sensilla found
in the cavity nesting A. mellifera and open nesting A. andreniformis. Due to lack
of data for other cavity nesting bees, thus, this study focused on A. cerana. This is
due to A. cerana has the most widely distributed species of cavity nesting honey
bee (Ruttner 1998).
Other question is the variations in the distributions and densities of sensilla
within the three castes of those species. There is no data between the anterior and
posterior sides of antennae and between the lower and mostly for upper ends of
each antenna within the three castes of both species. More over, there is no
ultramorphometric of antennal sensilla within the three castes of open nesting A.
andreniformis and cavity nesting A. cerana.
Objectives
The objectives of this research were to analyse: (1) the variations of the
type and density of antennal sensilla within the three castes of the open nesting A.
andreniformis and cavity nesting A. cerana, (2) the sensilla distribution between
the anterior and posterior sides of antenna and between lower and upper end of
each flagellar antenna within the three castes of A. andreniformis and A. cerana,
and (3) ultramorphometric of the length, width, and area of trichodea and placodea
sensilla in anterior and posterior sides of flagella within the three castes in both
species.
3
2 LITERATURE REVIEW
Honey Bees Evolution
There are nine species of honey bees, those are clustered in the three
groups based on the worker size and nesting type. There are two recognized
species of dwarf bees, i.e. A. florea and A. andreniformis. Five species of cavity
nester bees with medium-sized workers are A. mellifera, A. koschevnikovi, A.
nuluensis, A. nigrocincta, and A. cerana. At least two species of giant bees those
are A. dorsata and A. laboriosa (Oldroyd and Wogsiri 2006; Raffiudin and
Crozier 2007; Hepburn and Radloff 2011).
Among all nine species of honey bees, the dwarf bees is in the basal of the
molecular phylogenetic tree based on cox2 gene and the giant bees and cavity
nesters are more derive species (Figure 2) (Raffiudin and Crozier 2007). The
dwarf honey bees built a single comb around a small twig constructing a platform
on the top of the nest and develope a horizontal directed waggle. Furthermore, the
ancestor of giant bees turned to open nesting and contruct the single comb under a
large branch and the bees perform their waggle dances on the vertical surface. The
cavity nesters also perform a vertical dance in the vertical comb.
Figure 2
Bayesian consesus tree derived of Apis from the cox2 sequence
(Raffiudin and Crozier 2007)
4
Honey Bees Castes
Honey bees are social life insect and consist of three castes (queen, drone,
and worker). In one colony of A. cerana there are about 6,884 individuals (Ruttner
1988). Honey bees from different castes have different function in their colony.
Queen has the role in producing eggs and controlling pheromone, while drones
have the role as to mate the queen. The workers have the role based on age
polyethism (Figure 3), those are inside and outside colony. Inside tasks include
cell cleaning, cell capping, brood and queen tending, comb building, food
handling. Whereas outside task are ventilating, guarding, orientation flights,
foraging and any other activities (Winston 1991).
Distribution of Apis andreniformis and Apis cerana
The geographical distributions of A. cerana is more wide compare to A.
andreniformis. Open nesting A. andreniformis distribute in Southern China,
Thailand, Indonesia (Sumatera, Java, and Borneo), Malaysia, Philippines
(Palawan), Burma, Laos, and Vietnam (Figure 4) (Wongsiri et al. 1996; Hepburn
and Radloff 2011). Furthermore, the distribution of A. cerana in China, India,
Malaysia, Burma, Laos, Vietnam, Philippines, Japan, Iran, Pakistan, Nepal,
Bhutan, Afghanistan, North Korea, South Korea and Indonesia (Sumatera, Java,
Borneo and Sulawesi) (Figure 4). In Indonesia A. cerana is restricted at the west
of the Wallace line (Celebes-Timor). On the Moluku Islands (Buru, Obi, and
Batjan), there is no honey bees were found except on Ambon (Ruttner 1988).
Figure 3 Polyagethism of A. cerana workers (Darmayanti 2008)
5
Figure 4 Geographical distribution area of A. andreniformis (broken line) and
A. cerana (dotted line) (Ruttner 1988 and Wongsiri et al. 1996)
Nest Types
Based on the nest structures, honey bee nests consist of the single comb
open air nests and multiple comb cavity nests. The single comb open air nest
encircle a small branch or twig, those type of nest are a typical nest of A.
andreniformis (Figure 1a) and A. florea. In addition, the single comb open air
nests of A. dorsata builds the comb on the branch of a tree, an overhang on a tall
building, or on cliff faces. Meanwhile, multiple comb cavity nests honey bee
contruct the nest inside closed cavities such as hollow trees trunks, caves, cleft in
rocks and wall which are the characterization for A. cerana (Figure 1b), A.
koschevnikovi, A. nuluensis, A. nigrocincta, and A. mellifera nests (Smith 1991;
Oldroyd and Wongsiri 2006; Raffiudin and Crozier 2007).
Nest homeostasis such as the nest temperature, humidity, carbon dioxide
level, and other enviromental factors are maintenanced at relatively constant
levels regardless of external conditions. Nest homeostasis regulation are the
functions of honey bee worker, their were detected enviromental factors using
antennal sensilla (Chapman 1998).
Sex Determination and Sexual Dimorphism
The mechanism of caste determination in honey bees are depend on the
unfertilized and fertilized egg (Figure 5). If sperm are not released when the queen
lays an egg, the unfertilized egg has haploid number of chromosome. Thus,
develop into drone honey bee. Queen or worker honey bees produce from
fertilized eggs and has diploid number of chomosomes (Winston 1991).
6
Drones, queen, and workers have unique specialization, particularly
associated with reproduction. The reproduction organs of drone are designed for
mating. Furthermore, the male structures of reproduction organ, the penis consists
of an endophallus and copulatory claspers (Figure 6a). The sperm are transferred
during mating that are produced in the testes and stored in the seminal vesicles
until mating (Winston 1991).
Figure 5 Sex determination of queen eggs (Winston 1991)
a
Figure 6
b
c
Reproduction system structures of three castes of honey bees.
(a) queen, (b) drone, and (c) worker (Winston 1991).
7
Structure and Functions of Sensilla
The antenna are the noses of insect, consist of scape, pedicle, and flagella.
Antenna of worker A. mellifera consist of ten flagella (fl1-fl10) and cover by
sensilla (Chapman 1998). The structure of sensilla have external cuticular
component within which neural processes lie in close to pores in cuticle (Figure 7).
This organ is a developmental unit composed of neural cells and support cells that
secrete the cuticular component of the sensilla (Chapman 1998).
Antennal sensilla of A. andreniformis have four different types, those are
bassiconica, campaniform, placodea, and trichodea type with total 4,892 sensilla
per antenna (Gupta 1992; Suwannapong et al. 2012). Each of antennal sensilla
type have specific functions. These sensilla are important for perception of carbon
dioxide, humidity, taste, and temperature (Winston 1991). Bassiconica and
campaniform in A. mellifera workers are involved in hygroreception (Schneider
1964). The function of bassiconica sensilla is a tasting organ in A. mellifera
workers (Dostal 1958). Placodea are involved in detecting a range of sex
pheromones released by A. florea and A. mellifera queens (Brockman and
Brückner 2001) and to receive odor in worker A. mellifera (Erickson 1982). The
hair sensilla trichodea as touch receptor or mechanoreceptor in A. mellifera and
bassiconica are possibly as taste organ (Dostal 1958).
Several function of antenna are to locate the host or prey and to block
chemicals odor for the insect survival. This was shown by the study of fig
pollinating wasps Ceratosolen solmsi marchali (Hymenoptera: Agaonidae) (Li et
al. 2009). This phytophagous wasps mainly use sensilla to locate their fig Ficus
(Moraceae) hosts. Encarsia sophia (Hymenoptera: Aphelinidae) is a parasitoid use
for biological control of Bemisia tabaci. It select the prey aided by the sensilla
(chemoreception organs) (Zhang et al. (2014). Further, the sensilla of the fleshfly
Neobellieria bullata (Diptera: Sarcophagidae) is important structure to block the
nitric oxide synthase in plant derived odorants (Wasserman and Itagaki 2003).
Figure 7 Schematic of sensilla anatomy (Chapman 1998)
8
3 METHODS
Study Site and Time
This research was conducted at Division of Animal Function or Behavior,
Department of Biology, Bogor Agricultural University and at Scanning Electron
Microscope (SEM) Laboratory, Division of Zoology, Research Center for Biology
of Indonesian Institute of Sciences (LIPI) from June 2014 until June 2015.
Samples Collection
The honey bee specimens of A. andreniformis and A. cerana were
obtained from Padang Pariaman, West Sumatera (S 00° 32’ 75.0” and E 100° 09’
77.2”) in 104 m altitude and from Bogor, West Java (S 06° 33’ 24.9” and E 106°
43’ 29.7”) in 116 m altitude, respectively. Ten individuals of workers and drones
as well as one individual of queen of both species (Table 1) were taken and all
preserved in 70% ethanol (Goldstein et al. 1992). The antennae of individual
honey bees (Figure 8) were taken using insect pins under a stereomicroscope.
Table 1 The three castes of A. andreniformis and A. cerana
Species
Castes
Queen
Drone
A.a
A.c
Scale bar: 3 mm for all pictures (A.a: A. andreniformis and A.c: A. cerana)
Worker
9
0.5 mm
a
b
Figure 8 Morphology of honey bee antenna. (a) A. cerana worker and (b)
schematic of antenna honey bee (A: anterior side, P: posterior side
Antenna
Preparation
and SEM
Analysis
of flagella,
Sc: Scape,
Pd: Pedicle,
Fl: Flagella).
Antenna Preparation and SEM Analysis
Antenna preparations were conducted in several steps: (1) Antenna was
cleaned in caccodylate buffer at -4 oC for three days (Bozzola and Russell 1998).
(2) The prefixation of antenna was in 2.5 % glutaraldehyde for 24h at -4 oC and
fixation in 3 % tannic acid for one hour. (3) Antenna was dehydrated in a graded
alcohol series from 30, 50, 75, 95, and 99.9 % for twice, 15 min/time (Goldstein et
al. 1992). (4) The dehydrated antenna was dried in tert butanol for twice, 10
min/time, then it was freeze-dried for 45 minute at -20 oC (Bozzola and Russell
1998). (5) The freeze-dried antenna was mounted on a stub and was coated with
gold and examined by SEM type JSM-5310LV (Goldstein et al. 1992; Bozzola
and Russell 1998). The coated and examined was operation by SEM laboratory
tecnicians.
Each antenna of A. andreniformis and A. cerana caste was placed on six
stubs. (1) Queen: one of the left and right antenna of queen was placed at two
stubs. (2) Drones and (3) workers: four of the right part antenna of each caste was
put on two stubs. Antennal sensilla of three castes antenna of A. andreniformis
and A. cerana taken by using SEM were recorded using a computer-controller
microscope apparatus.
One of antenna per caste in both species was observed the anterior side or
the posterior to analysis type, density, and distribution of antennal sensilla. The
anterior side of antenna is the side in the same direction to the head and the
opposite direction of side antenna to the head as the posterior side (Figure 8a).
Data Analysis
The antennal sensilla types were characterized by ultramorphology with
2,000-3,500X and the density of antennal sensilla were analyzed with 350-750X
10
magnification of SEM. Numbers of antennal sensilla was calculated using a
manual counter and ImageJ program (http://www.rsbweb.nih.gov/ij) in areas
varied between 14,000-63,000 µm2 and the values are shown as density of sensilla
per 0.01 mm2 following Ågren and Hallberg (1996). Density and distribution of
antennal sensilla was counted on the flagella of anterior and posterior sides of
queen, drone, and worker A. andreniformis and A. cerana.
The ultramorphometric of trichodea and placodea sensilla were analysed
due to the most abundance in each flagella (Esslen and Kaissling 1976; Fang et al.
2012). Those were measured with the same magnification of SEM.
Ultramorphometric data are measurement of the length, width, and area of this
sensilla as examined using an imageJ program (Figure 9a-c). The data was
analyzed within three castes using ANOVA and data of sensilla in both flagella
sides with t-test was performed using R program version 3.1.3 (CRAN.Rproject.org).
a
b
c
Figure 9 Ultramorphometric measurement of honey bee sensilla. (a) length,
(b) width, and (c) area of sensilla.
4 RESULTS AND DISCUSSION
Results
Antennal structure and sensilla types of A. andreniformis and A. cerana, and
in the three castes
This study observed the antenna of A. andreniformis and A. cerana that
consist of scape, pedicel and flagella (Figure 10). In both bees species, the antenna
consist of 10 flagella (segments) in the queen and the worker, but 11 flagella in
the drone. The antennal sensilla only found in flagella and not in other part of
antenna (Appendix 2–7).
There are six sensilla types, trichodea, bassiconica, placodea, campaniform,
ampullacea and coeloconica, in the three castes of the open nesting A.
andreniformis (Figure 11). The first four types are in agreement with
Suwannapong et al. (2012). Therefore, ampullacea and coeloconica are the two
types of antennal sensilla that are new recorded in A. andreniformis from this
study. Accordingly, the three castes of A. cerana have six sensilla types the same
11
with those of A. andreniformis, except coeloconica and bassiconica, which is
absent on queen and drones, respectively (Appendix 1).
Further, this study found four variants (A, B, C, and D) of trichodea
sensilla, which is in agreement with Esslen and Kaissling (1976) and
Suwannapong et al. (2012). Trichodea A are hair-like sensilla with a saber-shape
and a pointed tip (Figure 11a), whereas trichodea B are long hair sensilla that are
slightly curved with sharpened tips (Figure 11b). Trichodea C and D are both hair
sensilla with nearly tapered tips and latter has an S-shape (Figure 11a).
Figure 10 Structure of A. cerana worker antenna
b
a
b
c
d
Figure 11 Antennal sensilla types in several flagella of A. andreniformis and
A. cerana. (a) fl2 of an A. andreniformis worker, (b) fl9 of an A.
cerana worker, (c) fl3 of an A. cerana queen (d) fl6 of an A.
andreniformis worker. (TA: trichodea A; TB: trichodea B; TC:
trichodea C; TD: trichodea D; BS: bassiconica; PL: placodea; CA:
campaniform; AM: ampullacea; CO: coeloconica).
12
This study observed the existence of bassiconica that are straight pegs with
a blunt tip of a relatively shorter length than trichodea (Figure 11b), and placodea
(plate-like sensilla) that were characterized by a radially striated oval plate
surrounded by a narrow membranous ring (Figure 11c-d). Several sensilla also
existed as campaniform (peg sunken in pit sensilla), which are characterized as
emerging from oval pits and having a bell-shape with no pores or openings.
Ampullacea (deep flask sensilla) and coeloconica (sensory pit peg) are pit organlike, with the ampullacea having a hole with a small opening in (Figure 11c) and
the coeloconica having a large opening (Figure 11d). These classifications
followed those of Esslen and Kaissling (1976) and Suwannapong et al. (2012).
Total density of sensilla/0.01mm2
Densities of total antennal sensilla in bees exhibiting different type of nest in
the three castes of A. andreniformis and A. cerana
The total densities of the six sensilla types in the cavity nesting A. cerana
are higher compare to the open nesting A. andreniformis (Figure 12). The density
of bassiconica in workers is the highest compare to the other castes in both species,
and the density of placodea in the drones is the highest as well (Figure 13).
However, the densities of certain sensilla such as bassiconica, campaniform,
ampullacea, and coeloconica are higher in A. andreniformis than those in A.
cerana (Figure 14). Several similar and different density patterns of each sensilla
type existed within castes of honey bees exhibiting both nesting behaviors. The
sensilla are varied among the three castes of bees (Figure 14).
Sensilla density of trichodea C, trichodea D, and bassiconica are more
abundant in worker flagella among other castes in both species, while placodea,
ampulacea, coeloconica sensilla are more abundant in drone among other castes
(Figure 14). Interestingly, this study did not find coeloconica in the queen of A.
cerana. Density of trichodea A has the highest in A. cerana queen and trichodea B
are more higher on in A. cerana worker. Furthermore, bassiconica were not found
in A. cerana drone (Figure 14; Appendix 1, 5, and 7).
4500
4000
3500
3000
2500
2000
1500
1000
500
0
A.a
A.c
Species
Figure 12 Total densities of antennal sensilla of A. andreniformis (A.a) and A.
cerana (A.c)
13
a
b
c
d
e
f
Figure 13 Sensilla distribution on the fl8 anterior side of the three castes of A.
andreniformis and A. cerana. (a) A. andreniformis queen; (b) A.
cerana queen; (c) A. andreniformis drone; (d) A. cerana drone; (e)
A. andreniformis worker; and (f) A. cerana worker.
70
Trichodea A (Aa)
60
50
40
30
20
10
Density of sensilla / 0.01
mm2
Density of sensilla / 0.01
mm²
14
0
60
50
40
30
20
10
50
40
30
20
10
1 2 3 4 5 6 7 8 9 10 11
Flagellomeres
Density of sensilla / 0.01
mm2
Density of sensilla / 0.01
mm²
Trichodea B (Aa)
0
70
60
40
30
20
10
0
50
40
30
20
10
1 2 3 4 5 6 7 8 9 10 11
Flagellomeres
Density of sensilla / 0.01
mm2
Density of sensilla / 0.01
mm²
Trichodea C (Aa)
60
0
70
50
40
30
20
10
0
Trichodea D (Aa)
60
50
40
30
20
10
0
1 2 3 4 5 6 7 8 9 10 11
Flagellomeres
Density of sensilla / 0.01
mm2
Density of sensilla / 0.01
mm²
Trichodea C (Ac)
60
1 2 3 4 5 6 7 8 9 10 11
Flagellomeres
70
Trichodea B (Ac)
50
1 2 3 4 5 6 7 8 9 10 11
Flagellomeres
70
Trichodea A (Ac)
60
0
1 2 3 4 5 6 7 8 9 10 11
Flagellomeres
70
70
70
Trichodea D (Ac)
60
50
40
30
20
10
0
1 2 3 4 5 6 7 8 9 10 11
Flagellomeres
1 2 3 4 5 6 7 8 9 10 11
Flagellomeres
5
Bassiconica (Aa)
4
3
2
1
Density of sensilla / 0.01
mm2
Density of sensilla / 0.01
mm²
15
0
Bassiconica (Ac)
5
4
3
2
1
0
1 2 3 4 5 6 7 8 9 10 11
1 2 3 4 5 6 7 8 9 10 11
Flagellomeres
70
Placodea (Aa)
60
50
40
30
20
10
Density of sensilla / 0.01
mm2
Density of sensilla / 0.01
mm²
Flagellomeres
70
60
50
40
30
20
10
0
0
1 2 3 4 5 6 7 8 9 10 11
1 2 3 4 5 6 7 8 9 10 11
Campaniform (Aa)
4
3
2
1
Flagellomeres
Density of sensilla / 0.01
mm2
Density of sensilla / 0.01
mm²
Flagellomeres
5
5
4
3
2
1
1 2 3 4 5 6 7 8 9 10 11
Flagellomeres
Ampullacea (Aa)
4
3
2
1
0
1 2 3 4 5 6 7 8 9 10 11
Flagellomeres
Density of sensilla / 0.01
mm2
1 2 3 4 5 6 7 8 9 10 11
Flagellomeres
Density of sensilla / 0.01
mm²
Campaniform (Ac)
0
0
5
Placodea (Ac)
5
Ampullacea (Ac)
4
3
2
1
0
1 2 3 4 5 6 7 8 9 10 11
Flagellomeres
5
Coeloconica (Aa)
4
3
2
1
Density of sensilla / 0.01
mm2
Density of sensilla / 0.01
mm2
16
0
5
Coeloconica (Ac)
4
3
2
1
0
1 2 3 4 5 6 7 8 9 10 11
1 2 3 4 5 6 7 8 9 10 11
Flagellomeres
Flagellomeres
Figure 14 Density of antennal sensilla in A. andreniformis (Aa) and A. cerana
(Ac) of queen ( ), drone ( ), and worker ( )
12
10
Anterior
Posterior
8
6
4
2
0
Queen
Drone Worker
Caste
a
Density of sensilla / 0.01 mm2
Density of sensilla / 0.01 mm2
Different distributions of antennal sensilla between flagellar sides, and
between the upper and lower ends of flagella
12
10
b
Anterior
Posterior
8
6
4
2
0
Queen Drone Worker
Caste
b
Figure 15 Density of antennal sensilla on the anterior and posterior side. (a) A.
andreniformis; (b) A. cerana.
The total sensilla number on the antennal anterior side is twice compared
with the posterior side, i.e. 68.4% and 31.6%, respectively, in A. andreniformis
(Figure 15a) and 60.5% and 39.5%, respectively, in A. cerana (Figure 15b).
However, there is no distribution pattern similarity of each sensilla type between
A. andreniformis and A. cerana (Figure 14; Appendix 2-7).
The distribution pattern of sensilla trichodea A are more abundant in lower
end of the antenna compare to upper end of antenna for both species (Figure 15).
Whereas, sensilla trichodea B, trichodea D, and bassiconica show the opposite
distribution pattern compare to trichodea A that are descend in lower end and
increase in upper end of flagella (Figure 15; Appendix 2-7). Furthermore, this
17
study found sensilla trichodea C, placodea, and ampullacea are distribute evenly
along the flagella of three caste in both species, except sparsely in fl1 and fl2 for
trichodea C and placodea and ampullaceal are not found on fl1and fl2 for.
Otherwise, sensilla campaniform and coeloconica do not show distribution pattern
(Figure 14).
Ultramorphometric variations of trichodea and placodea sensilla of three
castes in two sides of antenna in A. andreniformis and A. cerana
Ultramorphometric measurement of the length, width, and area of sensilla
of trichodea and placodea are significantly different (p < 0.05) among the three
caste of A. andreniformis, except trichodea C (Table 2). Significantly different of
all sensilla types measurements among the three castes are also found in A. cerana
(p < 0.05), except the length (p = 0.065) and area (p = 0.097) of trichodea A and
placodea (p = 0.079) (Table 2).
Table 2 Ultramorphometric of trichodea and placodea sensilla of the three castes
of A. andreniformis and A. cerana
Ultramorphometric (µm) ± SE
Queen
Drone
Worker
Apis andreniformis
Trichodea A
22.180ab ± 0.397
20.705a ± 0.643
23.335b ± 0.409
b
a
Trichodea B
16.265 ± 0.231 13.762 ± 0.338
13.807a ± 0.237
Length
a
a
Trichodea C
18.050 ± 0.589 16.667 ± 0.257
17.606a ± 0.356
(µm)
b
a
Trichodea D
15.860 ± 0.494 *12.217 ± 0.408
12.645a ± 0.265
a
ab
Placodea
14.692 ± 0.267 15.282 ± 0.143 16.146b ± 0.511
Trichodea A
3.357a ± 0.083
3.370a ± 0.165
3.612a ± 0.105
b
a
Trichodea B
1.614 ± 0.041
1.313 ± 0.045
1.434a ± 0.060
Width
a
a
Trichodea C
3.010 ± 0.067
2.608 ± 0.135
2.811a ± 0.174
(µm)
b
ab
Trichodea D
1.575 ± 0.071 *1.405 ± 0.079
1.236a ± 0.031
a
b
Placodea
8.965 ± 0.365 10.417 ± 0.151 10.403b ± 0.251
Trichodea A
49.497a ± 1.279 57.957a ± 4.352 58.720a ± 1.714
Trichodea B
12.355c ± 0.412 10.679b ± 0.513
8.604a ± 0.277
Area
a
a
Trichodea C
38.424 ± 1.687 33.462 ± 1.199 35.494a ± 2.338
(µm2)
Trichodea D
12.060b ± 0.761 *8.064a ± 0.516
7.202a ± 0.338
a
b
Placodea
121.594 ± 4.835 149.410 ± 3.647 154.816b ± 6.872
Trichodea A
25.339a ± 0.371 24.617a ± 0.762 23.484a ± 0.443
Trichodea B
18.726c ± 0.237 14.125a ± 0.561 16.294b ± 0.701
Length
Trichodea C
19.131b ± 0.441 15.749a ± 0.272 18.838b ± 0.365
(µm)
Trichodea D
20.965b ± 2.767 10.736a ± 0.522 15.494ab ± 0.141
Placodea
14.933a ± 0.255 14.622a ± 0.243 15.362a ± 0.158
Trichodea A
3.449a ± 0.120
4.405b ± 0.211
3.726a ± 0.077
b
ab
Trichodea B
1.930 ± 0.044
1.717 ± 0.099
1.674a ± 0.039
Width
a
a
Trichodea C
3.599 ± 0.083
2.792 ± 0.069
3.101b ± 0.075
(µm)
b
a
Trichodea D
1.830 ± 0.054
1.408 ± 0.062
1.759b ± 0.067
a
b
Placodea
9.077 ± 0.4725 10.324 ± 0.195
8.603a ± 0.1109
a
a
Trichodea A
57.733 ±1.831
69.626 ± 6.469 59.776a ± 2.196
c
Trichodea B
16.189 ± 0.492 10.854a ± 0.758 13.086b ± 0.194
Area
Trichodea C
38.730b ± 1.350 28.038a ± 0.916 34.317b ± 1.556
(µm2)
Trichodea D
14.039b ± 0.489
7.182a ± 0.445 12.807b ± 0.247
a
Placodea
126.036 ±6.1664 135.691a ± 4.9844 120.115a± 2.6416
Different letter in the same row indicate significant different (ANOVA, p < 0.05)
Data analysis using one way ANOVA and posthoc Tukey
Data are n = 20, except data with (*) is n = 10
Measure
Sensilla Type
Sig.
0.05
0.002
0.000
0.071
0.000
0.015
0.262
0.000
0.112
0.000
0.000
0.042
0.000
0.157
0.000
0.000
0.065
0.000
0.000
0.000
0.071
0.000
0.019
0.000
0.000
0.001
0.097
0.000
0.000
0.000
0.079
18
Ultramorphometric of trichodea B of queen in both species are the largest
among other castes. On the contrary, the measurement of trichodea C and
trichodea D of A. cerana drone are the smallest than other castes (Table 2). Thus,
ultramorphometrics sensilla show variation between the castes.
Among the three castes of A. andreniformis the areas measurement of
placodea between anterior and posterior are significantly different (p < 0.05). A.
andreniformis worker show ultramorphometric significantly different between
anterior and posterior (p < 0.05) of all sensilla types, except the length (t =
0.2277) and area (t = 0.7083) of trichodea A. The measurement of length and
width of all sensilla types of Apis. cerana queen on two sides of flagella are
significantly different (p < 0.05) (Table 3).
Table 3 Comparison ultramorphometric of trichodea and placodea sensilla on
anterior and posterior sides of the three castes of A. andreniformis and A.
cerana
Measure
Sensilla
Type
Trichodea A
Trichodea B
Length
(µm)
Trichodea C
Trichodea D
Placodea
Trichodea A
Trichodea B
Width
(µm)
Trichodea C
Trichodea D
Placodea
Trichodea A
Trichodea B
Area
(µm2)
Trichodea C
Trichodea D
Placodea
Ultramorphometric of sensilla on anterior : posterior side
t value
Queen
Drone
Worker
Apis andreniformis
21.238 : 23.123*
21.650 : 19.760ns
23.431 : 23.240ns
t = 2.758
t = 0.2277
t = 1.5195
15.819 : 16.712ns
13.965 : 13.560ns
13.157 : 14.457*
t = 2.0969
t = 0.5892
t = 3.4295
15.652 : 20.449*
15.942 : 17.392*
16.186 : 19.026*
t = 11.1115
t = 3.6099
t = 9.5902
15.180 : 16.540ns
13.550 : 11.740*
t = 1.4129
t = 5.3495
ns
15.131 : 14.254
15.147 : 15.417ns
13.990 : 18.302*
t = 1.7259
t = 0.9423
t = 16.5253
3.134 : 3.580*
3.470 : 3.270ns
3.314 : 3.910*
t = 3.335
t = 0.5961
t = 3.6292
1.520 : 1.708*
1.255 : 1.372ns
1.211 : 1.658*
t = 2.6145
t = 6.8896
t = 1.3401
2.778 : 3.188*
2.029 : 3.242*
2.076 : 3.547*
t = 5.5818
t = 22.1753
t =16.4161
1.580 : 1.570ns
1.159 : 1.314*
t = 0.0683
t = 2.9773
10.063 : 7.868*
10.620 : 10.214ns
9.557 : 11.249*
t = 4.033
t = 1.3797
t = 5.1782
48.656 : 50.339ns
69.140 : 46.775*
59.951 : 57.490ns
t = 0.6479
t = 3.0959
t = 0.7083
13.200 : 9.708*
11.650 : 11.510ns
7.862 : 9.346*
t = 2.2645
t = 2.046
t = 3.3128
31.489 : 45.360*
28.742 : 38.183*
30.461 : 40.527*
t = 8.9191
t = 12.046
t = 2.4098
11.280 : 12.840 ns
8.113 : 6.292*
t = 1.0263
t = 3.3368
156.787 : 105.894*
137.294 : 142.033*
128.022 : 181.610*
t = 4.7383
t = 2.2228
t = 8.4877
19
Table 3 (Continued)
Ultramorphometric of sensilla on anterior : posterior side
t value
Queen
Drone
Worker
Apis cerana
24.521 : 26.157*
27.325 : 21.910*
23.664 : 23.305ns
Trichodea A
t = 2.4907
t = 5.9615
t = 0.3958
19.468 : 17.984*
15.840 : 12.410*
15.298 : 17.291ns
Trichodea B
t = 4.3896
t = 4.1776
t = 1.463
Length
17.704 : 20.558*
14.774 : 16.724*
17.521 : 20.155*
Trichodea C
(µm)
t = 4.7025
t = 6.1395
t = 6.283
18.645 : 23.285*
8.6230 : 12.850ns
17.521 : 15.180*
Trichodea D
t = 0.8317
t = 10.6224
t = 2.5158
15.511 : 14.355*
13.696 : 15.549*
14.867 : 15.857*
Placodea
t = 2.5846
t = 7.6436
t = 4.3951
3.184 : 3.714*
5.100 : 3.710*
3.747 : 3.705ns
Trichodea A
t = 2.828
t = 4.9138
t = 0.2645
2.052 : 1.808*
2.050 : 1.384*
1.552 : 1.797*
Trichodea B
t = 3.5173
t = 5.1315
t = 4.4006
Width
3.293 : 3.905*
2.548 : 3.037*
2.816 : 3.386*
Trichodea C
( µm)
t = 6.843
t = 5.8728
t = 7.5507
1.958 : 1.703*
1.410 : 1.407ns
1.674 : 1.844ns
Trichodea D
t = 2.735
t = 0.0234
t = 1.2902
10.955 : 7.200*
9.552 : 11.096*
8.836 : 8.371*
Placodea
t = 9.4049
t = 9.0215
t = 2.326
56.070 : 59.396ns
92.790 : 46.463*
66.553 : 53.000*
Trichodea A
t = 0.904
t = 6.1126
t = 4.2525
16.820 : 15.558ns
13.400 : 8.308*
12.681 : 13.491*
Trichodea B
t = 5.131
t = 2.3163
t = 1.3069
Area
33.983 : 43.478*
24.404 : 31.673*
28.243 : 40.391*
Trichodea C
(µm2)
t = 5.7931
t = 9.3218
t = 8.5269
14.579 : 13.499ns
5.919 : 8.446*
12.410 : 13.205ns
Trichodea D
t = 1.1117
t = 3.6498
t = 1.6853
150.202 : 101.870*
116.544 : 154.839*
115.26 : 124.97ns
Placodea
t = 8.7129
t = 7.9125
t = 1.9729
Asterisk (*) in the same row indicate significant different (t-test, p < 0.05)
ns indicate not significant
(-) indicate no data
Measure
Sensilla
Type
Discussion
Density and distribution variations of sensilla types in open and cavity
nesting bees suggests an environmental adaptation
Previously, Suwannapong et al. (2012) found four sensilla types trichodea,
bassiconica, placodea, and campaniform in workers of A. andreniformis, was
collected in Thailand. In agreement with previous studies, the four described types
were found, along with two new sensilla types, ampullacea and coeloconica, in the
three castes of the open nesting A. andreniformis. These data are suggest because
inadvertence the types of sensilla in the previous studies.
Our findings corroborated that the cavity nesting honey bee A. cerana has
a higher total density of sensilla compared with the open air nesting A.
andreniformis. This is in agreement with the higher density of sensilla on cavity
nesting drones of A. melliferra than on the open nesting A. florea (Brockmann and
20
Brückner 2001). This phenomena suggests that cavity nesting honey bees, which
live in the dark (Oldroyd and Wongsiri 2006),
OF ANTENNAL SENSILLA BETWEEN Apis andreniformis AND
Apis cerana IN THE THREE CASTES OF HONEY BEES
CUT FERAWATI
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2015
STATEMENT LETTER
I hereby declare that this thesis entitled “Variations in the Density and
Ultramorphometric of Antennal Sensilla between Apis andreniformis and Apis
cerana in the Three Castes of Honey Bees” is the original result of my own
research under supervision of an advisory commitee and has never been submitted
in any form to any other institution before. All information from other authors
cited here are mentioned in the text and listed in the reference list at the end of the
thesis.
Bogor, October 2015
Cut Ferawati
Student ID G352130141
SUMMARY
CUT FERAWATI. Variations in the Density and Ultramorphometric of
Antennal Sensilla between Apis andreniformis and Apis cerana in the Three
Castes of Honey Bees. Supervised by RIKA RAFFIUDIN and BERRY
JULIANDI.
Honey bees live in two different types of nest, the single comb open air
nests of Apis andreniformis, A. florea and A. dorsata and multiple comb cavity
nests of A. cerana, A. koschevnikovi, A. nuluensis, A. nigrocincta, and Athra nests.
Honey bee that consist of three castes (queen, drone and worker caste) perform
complex communications. The communication mostly mediate by antennal
sensilla that receive odor or pheromone. However, the variations of the density,
distribution, and ultramorphometric of antennal sensilla of the open and cavity
nesting types is poorly understood and the available data is derived from the
worker caste.
The objectives of this research were to analyse: (1) the variations of the
type and density of antennal sensilla within the three castes of the open nesting A.
andreniformis and cavity nesting A. cerana, (2) the sensilla distribution between
the anterior and posterior sides of antenna and between lower and upper end of
each flagellar antenna within the three castes of A. andreniformis and A. cerana,
and (3) ultramorphometric of the length, width, and area of trichodea and
placodea sensilla in anterior and posterior sides of flagella within the three castes
in both species.
The honey bee samples of A. andreniformis and A. cerana were obtained
from Padang Pariaman (West Sumatera) and Bogor (West Java), Indonesia,
respectively. Antennal sensilla of honey bees were analysed by using scanning
electron microscope. The densities, distributions and ultramorphometric of
antennal sensilla were examined using the ImageJ program. Furthermore, the
ultramorphometric sensilla were analyzed with ANOVA and t-test.
This study observed six antennal sensilla types in A. andreniformis and A.
cerana. Those are trichodea (variants A, B, C, and D), bassiconica, placodea,
campaniform, ampullacea, and coeloconica. The last two types of sensilla are the
new report in the open nesting A. andreniformis.
Densities of sensilla in the cavity nesting honey bee A. cerana are higher
compare to the open air nesting A. andreniformis. This phenomena might be a
structure adaption due to the bees use antennal sensilla for communication inside
of dark cavity hive.
The density of sensilla is varied among the three castes of open nesting A.
andreniformis and cavity nesting A. cerana. The density of bassiconica in worker
caste is higher than in the other castes in both species. The density of placodea is
the highest in the drone caste of both species. Sensilla density of trichodea C,
trichodea D, and bassiconica are more abundant in worker flagella among other
castes in both species, while placodea, ampulacea, coeloconica sensilla are more
abundant in drone among other castes. Interestingly, this study did not find
coeloconica in the queen of A. cerana. Density of trichodea A is the highest in A.
cerana queen and trichodea B are more higher on in A. cerana worker.
Furthermore, bassiconica were not found in A. cerana drone.
This research also the first report of the variations of antennal sensilla
distribution found between the anterior and posterior sides, as well as between the
upper and the lower end of flagellar antenna. The number of sensilla on the
anterior side is twice compared to the posterior side of flagellar antenna in A.
andreniformis and A. cerana. This result suggests that anterior areas are more
prominent to receive odor comes from the frontal area of the bee head. This study
also observed that the sensilla distributions are more prominent on the upper end
of flagella compare to the lower end. This result might be due to the upper end of
antenna is directly contact to the substrate, thus serve as the chemo- and
mechanoreception.
Beside the variations of the density and distribution of antennal sensilla
types, almost all of ultramorphometrics of trichodea and placodea sensilla are
differ within the three castes of open nesting A. andreniformis and cavity nesting
A. cerana. Ultramorphometric measurements of A. andreniformis drone placodea
are the highest compare to other caste. This suggest that they need highly efficient
to detect the queen pheromones. The ultramorphometric sensilla on anterior and
posterior sides are also different, that might imply the functional of sensilla as an
effective ogan to capture the odor molecules exposure to the olfactory sensory
from the outside environment. Therefore, the diversity in the density and
distribution as well as the ultramorphometric of antennal sensilla might give an
indication of the adaption of honey bees to different nesting environments.
Key words: Olfactory sensory, antennal sensilla, open-nesting honey bee, cavitynesting honey bee, Apis andreniformis, Apis cerana
RINGKASAN
CUT FERAWATI. Variasi Densitas dan Ultramorphometrik dari Sensilla
Antena antara Apis andreniformis dan Apis cerana pada Ketiga Kasta Lebah
Madu. Dibimbing oleh RIKA RAFFIUDIN dan BERRY JULIANDI.
Lebah madu hidup pada dua tipe sarang yang berbeda, yaitu sarang
terbuka dengan satu sisir seperti sarang Apis andreniformis, A. florea and A.
dorsata dan sarang tertutup dengan beberapa sisir seperti A. cerana, A.
koschevnikovi, A. nuluensis, A. nigrocincta, dan A. mellifera. Lebah madu terdiri
atas tiga kasta (ratu, jantan, dan pekerja) yang melakukan komunikasi kompleks.
Komunikasi pada umumnya menggunakan sensila pada antenna untuk menerima
bau atau feromon. Namun, masih sedikit pengetahuan tentang variasi densitas,
distribusi, dan ultramorfometrik sensilla antena pada tipe sarang terbuka dan
tertutup dan data yang tersedia hanya kasta pekerja saja.
Tujuan penelitian ini adalah untuk menganalisis: (1) variasi tipe dan
densitas sensila antena pada ketiga kasta dari lebah sarang terbuka A.
andreniformis dan lebah sarang tertutup A. cerana, (2) distribusi sensila antara sisi
anterior dan posterior serta antara pangkal dan ujung dari masing-masing flagela
antena pada ketiga kasta A. andreniformis dan A. cerana, dan (3)
ultramorfometrik panjang, lebar dan area dari sensila trichodea dan placodea pada
sisi anterior and posterior flagela pada ketiga kasta dari kedua spesies.
Sampel lebah madu A. andreniformis dan A. cerana diperoleh dari Padang
Pariaman (Sumatera Barat) dan dari Bogor (Jawa Barat), Indonesia, secara
berurutan. Analisis Sensila antena lebah madu menggunakan scanning electron
microscope. Pengujian densitas, distribusi dan ultramorfometrik sensila antena
diuji menggunakan program ImageJ. Selanjutnya, data ultramorfometrik sensila
dianalisis menggunakan ANOVA dan uji t.
Pada penelitian ini ditemukan enam tipe sensila pada A. andreniformis dan
A. cerana, yaitu sensila trichodea (variasi A, B, C, dan D), bassiconica, placodea,
campaniform, ampullacea, dan coeloconica. Dua tipe sensilla terakhir merupakan
laporan baru pada lebah sarang terbuka A. andreniformis.
Total densitas sensila lebah sarang tertutup A. cerana memiliki yang lebih
banyak dibandingkan lebah sarang terbuka A. andreniformis. Fenomena ini
kemungkinan karena adanya adaptasi struktur karena lebah menggunakan sensilla
untuk berkomunikasi di dalam sarang yang gelap.
Densitas sensila bervariasi diantara ketiga kasta pada lebah sarang terbuka
A. andreniformis dan lebah sarang tertutup A. cerana. Densitas sensila bassiconica
pada kasta pekerja lebih tinggi dibandingkan kasta lainnya pada kedua spesies.
Densitas placodea paling tinggi ditemukan pada kasta jantan pada kedua spesies.
Densitas sensila trichodea C, trichodea D, dan bassiconica lebih banyak pada
flagela kasta pekerja dibandingkan kasta lainnya pada kedua spesies, sementara
itu sensila placodea, ampulacea, coeloconica lebih banyak pada kasta jantan
dibandingkan kasta lainnya. Menariknya, pada penelitian ini tidak ditemukan
sensila coeloconica pada kasta ratu A. cerana. Densitas trichodea A paling tinggi
ditemukan pada kasta ratu A. cerana dan trichodea B lebih banyak pada kasta
pekerja A. cerana. Selanjutnya, bassiconica tidak ditemukan pada kasta jantan A.
cerana.
Penelitian ini juga pertama kali mencatat adanya variasi distribusi sensila
antara sisi anterior dan posterior antena, sama halnya dengan sensila antara ujung
dan pangkal flagela antena. Densitas sensila pda sisi anterior dua kali lipat lebih
banyak dibandingkan sisi posterior dari flagela antena pada A. andreniformis dan
A. cerana. Hasil ini diperkirakan karena area anterior lebih dominan untuk
menerima bau dari area depan kepala lebah. Penelitian ini juga menemukan
bahwa distribusi sensila lebih dominan pada bagian ujung flagela dibandingkan
pangkal. Hasil tersebut diperkirakan karena bagian ujung antena kontak langsung
dengan substrat, dalam mengindera chemo dan mechanoreception.
Selain variasi densitas dan distribusi tipe sensila antena, hampir semua
ultramorfometrik sensila trichodea dan placodea berbeda pada ketiga kasta dari
lebah sarang terbuka A. andreniformis dan lebah sarang tertutup A. cerana. Hasil
pengukuran ultramorfometrik placodea pada kasta jantan A. andreniformis lebih
panjang dibandingkan kasta lainnya. Hal ini diperkirakan karena jantan
membutuhkan sensila tersebut agar lebih efisien dalam mendeteksi feromon ratu.
Ultramorfometrik sensila pada anterior dan posterior berbeda, diperkirakan fungsi
sensila agar efektif menangkap molekul bau dari lingkungan luar oleh sensorik
olfaktori. Oleh karena itu, keragaman densitas dan distribusi maupun
ultramorfometrik sensila antena mungkin mengindikasikan bahwa adaptasi lebah
madu terhadap perbedaan lingkungan di sekitar sarang.
Kata kunci: Sensorik olfaktori, sensilla antena, lebah madu sarang terbuka, lebah
madu sarang tertutup, Apis andreniformis, Apis cerana
Copyright © 2015 Bogor Agricultural University
Copyright are Protected By Law
It is prohibited to cite all or part of this thesis without referring to and
mentioning the sources. Citation only permitted for the sake of education,
research, scientific writing, report writing, critical writing or reviewing scientific
problem. Citation does not infict the name and honour of Bogor Agricultural
University
It is prohibited to publish or to reproduce all or part of thesis without the
written permission from Bogor Agricultural University
VARIATIONS IN THE DENSITY AND ULTRAMORPHOMETRIC
OF ANTENNAL SENSILLA BETWEEN Apis andreniformis AND
Apis cerana IN THE THREE CASTES OF HONEY BEES
CUT FERAWATI
Thesis
As a partial fulfilment of the requirements for a Master Degree
in Animal Biosciences Master Program of Graduate School of
Bogor Agricultural University
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2015
Non-Commitee Examiner: Dr. Ir. Sih Kahono, M.Sc
PREFACE
Praise and grateful to Allah the All Mighty, for the completion of this
Master Thesis entitled Variations in the Density and Ultramorphometric of
Antennal Sensilla between Apis andreniformis and Apis cerana in the Three
Castes of Honey Bees. The study was conducted from June 2014 to June 2015.
This research was supported by Master Program Scholarship (BPPDN) from the
Directorate of General Higher Education of Indonesia (DIKTI), grant no.
1094/E4.4/2013.
I thank profusely to Dr. Ir. Rika Raffiudin, M.Si and Dr. Berry Juliandi,
S.Si, M.Si as the supervising commitee, which have supervised and put so much
trust to me during my master study and the completion of the thesis. I sincerely
thank to Dr. Ir. Sih Kahono, M.Sc as the thesis defense examiner and to Dr. Ir.
R.R. Dyah Perwitasari, M.Sc as the Head of Animal Bioscience Postgraduate
Master Program, Department of Biology, for insightful comments and correction
of the thesis. My gratitude also to honey bee researcher Prof. Siti Salmah, Drs.
Mochammad Chandra Widjaja, MM and Dr. Jasmi, for the knowledge and
experience that you shared, that had raised my interest on honey bees.
I address my thankfulness also to staff Scanning Electron Microscope
Laboratory, Division of Zoology, Research Center for Biology of Indonesian
Institute of Sciences (LIPI) for their magnification technical assistance. I specially
thank to staff of National Beekeeping Center (PUSBAHNAS), sampling team in
Padang Pariaman (West Sumatra), and sampling team in Bogor (West Java) for
the invaluable assistance during field data collection.
I also would like to thank to all the lectures in Animal Bioscience Master
Program, who at the end shaping my knowledge up through their high dedication
into the research and teaching. The endless gratitude is addressed to my parents,
brothers and sister who have supported me by their own way during the study.
The least, but very important, I would like to thank to all my friends; class of BSH
2013, BSH students, Desmina Kristiani Hutabarat, Febri Ayu, and Rosi Fitri
Ramadani.
Hopefully this scientific work will be useful.
Bogor, October 2015
Cut Ferawati
CONTENT LIST
LIST OF TABLE
xv
LIST OF FIGURES
xv
LIST OF APPENDICES
xv
1 INTRODUCTION
Background
Objectives
1
1
2
2 LITERATURE REVIEW
Honey Bees Evolution
Honey Bees Castes
Distribution of Apis andreniformis and Apis cerana
Nest Types
Sex Determination and Sexual Dimorphism
Structure and Functions of Sensilla
3
3
4
4
5
5
7
3 METHODS
Study Site and Time
Samples Collection
Antenna Preparation and SEM Analysis
Data Analysis
8
8
8
9
9
4 RESULTS AND DISCUSSION
Results
Antennal structure and sensilla types of A. andreniformis and A.
cerana, and in the three castes
Densities of total antennal sensilla in bees exhibiting different type
of nest in the three castes of A. andreniformis and A. cerana
Different distributions of antennal sensilla between flagellar sides,
and between the upper and lower ends of flagella
Ultramorphometric variations of trichodea and placodea sensilla of
three castes in two sides of antenna in A. andreniformis and A.
cerana
Discussion
Density and distribution variations of sensilla types in open
and cavity nesting bees suggests an environmental adaptation
Antennal sensilla density varies among castes
Ultramorphometric sensilla are different among the three castes and
flagellar sides
10
10
10
12
16
17
19
19
20
21
5 CONCLUSSION
22
REFERENCES
22
APPENDICES
25
BIOGRAPHY
39
LIST OF TABLES
1 The three castes of A. andreniformis and A. cerana
2 Ultramorphometric of trichodea and placodea sensilla of the three
castes of A. andreniformis and A. cerana
3 Comparison ultramorphometric of trichodea and placodea sensilla on
anterior and posterior sides of the three castes of A. andreniformis and
A. cerana
8
17
18
LIST OF FIGURE
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
The type of honey bee nest
Bayesian consesus tree derived of Apis from the cox2 sequence
Polyagethism of A. cerana workers
Geographical distribution area of A. andreniformis and A. cerana
Sex determination of queen eggs
Reproduction system structures of three castes of honey bees
Schematic of sensilla anatomy
Morphology of honey bee antenna
Ultramorphometric measurement of honey bee sensilla
Structure of A. cerana worker antenna
Antennal sensilla types in several flagella of A. andreniformis and A.
cerana
Total densities of antennal sensilla of A. andreniformis and A. cerana
Sensilla distribution on the fl8 anterior side the three castes of A.
andreniformis and A. cerana
Density of antennal sensilla in A. andreniformis and A. cerana of queen,
drone, and worker
Density of antennal sensilla on the anterior and posterior side
1
3
4
5
6
6
7
9
10
11
11
12
13
16
16
LIST OF APPENDICES
1
2
3
4
5
6
Antennal sensilla of an A. andreniformis queen flagella
Antennal sensilla of an A. andreniformis drone flagella
Antennal sensilla of an A. andreniformis worker flagella
Antennal sensilla of an A. cerana queen flagella
Antennal sensilla of an A. cerana drone flagella
Antennal sensilla of an A. cerana worker flagella
25
27
30
32
34
37
1
1 INTRODUCTION
Background
Honey bees (Hymenoptera: Apidae) live in two different types of nest, the
single comb open air nests of Apis andreniformis (Figure 1a), A. florea and A.
dorsata and multiple comb cavity nests of A. cerana (Figure 1b), A. koschevnikovi,
A. nuluensis, A. nigrocincta, and A. mellifera nests (Smith 1991; Oldroyd and
Wongsiri 2006; Raffiudin and Crozier 2007). Honey bee in both nest types have
different dance behaviors, which are A. andreniformis perform a horizontal dance
compare to vertical dance of A. cerana (Winston 1991; Dyer 2002). The different
dance behaviors might adapt different nest environment (Raffiudin and Crozier
2007; Hepburn and Radloff 2011).
Social life is one of the characteristics of honey bee. A colony of honey
bee consist of three castes based on their tasks (Ruttner 1988). However, the
division of honey bee caste can also based on the reproductive ability. The
reproductive caste consist of queen and drones, whereas the non-reproductive
caste all workers (Winston 1991). Each caste of honey bees have spesific function
or task in the colony. The role of queen honey bee has the role in produce eggs
and control the colony using queen pheromone. Drones have the role to mate the
queen.
The inside role of workers in the colony are cleaning the cell, tending the
brood, receiving nectar, capping the cell, attending the queen, building the comb,
food handling, and cleaning debris. The outside role of worker are ventilation,
guarding, and foraging (Winston 1991). Therefore, honey bees need the spesific
communication to carry out vary kind activity between or within castes.
Those communications use odor or pheromones and are received by the
antennal sensilla. Antennal sensilla of the queen receive odor or pheromones that
mediate communications with the workers in the hive and with the drones during
mating flight. Drones use these sensilla to detect queen and workers pheromones
a
b
Figure 1 The type of honey bee nest. (a) open nesting A. andreniformis and (b)
cavity nesting A. cerana (Figure b from Suwannapong et al. 2011).
2
(Winston 1991). Workers use olfactory sensilla to detect odors inside the colony
such as brood pheromone, queen pheromone, and outside the colony for detected
floral odors (Chapman 1998). Besides chemoreceptor, the antennal sensilla
facilitates also as mechanoreceptor, hygroreceptor, thermoreceptor, and gustatory
receptor (Chapman 1998).
Four antennal sensilla types of open nesting A. andreniformis workers are
trichodea, bassiconica, placodea, and campaniform and 81.6% of the total sensilla
is trichodea (Suwannapong et al. 2012). In cavity nesting A. mellifera there are
five types of sensilla, those are trichodea, bassiconica, placodea, ampulacea, and
coeloconica (Dostal 1958). Placodea sensilla in the drones are higher than in the
workers in A. mellifera (Esslen and Kaissling 1976). The total number of hair-like
sensilla (trichodea) per antenna in workers is higher than the queens and drones in
(Fang et al. 2012).
Further examinations showed that the anterior side of A. mellifera worker
antennae has higher number of antennal sensilla compare to the posterior side
(Esslen and Kaissling 1976). The ultramorphometric analysis of antennal sensilla
are different between A. florea and A. mellifera worker. The average length of
bassiconica, area of placodea, and diameter of coeloconica of open nesting A.
florea worker are higher than cavity nesting A. mellifera African race worker (Al
Ghamdi 2006). This suggest that to the length of antennal sensilla is adapt to
maximize the effective to capture the odor molecules from the outside
environment (Wyatt 2003).
There is no information on the variations in antennal sensilla among
queen and drone castes of the open nesting A. andreniformis. This study
questioned other types of sensilla besides the four known types of sensilla found
in the cavity nesting A. mellifera and open nesting A. andreniformis. Due to lack
of data for other cavity nesting bees, thus, this study focused on A. cerana. This is
due to A. cerana has the most widely distributed species of cavity nesting honey
bee (Ruttner 1998).
Other question is the variations in the distributions and densities of sensilla
within the three castes of those species. There is no data between the anterior and
posterior sides of antennae and between the lower and mostly for upper ends of
each antenna within the three castes of both species. More over, there is no
ultramorphometric of antennal sensilla within the three castes of open nesting A.
andreniformis and cavity nesting A. cerana.
Objectives
The objectives of this research were to analyse: (1) the variations of the
type and density of antennal sensilla within the three castes of the open nesting A.
andreniformis and cavity nesting A. cerana, (2) the sensilla distribution between
the anterior and posterior sides of antenna and between lower and upper end of
each flagellar antenna within the three castes of A. andreniformis and A. cerana,
and (3) ultramorphometric of the length, width, and area of trichodea and placodea
sensilla in anterior and posterior sides of flagella within the three castes in both
species.
3
2 LITERATURE REVIEW
Honey Bees Evolution
There are nine species of honey bees, those are clustered in the three
groups based on the worker size and nesting type. There are two recognized
species of dwarf bees, i.e. A. florea and A. andreniformis. Five species of cavity
nester bees with medium-sized workers are A. mellifera, A. koschevnikovi, A.
nuluensis, A. nigrocincta, and A. cerana. At least two species of giant bees those
are A. dorsata and A. laboriosa (Oldroyd and Wogsiri 2006; Raffiudin and
Crozier 2007; Hepburn and Radloff 2011).
Among all nine species of honey bees, the dwarf bees is in the basal of the
molecular phylogenetic tree based on cox2 gene and the giant bees and cavity
nesters are more derive species (Figure 2) (Raffiudin and Crozier 2007). The
dwarf honey bees built a single comb around a small twig constructing a platform
on the top of the nest and develope a horizontal directed waggle. Furthermore, the
ancestor of giant bees turned to open nesting and contruct the single comb under a
large branch and the bees perform their waggle dances on the vertical surface. The
cavity nesters also perform a vertical dance in the vertical comb.
Figure 2
Bayesian consesus tree derived of Apis from the cox2 sequence
(Raffiudin and Crozier 2007)
4
Honey Bees Castes
Honey bees are social life insect and consist of three castes (queen, drone,
and worker). In one colony of A. cerana there are about 6,884 individuals (Ruttner
1988). Honey bees from different castes have different function in their colony.
Queen has the role in producing eggs and controlling pheromone, while drones
have the role as to mate the queen. The workers have the role based on age
polyethism (Figure 3), those are inside and outside colony. Inside tasks include
cell cleaning, cell capping, brood and queen tending, comb building, food
handling. Whereas outside task are ventilating, guarding, orientation flights,
foraging and any other activities (Winston 1991).
Distribution of Apis andreniformis and Apis cerana
The geographical distributions of A. cerana is more wide compare to A.
andreniformis. Open nesting A. andreniformis distribute in Southern China,
Thailand, Indonesia (Sumatera, Java, and Borneo), Malaysia, Philippines
(Palawan), Burma, Laos, and Vietnam (Figure 4) (Wongsiri et al. 1996; Hepburn
and Radloff 2011). Furthermore, the distribution of A. cerana in China, India,
Malaysia, Burma, Laos, Vietnam, Philippines, Japan, Iran, Pakistan, Nepal,
Bhutan, Afghanistan, North Korea, South Korea and Indonesia (Sumatera, Java,
Borneo and Sulawesi) (Figure 4). In Indonesia A. cerana is restricted at the west
of the Wallace line (Celebes-Timor). On the Moluku Islands (Buru, Obi, and
Batjan), there is no honey bees were found except on Ambon (Ruttner 1988).
Figure 3 Polyagethism of A. cerana workers (Darmayanti 2008)
5
Figure 4 Geographical distribution area of A. andreniformis (broken line) and
A. cerana (dotted line) (Ruttner 1988 and Wongsiri et al. 1996)
Nest Types
Based on the nest structures, honey bee nests consist of the single comb
open air nests and multiple comb cavity nests. The single comb open air nest
encircle a small branch or twig, those type of nest are a typical nest of A.
andreniformis (Figure 1a) and A. florea. In addition, the single comb open air
nests of A. dorsata builds the comb on the branch of a tree, an overhang on a tall
building, or on cliff faces. Meanwhile, multiple comb cavity nests honey bee
contruct the nest inside closed cavities such as hollow trees trunks, caves, cleft in
rocks and wall which are the characterization for A. cerana (Figure 1b), A.
koschevnikovi, A. nuluensis, A. nigrocincta, and A. mellifera nests (Smith 1991;
Oldroyd and Wongsiri 2006; Raffiudin and Crozier 2007).
Nest homeostasis such as the nest temperature, humidity, carbon dioxide
level, and other enviromental factors are maintenanced at relatively constant
levels regardless of external conditions. Nest homeostasis regulation are the
functions of honey bee worker, their were detected enviromental factors using
antennal sensilla (Chapman 1998).
Sex Determination and Sexual Dimorphism
The mechanism of caste determination in honey bees are depend on the
unfertilized and fertilized egg (Figure 5). If sperm are not released when the queen
lays an egg, the unfertilized egg has haploid number of chromosome. Thus,
develop into drone honey bee. Queen or worker honey bees produce from
fertilized eggs and has diploid number of chomosomes (Winston 1991).
6
Drones, queen, and workers have unique specialization, particularly
associated with reproduction. The reproduction organs of drone are designed for
mating. Furthermore, the male structures of reproduction organ, the penis consists
of an endophallus and copulatory claspers (Figure 6a). The sperm are transferred
during mating that are produced in the testes and stored in the seminal vesicles
until mating (Winston 1991).
Figure 5 Sex determination of queen eggs (Winston 1991)
a
Figure 6
b
c
Reproduction system structures of three castes of honey bees.
(a) queen, (b) drone, and (c) worker (Winston 1991).
7
Structure and Functions of Sensilla
The antenna are the noses of insect, consist of scape, pedicle, and flagella.
Antenna of worker A. mellifera consist of ten flagella (fl1-fl10) and cover by
sensilla (Chapman 1998). The structure of sensilla have external cuticular
component within which neural processes lie in close to pores in cuticle (Figure 7).
This organ is a developmental unit composed of neural cells and support cells that
secrete the cuticular component of the sensilla (Chapman 1998).
Antennal sensilla of A. andreniformis have four different types, those are
bassiconica, campaniform, placodea, and trichodea type with total 4,892 sensilla
per antenna (Gupta 1992; Suwannapong et al. 2012). Each of antennal sensilla
type have specific functions. These sensilla are important for perception of carbon
dioxide, humidity, taste, and temperature (Winston 1991). Bassiconica and
campaniform in A. mellifera workers are involved in hygroreception (Schneider
1964). The function of bassiconica sensilla is a tasting organ in A. mellifera
workers (Dostal 1958). Placodea are involved in detecting a range of sex
pheromones released by A. florea and A. mellifera queens (Brockman and
Brückner 2001) and to receive odor in worker A. mellifera (Erickson 1982). The
hair sensilla trichodea as touch receptor or mechanoreceptor in A. mellifera and
bassiconica are possibly as taste organ (Dostal 1958).
Several function of antenna are to locate the host or prey and to block
chemicals odor for the insect survival. This was shown by the study of fig
pollinating wasps Ceratosolen solmsi marchali (Hymenoptera: Agaonidae) (Li et
al. 2009). This phytophagous wasps mainly use sensilla to locate their fig Ficus
(Moraceae) hosts. Encarsia sophia (Hymenoptera: Aphelinidae) is a parasitoid use
for biological control of Bemisia tabaci. It select the prey aided by the sensilla
(chemoreception organs) (Zhang et al. (2014). Further, the sensilla of the fleshfly
Neobellieria bullata (Diptera: Sarcophagidae) is important structure to block the
nitric oxide synthase in plant derived odorants (Wasserman and Itagaki 2003).
Figure 7 Schematic of sensilla anatomy (Chapman 1998)
8
3 METHODS
Study Site and Time
This research was conducted at Division of Animal Function or Behavior,
Department of Biology, Bogor Agricultural University and at Scanning Electron
Microscope (SEM) Laboratory, Division of Zoology, Research Center for Biology
of Indonesian Institute of Sciences (LIPI) from June 2014 until June 2015.
Samples Collection
The honey bee specimens of A. andreniformis and A. cerana were
obtained from Padang Pariaman, West Sumatera (S 00° 32’ 75.0” and E 100° 09’
77.2”) in 104 m altitude and from Bogor, West Java (S 06° 33’ 24.9” and E 106°
43’ 29.7”) in 116 m altitude, respectively. Ten individuals of workers and drones
as well as one individual of queen of both species (Table 1) were taken and all
preserved in 70% ethanol (Goldstein et al. 1992). The antennae of individual
honey bees (Figure 8) were taken using insect pins under a stereomicroscope.
Table 1 The three castes of A. andreniformis and A. cerana
Species
Castes
Queen
Drone
A.a
A.c
Scale bar: 3 mm for all pictures (A.a: A. andreniformis and A.c: A. cerana)
Worker
9
0.5 mm
a
b
Figure 8 Morphology of honey bee antenna. (a) A. cerana worker and (b)
schematic of antenna honey bee (A: anterior side, P: posterior side
Antenna
Preparation
and SEM
Analysis
of flagella,
Sc: Scape,
Pd: Pedicle,
Fl: Flagella).
Antenna Preparation and SEM Analysis
Antenna preparations were conducted in several steps: (1) Antenna was
cleaned in caccodylate buffer at -4 oC for three days (Bozzola and Russell 1998).
(2) The prefixation of antenna was in 2.5 % glutaraldehyde for 24h at -4 oC and
fixation in 3 % tannic acid for one hour. (3) Antenna was dehydrated in a graded
alcohol series from 30, 50, 75, 95, and 99.9 % for twice, 15 min/time (Goldstein et
al. 1992). (4) The dehydrated antenna was dried in tert butanol for twice, 10
min/time, then it was freeze-dried for 45 minute at -20 oC (Bozzola and Russell
1998). (5) The freeze-dried antenna was mounted on a stub and was coated with
gold and examined by SEM type JSM-5310LV (Goldstein et al. 1992; Bozzola
and Russell 1998). The coated and examined was operation by SEM laboratory
tecnicians.
Each antenna of A. andreniformis and A. cerana caste was placed on six
stubs. (1) Queen: one of the left and right antenna of queen was placed at two
stubs. (2) Drones and (3) workers: four of the right part antenna of each caste was
put on two stubs. Antennal sensilla of three castes antenna of A. andreniformis
and A. cerana taken by using SEM were recorded using a computer-controller
microscope apparatus.
One of antenna per caste in both species was observed the anterior side or
the posterior to analysis type, density, and distribution of antennal sensilla. The
anterior side of antenna is the side in the same direction to the head and the
opposite direction of side antenna to the head as the posterior side (Figure 8a).
Data Analysis
The antennal sensilla types were characterized by ultramorphology with
2,000-3,500X and the density of antennal sensilla were analyzed with 350-750X
10
magnification of SEM. Numbers of antennal sensilla was calculated using a
manual counter and ImageJ program (http://www.rsbweb.nih.gov/ij) in areas
varied between 14,000-63,000 µm2 and the values are shown as density of sensilla
per 0.01 mm2 following Ågren and Hallberg (1996). Density and distribution of
antennal sensilla was counted on the flagella of anterior and posterior sides of
queen, drone, and worker A. andreniformis and A. cerana.
The ultramorphometric of trichodea and placodea sensilla were analysed
due to the most abundance in each flagella (Esslen and Kaissling 1976; Fang et al.
2012). Those were measured with the same magnification of SEM.
Ultramorphometric data are measurement of the length, width, and area of this
sensilla as examined using an imageJ program (Figure 9a-c). The data was
analyzed within three castes using ANOVA and data of sensilla in both flagella
sides with t-test was performed using R program version 3.1.3 (CRAN.Rproject.org).
a
b
c
Figure 9 Ultramorphometric measurement of honey bee sensilla. (a) length,
(b) width, and (c) area of sensilla.
4 RESULTS AND DISCUSSION
Results
Antennal structure and sensilla types of A. andreniformis and A. cerana, and
in the three castes
This study observed the antenna of A. andreniformis and A. cerana that
consist of scape, pedicel and flagella (Figure 10). In both bees species, the antenna
consist of 10 flagella (segments) in the queen and the worker, but 11 flagella in
the drone. The antennal sensilla only found in flagella and not in other part of
antenna (Appendix 2–7).
There are six sensilla types, trichodea, bassiconica, placodea, campaniform,
ampullacea and coeloconica, in the three castes of the open nesting A.
andreniformis (Figure 11). The first four types are in agreement with
Suwannapong et al. (2012). Therefore, ampullacea and coeloconica are the two
types of antennal sensilla that are new recorded in A. andreniformis from this
study. Accordingly, the three castes of A. cerana have six sensilla types the same
11
with those of A. andreniformis, except coeloconica and bassiconica, which is
absent on queen and drones, respectively (Appendix 1).
Further, this study found four variants (A, B, C, and D) of trichodea
sensilla, which is in agreement with Esslen and Kaissling (1976) and
Suwannapong et al. (2012). Trichodea A are hair-like sensilla with a saber-shape
and a pointed tip (Figure 11a), whereas trichodea B are long hair sensilla that are
slightly curved with sharpened tips (Figure 11b). Trichodea C and D are both hair
sensilla with nearly tapered tips and latter has an S-shape (Figure 11a).
Figure 10 Structure of A. cerana worker antenna
b
a
b
c
d
Figure 11 Antennal sensilla types in several flagella of A. andreniformis and
A. cerana. (a) fl2 of an A. andreniformis worker, (b) fl9 of an A.
cerana worker, (c) fl3 of an A. cerana queen (d) fl6 of an A.
andreniformis worker. (TA: trichodea A; TB: trichodea B; TC:
trichodea C; TD: trichodea D; BS: bassiconica; PL: placodea; CA:
campaniform; AM: ampullacea; CO: coeloconica).
12
This study observed the existence of bassiconica that are straight pegs with
a blunt tip of a relatively shorter length than trichodea (Figure 11b), and placodea
(plate-like sensilla) that were characterized by a radially striated oval plate
surrounded by a narrow membranous ring (Figure 11c-d). Several sensilla also
existed as campaniform (peg sunken in pit sensilla), which are characterized as
emerging from oval pits and having a bell-shape with no pores or openings.
Ampullacea (deep flask sensilla) and coeloconica (sensory pit peg) are pit organlike, with the ampullacea having a hole with a small opening in (Figure 11c) and
the coeloconica having a large opening (Figure 11d). These classifications
followed those of Esslen and Kaissling (1976) and Suwannapong et al. (2012).
Total density of sensilla/0.01mm2
Densities of total antennal sensilla in bees exhibiting different type of nest in
the three castes of A. andreniformis and A. cerana
The total densities of the six sensilla types in the cavity nesting A. cerana
are higher compare to the open nesting A. andreniformis (Figure 12). The density
of bassiconica in workers is the highest compare to the other castes in both species,
and the density of placodea in the drones is the highest as well (Figure 13).
However, the densities of certain sensilla such as bassiconica, campaniform,
ampullacea, and coeloconica are higher in A. andreniformis than those in A.
cerana (Figure 14). Several similar and different density patterns of each sensilla
type existed within castes of honey bees exhibiting both nesting behaviors. The
sensilla are varied among the three castes of bees (Figure 14).
Sensilla density of trichodea C, trichodea D, and bassiconica are more
abundant in worker flagella among other castes in both species, while placodea,
ampulacea, coeloconica sensilla are more abundant in drone among other castes
(Figure 14). Interestingly, this study did not find coeloconica in the queen of A.
cerana. Density of trichodea A has the highest in A. cerana queen and trichodea B
are more higher on in A. cerana worker. Furthermore, bassiconica were not found
in A. cerana drone (Figure 14; Appendix 1, 5, and 7).
4500
4000
3500
3000
2500
2000
1500
1000
500
0
A.a
A.c
Species
Figure 12 Total densities of antennal sensilla of A. andreniformis (A.a) and A.
cerana (A.c)
13
a
b
c
d
e
f
Figure 13 Sensilla distribution on the fl8 anterior side of the three castes of A.
andreniformis and A. cerana. (a) A. andreniformis queen; (b) A.
cerana queen; (c) A. andreniformis drone; (d) A. cerana drone; (e)
A. andreniformis worker; and (f) A. cerana worker.
70
Trichodea A (Aa)
60
50
40
30
20
10
Density of sensilla / 0.01
mm2
Density of sensilla / 0.01
mm²
14
0
60
50
40
30
20
10
50
40
30
20
10
1 2 3 4 5 6 7 8 9 10 11
Flagellomeres
Density of sensilla / 0.01
mm2
Density of sensilla / 0.01
mm²
Trichodea B (Aa)
0
70
60
40
30
20
10
0
50
40
30
20
10
1 2 3 4 5 6 7 8 9 10 11
Flagellomeres
Density of sensilla / 0.01
mm2
Density of sensilla / 0.01
mm²
Trichodea C (Aa)
60
0
70
50
40
30
20
10
0
Trichodea D (Aa)
60
50
40
30
20
10
0
1 2 3 4 5 6 7 8 9 10 11
Flagellomeres
Density of sensilla / 0.01
mm2
Density of sensilla / 0.01
mm²
Trichodea C (Ac)
60
1 2 3 4 5 6 7 8 9 10 11
Flagellomeres
70
Trichodea B (Ac)
50
1 2 3 4 5 6 7 8 9 10 11
Flagellomeres
70
Trichodea A (Ac)
60
0
1 2 3 4 5 6 7 8 9 10 11
Flagellomeres
70
70
70
Trichodea D (Ac)
60
50
40
30
20
10
0
1 2 3 4 5 6 7 8 9 10 11
Flagellomeres
1 2 3 4 5 6 7 8 9 10 11
Flagellomeres
5
Bassiconica (Aa)
4
3
2
1
Density of sensilla / 0.01
mm2
Density of sensilla / 0.01
mm²
15
0
Bassiconica (Ac)
5
4
3
2
1
0
1 2 3 4 5 6 7 8 9 10 11
1 2 3 4 5 6 7 8 9 10 11
Flagellomeres
70
Placodea (Aa)
60
50
40
30
20
10
Density of sensilla / 0.01
mm2
Density of sensilla / 0.01
mm²
Flagellomeres
70
60
50
40
30
20
10
0
0
1 2 3 4 5 6 7 8 9 10 11
1 2 3 4 5 6 7 8 9 10 11
Campaniform (Aa)
4
3
2
1
Flagellomeres
Density of sensilla / 0.01
mm2
Density of sensilla / 0.01
mm²
Flagellomeres
5
5
4
3
2
1
1 2 3 4 5 6 7 8 9 10 11
Flagellomeres
Ampullacea (Aa)
4
3
2
1
0
1 2 3 4 5 6 7 8 9 10 11
Flagellomeres
Density of sensilla / 0.01
mm2
1 2 3 4 5 6 7 8 9 10 11
Flagellomeres
Density of sensilla / 0.01
mm²
Campaniform (Ac)
0
0
5
Placodea (Ac)
5
Ampullacea (Ac)
4
3
2
1
0
1 2 3 4 5 6 7 8 9 10 11
Flagellomeres
5
Coeloconica (Aa)
4
3
2
1
Density of sensilla / 0.01
mm2
Density of sensilla / 0.01
mm2
16
0
5
Coeloconica (Ac)
4
3
2
1
0
1 2 3 4 5 6 7 8 9 10 11
1 2 3 4 5 6 7 8 9 10 11
Flagellomeres
Flagellomeres
Figure 14 Density of antennal sensilla in A. andreniformis (Aa) and A. cerana
(Ac) of queen ( ), drone ( ), and worker ( )
12
10
Anterior
Posterior
8
6
4
2
0
Queen
Drone Worker
Caste
a
Density of sensilla / 0.01 mm2
Density of sensilla / 0.01 mm2
Different distributions of antennal sensilla between flagellar sides, and
between the upper and lower ends of flagella
12
10
b
Anterior
Posterior
8
6
4
2
0
Queen Drone Worker
Caste
b
Figure 15 Density of antennal sensilla on the anterior and posterior side. (a) A.
andreniformis; (b) A. cerana.
The total sensilla number on the antennal anterior side is twice compared
with the posterior side, i.e. 68.4% and 31.6%, respectively, in A. andreniformis
(Figure 15a) and 60.5% and 39.5%, respectively, in A. cerana (Figure 15b).
However, there is no distribution pattern similarity of each sensilla type between
A. andreniformis and A. cerana (Figure 14; Appendix 2-7).
The distribution pattern of sensilla trichodea A are more abundant in lower
end of the antenna compare to upper end of antenna for both species (Figure 15).
Whereas, sensilla trichodea B, trichodea D, and bassiconica show the opposite
distribution pattern compare to trichodea A that are descend in lower end and
increase in upper end of flagella (Figure 15; Appendix 2-7). Furthermore, this
17
study found sensilla trichodea C, placodea, and ampullacea are distribute evenly
along the flagella of three caste in both species, except sparsely in fl1 and fl2 for
trichodea C and placodea and ampullaceal are not found on fl1and fl2 for.
Otherwise, sensilla campaniform and coeloconica do not show distribution pattern
(Figure 14).
Ultramorphometric variations of trichodea and placodea sensilla of three
castes in two sides of antenna in A. andreniformis and A. cerana
Ultramorphometric measurement of the length, width, and area of sensilla
of trichodea and placodea are significantly different (p < 0.05) among the three
caste of A. andreniformis, except trichodea C (Table 2). Significantly different of
all sensilla types measurements among the three castes are also found in A. cerana
(p < 0.05), except the length (p = 0.065) and area (p = 0.097) of trichodea A and
placodea (p = 0.079) (Table 2).
Table 2 Ultramorphometric of trichodea and placodea sensilla of the three castes
of A. andreniformis and A. cerana
Ultramorphometric (µm) ± SE
Queen
Drone
Worker
Apis andreniformis
Trichodea A
22.180ab ± 0.397
20.705a ± 0.643
23.335b ± 0.409
b
a
Trichodea B
16.265 ± 0.231 13.762 ± 0.338
13.807a ± 0.237
Length
a
a
Trichodea C
18.050 ± 0.589 16.667 ± 0.257
17.606a ± 0.356
(µm)
b
a
Trichodea D
15.860 ± 0.494 *12.217 ± 0.408
12.645a ± 0.265
a
ab
Placodea
14.692 ± 0.267 15.282 ± 0.143 16.146b ± 0.511
Trichodea A
3.357a ± 0.083
3.370a ± 0.165
3.612a ± 0.105
b
a
Trichodea B
1.614 ± 0.041
1.313 ± 0.045
1.434a ± 0.060
Width
a
a
Trichodea C
3.010 ± 0.067
2.608 ± 0.135
2.811a ± 0.174
(µm)
b
ab
Trichodea D
1.575 ± 0.071 *1.405 ± 0.079
1.236a ± 0.031
a
b
Placodea
8.965 ± 0.365 10.417 ± 0.151 10.403b ± 0.251
Trichodea A
49.497a ± 1.279 57.957a ± 4.352 58.720a ± 1.714
Trichodea B
12.355c ± 0.412 10.679b ± 0.513
8.604a ± 0.277
Area
a
a
Trichodea C
38.424 ± 1.687 33.462 ± 1.199 35.494a ± 2.338
(µm2)
Trichodea D
12.060b ± 0.761 *8.064a ± 0.516
7.202a ± 0.338
a
b
Placodea
121.594 ± 4.835 149.410 ± 3.647 154.816b ± 6.872
Trichodea A
25.339a ± 0.371 24.617a ± 0.762 23.484a ± 0.443
Trichodea B
18.726c ± 0.237 14.125a ± 0.561 16.294b ± 0.701
Length
Trichodea C
19.131b ± 0.441 15.749a ± 0.272 18.838b ± 0.365
(µm)
Trichodea D
20.965b ± 2.767 10.736a ± 0.522 15.494ab ± 0.141
Placodea
14.933a ± 0.255 14.622a ± 0.243 15.362a ± 0.158
Trichodea A
3.449a ± 0.120
4.405b ± 0.211
3.726a ± 0.077
b
ab
Trichodea B
1.930 ± 0.044
1.717 ± 0.099
1.674a ± 0.039
Width
a
a
Trichodea C
3.599 ± 0.083
2.792 ± 0.069
3.101b ± 0.075
(µm)
b
a
Trichodea D
1.830 ± 0.054
1.408 ± 0.062
1.759b ± 0.067
a
b
Placodea
9.077 ± 0.4725 10.324 ± 0.195
8.603a ± 0.1109
a
a
Trichodea A
57.733 ±1.831
69.626 ± 6.469 59.776a ± 2.196
c
Trichodea B
16.189 ± 0.492 10.854a ± 0.758 13.086b ± 0.194
Area
Trichodea C
38.730b ± 1.350 28.038a ± 0.916 34.317b ± 1.556
(µm2)
Trichodea D
14.039b ± 0.489
7.182a ± 0.445 12.807b ± 0.247
a
Placodea
126.036 ±6.1664 135.691a ± 4.9844 120.115a± 2.6416
Different letter in the same row indicate significant different (ANOVA, p < 0.05)
Data analysis using one way ANOVA and posthoc Tukey
Data are n = 20, except data with (*) is n = 10
Measure
Sensilla Type
Sig.
0.05
0.002
0.000
0.071
0.000
0.015
0.262
0.000
0.112
0.000
0.000
0.042
0.000
0.157
0.000
0.000
0.065
0.000
0.000
0.000
0.071
0.000
0.019
0.000
0.000
0.001
0.097
0.000
0.000
0.000
0.079
18
Ultramorphometric of trichodea B of queen in both species are the largest
among other castes. On the contrary, the measurement of trichodea C and
trichodea D of A. cerana drone are the smallest than other castes (Table 2). Thus,
ultramorphometrics sensilla show variation between the castes.
Among the three castes of A. andreniformis the areas measurement of
placodea between anterior and posterior are significantly different (p < 0.05). A.
andreniformis worker show ultramorphometric significantly different between
anterior and posterior (p < 0.05) of all sensilla types, except the length (t =
0.2277) and area (t = 0.7083) of trichodea A. The measurement of length and
width of all sensilla types of Apis. cerana queen on two sides of flagella are
significantly different (p < 0.05) (Table 3).
Table 3 Comparison ultramorphometric of trichodea and placodea sensilla on
anterior and posterior sides of the three castes of A. andreniformis and A.
cerana
Measure
Sensilla
Type
Trichodea A
Trichodea B
Length
(µm)
Trichodea C
Trichodea D
Placodea
Trichodea A
Trichodea B
Width
(µm)
Trichodea C
Trichodea D
Placodea
Trichodea A
Trichodea B
Area
(µm2)
Trichodea C
Trichodea D
Placodea
Ultramorphometric of sensilla on anterior : posterior side
t value
Queen
Drone
Worker
Apis andreniformis
21.238 : 23.123*
21.650 : 19.760ns
23.431 : 23.240ns
t = 2.758
t = 0.2277
t = 1.5195
15.819 : 16.712ns
13.965 : 13.560ns
13.157 : 14.457*
t = 2.0969
t = 0.5892
t = 3.4295
15.652 : 20.449*
15.942 : 17.392*
16.186 : 19.026*
t = 11.1115
t = 3.6099
t = 9.5902
15.180 : 16.540ns
13.550 : 11.740*
t = 1.4129
t = 5.3495
ns
15.131 : 14.254
15.147 : 15.417ns
13.990 : 18.302*
t = 1.7259
t = 0.9423
t = 16.5253
3.134 : 3.580*
3.470 : 3.270ns
3.314 : 3.910*
t = 3.335
t = 0.5961
t = 3.6292
1.520 : 1.708*
1.255 : 1.372ns
1.211 : 1.658*
t = 2.6145
t = 6.8896
t = 1.3401
2.778 : 3.188*
2.029 : 3.242*
2.076 : 3.547*
t = 5.5818
t = 22.1753
t =16.4161
1.580 : 1.570ns
1.159 : 1.314*
t = 0.0683
t = 2.9773
10.063 : 7.868*
10.620 : 10.214ns
9.557 : 11.249*
t = 4.033
t = 1.3797
t = 5.1782
48.656 : 50.339ns
69.140 : 46.775*
59.951 : 57.490ns
t = 0.6479
t = 3.0959
t = 0.7083
13.200 : 9.708*
11.650 : 11.510ns
7.862 : 9.346*
t = 2.2645
t = 2.046
t = 3.3128
31.489 : 45.360*
28.742 : 38.183*
30.461 : 40.527*
t = 8.9191
t = 12.046
t = 2.4098
11.280 : 12.840 ns
8.113 : 6.292*
t = 1.0263
t = 3.3368
156.787 : 105.894*
137.294 : 142.033*
128.022 : 181.610*
t = 4.7383
t = 2.2228
t = 8.4877
19
Table 3 (Continued)
Ultramorphometric of sensilla on anterior : posterior side
t value
Queen
Drone
Worker
Apis cerana
24.521 : 26.157*
27.325 : 21.910*
23.664 : 23.305ns
Trichodea A
t = 2.4907
t = 5.9615
t = 0.3958
19.468 : 17.984*
15.840 : 12.410*
15.298 : 17.291ns
Trichodea B
t = 4.3896
t = 4.1776
t = 1.463
Length
17.704 : 20.558*
14.774 : 16.724*
17.521 : 20.155*
Trichodea C
(µm)
t = 4.7025
t = 6.1395
t = 6.283
18.645 : 23.285*
8.6230 : 12.850ns
17.521 : 15.180*
Trichodea D
t = 0.8317
t = 10.6224
t = 2.5158
15.511 : 14.355*
13.696 : 15.549*
14.867 : 15.857*
Placodea
t = 2.5846
t = 7.6436
t = 4.3951
3.184 : 3.714*
5.100 : 3.710*
3.747 : 3.705ns
Trichodea A
t = 2.828
t = 4.9138
t = 0.2645
2.052 : 1.808*
2.050 : 1.384*
1.552 : 1.797*
Trichodea B
t = 3.5173
t = 5.1315
t = 4.4006
Width
3.293 : 3.905*
2.548 : 3.037*
2.816 : 3.386*
Trichodea C
( µm)
t = 6.843
t = 5.8728
t = 7.5507
1.958 : 1.703*
1.410 : 1.407ns
1.674 : 1.844ns
Trichodea D
t = 2.735
t = 0.0234
t = 1.2902
10.955 : 7.200*
9.552 : 11.096*
8.836 : 8.371*
Placodea
t = 9.4049
t = 9.0215
t = 2.326
56.070 : 59.396ns
92.790 : 46.463*
66.553 : 53.000*
Trichodea A
t = 0.904
t = 6.1126
t = 4.2525
16.820 : 15.558ns
13.400 : 8.308*
12.681 : 13.491*
Trichodea B
t = 5.131
t = 2.3163
t = 1.3069
Area
33.983 : 43.478*
24.404 : 31.673*
28.243 : 40.391*
Trichodea C
(µm2)
t = 5.7931
t = 9.3218
t = 8.5269
14.579 : 13.499ns
5.919 : 8.446*
12.410 : 13.205ns
Trichodea D
t = 1.1117
t = 3.6498
t = 1.6853
150.202 : 101.870*
116.544 : 154.839*
115.26 : 124.97ns
Placodea
t = 8.7129
t = 7.9125
t = 1.9729
Asterisk (*) in the same row indicate significant different (t-test, p < 0.05)
ns indicate not significant
(-) indicate no data
Measure
Sensilla
Type
Discussion
Density and distribution variations of sensilla types in open and cavity
nesting bees suggests an environmental adaptation
Previously, Suwannapong et al. (2012) found four sensilla types trichodea,
bassiconica, placodea, and campaniform in workers of A. andreniformis, was
collected in Thailand. In agreement with previous studies, the four described types
were found, along with two new sensilla types, ampullacea and coeloconica, in the
three castes of the open nesting A. andreniformis. These data are suggest because
inadvertence the types of sensilla in the previous studies.
Our findings corroborated that the cavity nesting honey bee A. cerana has
a higher total density of sensilla compared with the open air nesting A.
andreniformis. This is in agreement with the higher density of sensilla on cavity
nesting drones of A. melliferra than on the open nesting A. florea (Brockmann and
20
Brückner 2001). This phenomena suggests that cavity nesting honey bees, which
live in the dark (Oldroyd and Wongsiri 2006),