III. MA TERIALS AND METHODS
111.1. Study location
The study location is covered by the tribal belt of the high ranges of
the Western Ghats in Thiruvananthapuram district between north latitude
8O17'50"and 8"53'42" and east longitudes 76"40'24" and 70°17'00", the
Southern most administrative unit of the state with Thiruvananthapuram as
the capital. The district is bounded by the Arabian Sea in the west and the
Districts of the state of Tamil Nadu, namely Kanyakumari in the South and
Tirunelveli in the east and north by Kollam district (Fig.2).
While Thiruvananthapuram district has three physiographic zones
namely the highlands, midlands and low lands (Sreedhara Menon, 1962), it is
only the high land and mid land region is relevant in the present study. This
mountainous zone consists of hills, which form the southern most part of the
Western Ghats. The main Ghats stretch along the eastern border with
elevations varying between 1000 and 1869 m. The top and higher slopes of
the Ghats are very steep and rocky and in many places inaccessible. Such
places are usually barren. The lower slopes of the Ghats on the other hand
are covered with dense vegetation.
The climate of the district varies with the topography, but in general
the typical tropical humid climate with the two monsoons (north west and
north east) exists all over. Temperature and other meteorological conditions
are fairly uniform throughout the district except on the Ghats.
III.l.l. Field survey and collection
A field survey on beekeeping in the district was conducted in order to
identify the zones of the district where the beekeepers depend on for keeping
bees. The potential sites were identified and sample collection was made
from the different apiaries of the district. The tribal settlements of the district
were surveyed for the dependency of the tribes for honey as a Non- wood
forest product. A case study was conducted in selected settlements.
Household level interview was conducted by a questionnaire prepared as per
the format in Annexure 1.
The sample collection at different forest areas was done with
assistance from selected members of the tribal community. Information on
the materials analysed is given in Table 1.
111.2. Methodology
111.2.1. Preparation of acetolysis mixture
9ml of acetic anhydride was mixed with l m l of concentrated Sulphuric
acid, which was added as drops to the acetic anhydride.
111.2.2. Preparation of glycerine jelly
Glycerine Jelly was used as the mounting medium for acetolysed
pollen grains from honey, pollen load and anthers of individual flowers.
Reagents
Gelatin 59
Water 30ml
Glycerine 3511-11
Phenol Crystals 39
Tablel. Materials analysed for nectar and pollen sources
2 . 1 ~ p ~ r a n a indica / Wild / p a l a d e l
SI.
No.
1 YNY -3 I Apis cerana indica rd Pabde 1 HNY -4 -+- -- Trigona irridipennis Palode
/ 5 / HNY -5 1 PL-5 1 Apis cerana indica / Wild I Palode I
Honey I Pollen
Sample Loads
1 6 ~ H N Y - ~ i 1 Trigona irridipennis / Wild I Amboori, Kottur I
-8 - t ~ ~ ~ - r ~ z i r r i d i ~ e n n i s j Wild j palo-1
Bee species
1 12 1 HNY -12 1 1 Trigona inidipennis 1 Wild 1 Njaraneeli I
- b z a n a indica / p i / C h e r a v I
Nature
115 HNY-15 1 1 Apis cerana indica Apiary / Cherappally i
Locality
I I
Apis cerana indica ( Market ( Thiruvananthapuram
118 / HNY-18 1 I Apis cerana indica I Apiary 1 Cherappally,Parandode I I I
HNY -19
HNY -20
HNY -21
HNY -22
HNY -23
HNY -24
HNY -25
HNY -26
HNY -27
HNY -28
HNY -29
HNY -30
HNY -31
HNY -32
HNY -33
HNY -34
HNY -35
HNY -36
HNY -37
HNY -38
HNY -39
I Apis cerana indica Wild
/ Apis cerana indica
I Apis cerana indica
1 Apis cerana indica
PL-24 ) Trigona irridipennis
PL-25 Trigona irridipennis - --t- PL-26 / Trigona irridipennis
I Trigona irridipennis
- 7 A ~ i a n a indica
1 Apis cerana indica
PL-30 Trigona imdipennis ----I-- PL-31 Ap~s cerana indica I
1 Apis dorsata
I Trigona irridipennis
I Apis cerana indica
) Apis cerana indica
( Apis cerana indica
PL-37 Apis cerana indica
PL-38 Apis cerana indica - --I-
I Apis cerana indica
Apiary
Market
Market
Wild
Wild
Wild
Wild
Wild
Apiary
Apiary
Wild
Wild
Wild
Wild
Apiary
Apiary
Apiary
Apiary
Apiary
Apiary
- Amboori, Kottur
Marthandam - Marthandam - Sanghili,Madathara
Peringamala
Peringamala - Cheenikkala, Sanghili
Cheenikkala, Sanghili
Cheenikkala, Sanghili
Parandode
Cherappally ,Parandode
Kesaripuram, Peringamala
Pullivattom, Peringamala
Peringamala
w-40 T -7-erana indica 1 Market (Bt Thiruvananthapuram
142 / HNY -42-t --
I Apis cerana indica Market Thiruvananthapuram i
145 1 HNY -45 1 I Apis cerana indica I Apiary 1 Kesaripuram, Peringamala I
WE7 ~ ~ 4 i b ~ a n a indica / Apiary / C h a l l i m u ~ ~ u j
~ ~ ~ ~ - ~ ~ ~ ~ ~ n a indica A / Arippa. Sanghili
1 1; 1 HNY -50 1 1 Apiscerana indica Apiary 1 Kulathuppuzha 1 HNY -51 Apis cerana indica Apiary Kesarrpuram, Peringamala
5 2 1 HNY -52 cerana indica 1 :;a 'lode 1 - - - - - --
53 HNY -53 Apis cerana indica Elavupalam
1 57 1 HNY -57 I I Apis cerana indica I Wild / Palode 1 58 I HNY -58 Apisceranaindica :I 1 Palode 1 59 HNY -59 PL-59 Yrigona irridipennis Ponmudi
1 60 1 HNY -60 / PL-60 I Trigona irridipennis I Wild I Ponmudi I
-- . . -- - T - 6 1 T PL-61 1 Trigona irridipennis i Wild
62 / HNY -62 I PL-62 Trigona irridipennis 1 Wild 1 Ponmudi I
-4s- t ~ l ? & n a irridipennis Wild 1 ~ o n m u d i l
64 / HNY -64 1 PL-64 1 Trigona irridipennis 1 Wild 1 Ponmudi 1
66 1 HNY -66 / PL-66 / Apis cerana indica I Wild / Kallar, Vithura I
I 68 1 HNY -68 i I Apis cerana indica I Apiary 1 Palode I
i
69 1 HNY -69 1 / Apis dorsata I Wild / Adiparambu, Madathara I
67
70 ) HNY - ~ ~ ~ ~ ~ - / ~ ~ r s ~ t ~ Wild
HNY --67 1
71 1 HNY I 1 1 Apis cerana indica 1 Apiary 1 Palode 1 .-
72 HNY -72 PL-72 Apis cerana indica Wild Palode
73 1 HNY --73 / PL-73 I Trigona irridipennis I Wild Palode I 74 ( HNY -74 1 1 Apis cerana indica 1 Wild 1 Vithura 1
*Y~ i s t -t=rana indica / Market / Peringamala
76 1 HNY -78 1 I Apis cerana indica I Wild / Chonampara, Kottur I
78 / HNY -80 1 I Apis cerana indica / Apiary I Amboori, Kottur I
I
HNY -81 1 PL-81 Trigona irridipennis Apiary
kii-~ \~-~~-jGin*&l
HNY -79 PL-79 Trigona irridipennis
8 1 / ~ ~ y - 8 3 1 1 Trigona irridipennis 1 Wild I K a l t . Vithura I
Wild 17- t--I--
HNY -84
HNY -85
HNY -86
HNY -87
HNY -88
HNY -91
HNY -92
HNY -93
HNY -97
HNY -98
HNY -99
HNY -100
HNY -101
Trigona irridipennis
Apis cerana indica
Apis cerana indica
Jrigona irridipennis
Trigona irridipennis
Trigona irridipennis
Trigona irridipennis
Apis dorsata
Apis cerana indica
Apis cerana indica
Apis cerana indica
Trigona irridipennis
Trigona irridipennis
Trigona irridipennis
Trigona irridipennis .-
Apis cerana indica
Apis florea
Apis cerana indica
Wild
Apiary
Apiary
Wild
Wild
Wild
Wild
Wild
Apiary
Wild
Wild
Wild
Wild
Wild
Wild - Apiary
Wild
Wild -
-
Peringamala
Amboori, Kottur
Chonampara, Kottur
Chonampara, Kottur
Chonampara, Kottur
Chonampara, Kottur
Amboori, Kottur
Peringamala
Agastyamala, Bonnoccord
Bonnoccord
Agastyamala, Bonnoccord
Agastyamala, Bonnoccord - Agastyamala, Bonnoccord
Agastyamala, Bonnoccord - Peringamala
Bonnoccord
Agastyamala, Bonnoccord -
The gelatin is dissolved in warm water with continuous stirring. The
other components are added to it and then filtered hot. It is kept in a closed
bottle. Phenol Crystals are added as a preservative to avoid growth of fungal
elements.
111.2.3. Preparation of pollen: Acetolysis Method (Erdtmann, 1960)
A. From honey
I rnl of honey sample was taken in a test tube and diluted to 10ml by
hot distilled water of 40" C. The diluted honey is sieved through a mesh of
100p rn. The suspension thus obtained was centrifuged at 3000 rpm. for 5
minutes. The supernatant was decanted. The pellet of pollen sediment was
subjected for acetolysis (Erdtman, 1960). Detailed procedure is outlined
below.
Pollen material from honey
& Washed with 51~1170% alcohol Centrifugation
4 Decanted the alcohol Sediment
& Wash with 5ml glacial acetic acid Centrifugation
4 Decanted glacial acetic acid Sediment
4 (9:l acetic anhydride and Con.H2S04) Coloured medium
& Centrifugation Sediment
& Wash and centrifuge in distilled water (3 times)
Sediment
Mounted in Glycerine jelly
B. From pollen load
Individual pollen loads were separated from the beehive and
dispersed directly in 70% alcohol and analysed by acetolysis as above.
C. From anther
For identification of pollen grains isolated from honey and bee pollen
loads, pollen slides from flowering species were prepared as reference
slides. For this, anthers separated from filaments were put in distilled water
and crushed with a glass rod. It is then sieved through a mesh of 100pm
size. The pollen suspension was centrifuged at 3000 rpm for 3 min. and
decanted the supernatant. Pollen pellet was subjected for acetolysis
(Erdtman, 1960) as detailed above.
111.2.4. Field notes on forage preference by Apis cerana indica F.
For the elucidation of the dietary preference shown by the commercial
bee A. cerana indica, three different colonies of this bee were located
between 8"45' and 8"47' north latitudes and 77" 1' and 77" 4' east longitude,
in Thiruvananthapuram district. The colonies were in three different
vegetational patches, one in open cultivated land of mixed vegetation,
second in dry deciduous forest and the third in a transient zone between
introduced exotic vegetation and cultivated land of mixed crops. The bee
colonies were nearly lOOOm apart from each other. The beehive in the forest
patch was a natural one, at the base of a tree trunk of Aporosa lindeliyana L.
(Euphorbiaceae) and the other two were artificial Newton type hives
established for the experiment.
The hives at the three sites were sampled for the corbicular pollen
loads at an interval of 6 days by using pollen traps attached to the entrance
of the hives for a period of 3 months from September-November, which forms
the critical brood rearing season of the honeybee colonies at the area under
investigation. The corbicular pollen loads were acetolysed (Erdtmann, 1960)
and studied by means of Nikon Labophot light microscope-I.
111.2.5. Measurement of pollen volume (Buchman eta/, 1990)
The acetolysed pollen grains (Erdtmann, 1960) were mounted
permanently in glycerine jelly and a Nikon Labophot microscope with
micrometer was used to measure grain diameter in circular grains or length of
long and short axes in elliptical grains. Grain volume was calculated as
nab2/6 where 'a' is grain length and 'b' is grain width. (For circular grains
a=b).
111.2.6. Microphotography
Microphotographs were taken on Nikon-Labophot-l photomicroscope
using lllford NP 55. 35 lnm black and white film.
111.2.7. Nomenclature
Taxonomic revisions and recent local Floras were consulted to bring
the nomenclature of plants up-to-date. Only the legitimate name in
accordance with the International Code of Botanical Nomenclature (1983)
has been followed avoiding synonyms.
111.2.8. Terminology
In general, terms used for describing the pollen grains were that of
Erdtman (1952) incorporating the suggestions made by Reitsma (1970). For
describing the exine stratification, Faegri and lverson (1975) and for amb,
size and shape classes, Walker and Doyle (1975) were followed. Regarding
shapes of apertures, the terminology of simple symmetrical plane shapes
suggested by Systematic Association Committee (1962:) is adopted.
111.2.9. Pollen descriptions
Pollen grains are described in the following sequence; nature of
occurrence, polar outline, equatorial outline, apertures, exine and
sculpturing.
111.2.10. Cluster Analysis
Pollen grains isolated from individual honey samples were acetolysed
and the slides were observed under Nikon Labophot light microscope. The
species identification was done with reference slides. The palynological data
recorded for individual samples were analysed by cluster analysis using
NTSYS-pc package for similarity index between honey collected from
different honeybees and from different floristic locations. The dominant
species represented in all the honey samples were scored as present (1) or
absent (0) across all the honey samples. The two-way data matrix of samples
x species present was subjected for standardization.
The standardized data matrix was used to calculate the similarity
dissimilarity distance using the function 'simint'. This matrix of similarity
coefficients was subjected to unweighted pair group method analysis
(UPGMA) to generate a dendrogram using average linkage procedure. The
similarity matrix coefficients were also analysed by single link and complete
link methods to see the real clustering in the samples based on the
palynological data. All these analysis were conducted using the computer
programme, NTSYS-pc:, version 1.80 (Exter Software, New York). The results
obtained were represented by constructing a dendrogram.
111.2.11. Foraging studies
For the elucidation of foraging behaviour of hive bee A. c. indica, two
species were selected based on the nectar and pollen preference of this bee
species. Bombax ceiba I.. (Bombacaceae) was selected for the behavioural
study of A. c. indica in nectar foraging and Dillenia pentagyna L.
(Dilleniaceae), a major pollen resource for pollen collection.
111.2.11.1. Nectar foraging in Bombux ceiba L.
A sample tree in full bloom was selected and 5 different flowers were
marked. Two of them were bagged using muslin cloth bags and 3 were left
open for bee v~sits. The quantification of nectar productivity of individual
flowers were carried out using micropipette at an interval of I hour.
The frequency of bee visits to individual flowers, time spend by each
bee on individual flower and number of bees visiting marked flowers were
recorded. The diurnal variation in bee visit to this species was also recorded
from 5 AM in the morning when flowers are in bud condition to 6 PM in the
evening till the sunset. Quantification of nectar productivity of individual tree
was assessed by measuring number of branches, branch lets, inflorescences
and individual flowers.
111.2.1 1.2. Pollen foraging in Dillenia pentagyna L.
An individual Willenia tree in full bloom with aggressive foraging by
A. c. indica bees was selected. The quantification of flower production /day
was assessed by counting branchesltree, number of branch letslbranch,
number of inflorescences I branch lets, number of flowerslinflorescences and
number of antherslflower. The data were recorded on the number of bee
visits to individual flower, time spend by each bee in single flower, time taken
by individual bee to load one complete pollen load and diurnal variation in
pollen foraging in this species from dawn to dusk.
111.2.12. Pollen frequency determination (Louveaux's method, 1978)
The honey samples were subjected for pollen frequency determination
by the method of Louveaux (1978). Im l honey was diluted to 10ml by 60' C
water and centrifuged. The pollen sediment was stained by safrannin and
the grains were made into pollen slides. Strip count method was adopted for
counting, as there was not much variation from point counting and total slide
count. Three complete strips were counted across the slide. That sample
where the grain count was very less, total slide count was taken for accurate
frequency of individual species. The results were presented as pollen spectra
of individual samples (Fig. 3 - Fig. 66).
111.2.13. Studies on honey as an NWFP
For the elucidation of the utilization of honey as a non-wood forest
product, five different settlements have been selected at the extreme south
of southern Western Ghats in Thiruvananthapuram district between 8"34
'999 and 8"49'178"N latitude and 77"02'96OU and 7710'083"E longitude.
56
The settlements are potomav in Sanghili forest, Mottamood near
Kallar, Chathancode and Chemmankala at Bonnoccord and Chonampara in
Kottur reserve forest. These settlements were with varying population,
literary level and with different man-day utilization for different jobs. A
questionnaire has been prepared (Annexure 1) and a household level
interview was conducted.
In addition, a seasonal monitoring for traditional apiculture and honey
collection from forests were also made in all these settlements.