entre for pollination studies: project protocols long term...

Centre for Pollination Studies: Project Protocols

Long-term monitoring protocol

Title of the protocol: Pollinator survey for long term monitoring of pollinator diversity in the

crop fields of two Eastern state of India.

Statement of Purpose: The aim is to monitor the diversity of wild bees along agricultural

intensification gradient.

Background:

Quantification the pollinator diversity at the study sites in Orissa and Tripura will be done.

General diversity of pollinator in agricultural fields which will also include a focal crop plant

e.g. brinjal will be assessed. Sampling will be done most of the year except during the rainy

season. The aim of this extensive sampling over many months is to find out whether the

pollinators are around even during non-flowering period of the focal plant. Three agricultural

intensification zones (nodes) will be investigated. Refer to Table 1 for characteristic of each

intensification type.

About sampling methods:

Three separate sampling methods will be employed to obtain the most accurate estimate of

species diversity and abundance at the study sites. These are pan traps, transect walk and

focal observations. Focal observations will be designed to capture the behaviour of insects;

this will help classify the insects observed on plants into visitors and pollinators. Sweep

netting will also be employed if feasible.

Table 1: The characteristics of each intensification zone (node)

Intensification /

Habitat characteristics

Low Medium High

Nearness to natural

vegetation

On the forest edge Away from forest,

thickets and groves

may be present

Away from forest,

fewer thickets and

groves, vast plots

of agricultural

fields

Agrochemicals (pesticide) Zero or low Medium to High High

Farming practice Subsistence Subsistence +

commercial

Commercial

Cropping Mixed Mixed or a few

crop species

Mostly

monoculture

Farm size Small Small/medium Medium to large

Pan trap

Pan traps are passive way of collecting aerial insects. This method is relatively easy and

inexpensive. The choice of bowl colour is important for trapping. Yellow, blue and white

have been shown to be good in capturing pollinators, especially yellow colour. However, the

drawback of pan traps could be that large bodied pollinators may not fall into the trap.

Protocol:

1. All three intensification zones (nodes) will have five villages.

2. Villages which are at least 1 km apart will be chosen.

3. A pan trap cluster will be set up within a focal crop plant plot if feasible. (See Setting

the Pan traps for details on pan trap.)

4. Additional sets of three traps each will be placed on both sides of the trap cluster at a

distance of 50 m from the centre of the cluster up to 100 m on both sides of cluster

(Figure 1). Therefore we will have five trap sets in the centre and six sets on the

wings. The total spread of the traps will cover up to 200 m.

5. A pan trap set (cluster of three) will also be placed at a height of at least 10 feet from

ground level on 2 trees on each side of the trap, thus there will be a total of 4 trap sets

on the trees present in thicket or grove surrounding the crop field subject to

availability of trees.

6. Insects will be collected after 24 hours of after setting up of traps in the field.

7. Trapping will be done once a month at each of the trapping site/village.

8. Thus for each village/node we will have approximately 33 traps (15 centre + 6 wings

+ 12 on trees).

Figure 1. Schematic diagram of a trap cluster with additional traps on the wings.

Setting pan traps:

1. Pan traps will be of dimension (18 cm X 7.5 cm) and will be painted with UV bright

paint.

Central trap cluster

50 m

2. Traps will be placed on a trap holder (a metal wire frame) and fixed on a bamboo

pole.

3. Inter-trap distance will be 1 m.

4. Traps will be filled with water and a few grains of locally available washing powder

will be added.

5. Washing powder will break the surface tension and allow the insect to sink into the

water when it’s fallen into the trap.

Choosing a site and sampling of same site every month:

1. Before the traps are set up decide on the location of pan trap station.

2. Use a GIS map to locate the areas with the habitat characteristics mentioned above.

3. Mark as many locations within a village as possible and randomly choose one

location (habitat type). Note the co-ordinates.

4. Obtain the co-ordinates for the pan trap station using the map.

5. Locate the co-ordinate in the crop field using a GPS.

6. Use the first co-ordinate as the tip of pan trap

7. Using a measuring tape measure the distance for placement of individual traps.

6. Record and save each pan trap location on a GPS.

7. For the next month use the same points to put the traps.

8. Weather parameters will be recorded/ collected for the sampling days.

Please note:

1. GPS will have an inbuilt error for estimation of location. It could be ± 3 m for Garmin

etrex. Therefore, setting up of traps at the same location each month should be based

on observer’s spatial memory and GPS. If required, fix some poles which will mark

the location permanently.

Materials for pan trapping:

Measuring tape, GPS, pocket weather station, pan trap stands, trap holders, pan trap (blue,

white and yellow colour), rope, water, washing powder, extra sticks or flags for marking,

insect collection vials, ethanol (70%), strainer/sieve (to filter out the trapped insects), labels,

marker, pencil, pan trap datasheet, UV-bright paint.

Transect walk

Objective: To get an estimate of insects present in the study area through visual recording.

These insects could be visiting the crops but might have low or no capture rate on pan traps.

This will also supplement the pan trap data. This protocol is adapted from Westphal et al.

(2008)1.

Protocol:

1. On either side of each trap station (i.e. cluster of 15 bowls) at a distance of 5 m from

the edge of the cluster there will be a line transect of length 200 m, thus the total

transect length is 400 m will be laid.(Figure 2)

2.

1 Westphal, C., R. Bommarco, G. Carré, E. Lamborn, N. Morison, T. Petanidou, Simon G. Potts, S. P. M.

Roberts, H. Szentgyörgyi, T. Tscheulin, B. E. Vaissière, M. Woyciechowski, J. C. Biesmeijer, W. E. Kunin, J.

Settele, and I. Steffan-Dewenter.(2008). Measuring bee diversity in different European habitats and

biogeographical regions. Ecological Monographs 78:653–671.

2. 5 m

5 m

Figure 2. Schema for transect walk and focal plot sampling. The solid line indicates the path of

transect walk. Dashed line indicates the observation distance on both sides of the line. Another set of

lines will also be marked below the pan trap cluster. The yellow boxes indicate focal observation plots

(1m X 1m). See behavioral observations for focal observations. This figure is not to scale.

3. An Observer will walk at a slow speed and note all the pollinator species as well as

flower and foliage visitors to the vegetation 2.5 meters on each side of the observer

(demarcated using a strip of ribbon, see Figure 2). Observer should try not to disturb

the insects while walking. The name of the crop plant on which the insect is observed

should also be noted (in case of presence of more than one crop type in the same

field). The name of the insect as well the number of individuals observed will be

noted.

4. Step 2 will be performed at two different time points of the day (08:00/ 09:00 and

11:00/ 12:00 hrs respectively) coinciding with pollinator activity.

5. If the insect is unidentified a photograph will be taken, if possible it will be collected

and identified later.

6. Within one observation period, example, 07:00 hrs, step 2 will be repeated once more

but on the other side of the trap.

7. Thus, there will be two transect walks per time session. We will use either the average

value or the total number of insects obtained from transect walk for our analyses.

Materials for transect walk:

Measuring tape, ribbon, camera, transect walk datasheet, pencil/pen, collection vial, label,

marker.

Behavioural observation

This will be performed to understanding the value of small scale observations in knowing the

pollinator diversity. Also, this will help in separating the pollinators from visitors.

Protocol:

1. Four focal sampling plots (1 m X 1 m) will be identified along the length of transect,

two on each side of the pan trap cluster (a total of 4 focal observation plots). Visually,

these would be present parallel to the pan traps placed 50 m away from the cluster and

will fall on the transect walk line These focal plots are permanent focal observation

plots, therefore, every month the same four focal plots will be used for observations.

2. Co-ordinates would be noted using GPS.

3. Before the observations each plot will be demarcated using a ribbon.

4. Observations will be taken for 10 minutes at each plot at 07:00/08:00 and 10:00/11:00

hrs respectively.

5. The order of focal observations should be randomly decided (write 1-4 on a piece of

paper, shuffle it. Pick one, the first number picked is the first observation plot, this

way decide the order the remaining three plots).

6. Insect behaiour especially the behaviour of bees will be noted. If any other insects are

present their activities will also be noted.

7. The focal observations will be done before transect walk to minimize disturbance.

8. If the insect is new to the observer it will be collected with aspirator/vials.

Materials for behavioural observations:

Measuring tape, ribbon (to mark 1m X 1 m area), focal observation datasheet, stop watch,

labels, pencil, insect vials, GPS.

Sweep netting

Insects at the top layer of the crop can be collected using sweep nets. This would include the

insects on flowers and foliage. We will use this method if it is feasible at the study sites. If

this is performed, this will be at the end of sampling since sweeping can disturb the fauna

which might affect the focal observations as well as transect walks. Some fast flying insects will

not be captured in this method.

Protocol (Adapted from South Sue):

1. The observer will walk on the belt (transect walk belt) with sweep net.

2. Sampling using sweep nets cover a certain number of sweeps rather than a distance.

3. Sweeps will be conducted from one side to other. The net should be moved from one

side to another gently as far as the net can reach. However, this should be done gently

without damaging the flowers.

4. The handle of the net should be turned to sweep from one side to other.

5. The sweeping should be done such that the insects are allowed to enter the net but

prevented from escaping.

6. One sweep is sweeping of net on one direction. One sweeping sample would comprise

of all sweeps along each 100 m segment. At the end of each sample/ sweep (which

ever is convenient) shake the insects will be shaken to the bottom of the net.

7. This will be performed on the both the transect belts.

8. Each insect will be identified after each sweep.

9. Since most of the insects will already have been recorded during behavioural

observation and transect walk only those insects which have not been encountered

earlier will be recorded.

10. The name of the insect and number of individuals collected in the sweep will be

noted.

11. Unidentified insects will be kept at voucher specimens inside insect collection vials

and will be identified later.

Materials for sweep netting:

Sweep net, spare net, labels, pencil, insect vials, killing jars, sweep netting datasheet

Please note:

1. After one transect walk a gap of 60 min will be provided before the beginning of the

next focal observations. This will be done to allow the plant-insect interactions to

recover after the transect walk.

2. Sweeping (if done) will be performed at the end because sweeping causes a lot of

disturbance to vegetation.

Plant phenology

Information on plant phenology including floral phenology will be important for data

analysis. For example, a correlation can be drawn between increase in flower numbers and

pollinator abundance. Along data on dominant crop present in the sampling area a record of

the phenology of the plants in the same area will be collected.

Protocol

1. The area of the plot for which phenology will be done will be measured.

2. A quadrat 5 X 5 m will be laid randomly within the plot.

3. Number of plants within the quadrat will be counted.

4. For ten plants the total number of inflorescence will also be counted. If it is not

possible to count the total number of inflorescence present, inflorescence for one

quarter of the plant will be counted and multiplied with four to get the total

inflorescence number.

5. For each plant 2 inflorescence will be chosen randomly and the total number of

flowers, buds etc. will be counted for these two inflorescence.

6. Using these numbers the total number of flowers, buds etc. for the plot under

observation will be calculated.

7. Once the quadrat is chosen, it will be observed every month for inflorescence count.

8. For rare plants defined as plants which have only 10 or less individuals growing

within the field two random inflorescences for all the ten individuals will be counted.

Therefore for these plants we will have an absolute count.

Materials for phenology:

Camera, pencil, measuring tape, phenology datasheet

Summary of the observation schedule:

Summer observation

periods (hrs)

Winter observation

periods (hrs)

Sampling activity

07:00–08:00 08:00–09:00 Focal observations (4 numbers)

If extra time is left after dedicated

focal observations, opportunistic

focal observations on focal crop

plants will be conducted.

08:00–09:00 09:00–10:00 Transect walk (2 numbers)

09:00–10:00 10:00–11:00 Break to let the insects settle after

transect walk.

This period will be used for some

more opportunistic focal

observations on focal crop plants. If

samples need to be labeled this time

will be used.

10:00–11:00 11:00–12:00 Focal observations (4 numbers)

11:00–12:00 12:00–13:00 Transect walk (2 numbers)

12:00–13:00 13:00–14:00 Sweeping (2 numbers)

Anytime Anytime Phenology

PhD Programme: Plant-Pollinator Network along a Gradient of Agricultural Intensification in Tripura

Name: Pushan Chakraborty

Supervisor: Dr. Parthiba Basu

Associate Proffesssor

Department of Zoology

University of Calcutta

35, Ballygunge Circular Road

Kolkata – 700019

Associate Supervisor: Dr. V. P. Uniyal

Scientist – E

Wildlife Institute of India

Chandrabni, Dehradun

Uttarkhand

Abstract: The simplification of ecosystems due to agricultural intensification is

believed to be a major cause of decline of pollinators. In our proposed study, local

plant-pollinator networks will be studied along a gradient of agricultural

intensification (low, medium and high levels of the same). The effectiveness of each

pollinator taxa and the seasonal and temporal variations of the network, also will be

worked out. Non crops are supposed to sustain the network by providing food and

refuge year round. But, some non crops may have competitive roles for the

pollinators. In our study, we will focus in this aspect also.

Keywords: Pollinators, Agricultural intensification, Network, Effectiveness, Pollen,

Non crops.

1. Introduction

Pollination is a very important free ecosystem service, mediated mostly

by insects. Animal mediated transfer of pollen from one flower to another is a

necessary service for majority of the angiosperms. About 70% of all tropical

crops depend on insect mediated pollination (Roubik 1995).

A network can be defined as representation of the interrelations between

different nodes interacting among each other in a given system. In a network,

the nodes are the elementary components of the system and the edges connect

different nodes (Crucitti et al, 2004). The mutualistic relations between plants

and pollinators can also be represented as a network structure where plants and

pollinators form the two nodes. In recent times, there is a trend to study plant-

pollinator communities in a network approach (Baldock et al, 2011). After

thorough study, it has been established that generalization along with

specialization in the plant-pollinator systems are widespread. The network

approach represents these interconnections between plants and their pollinators

in a simplified fashion.

In the network approach, the pollination systems are presented in a

bipartite matrix where plants and pollinators form the two layers which are

linked in between. The linkage density is the mean number of links per species,

whereas, connectance is the proportion of actual and possible links in the

system.

The complex nature of the functional diversity of the plant-pollinator

systems, in terms of a network, prevents the decline of any one of them upon

the loss of the other (Fontaine at el, 2006). In the simplified monoculture

scenario, this is very likely to be seen as the weeds are usually excluded from

crop fields. In the non flowering season of the dicotyledonous crops, pollinators

either die or move outwards. So, a more diverse community (including both

crops and weeds) may provide more tolerance to the attacks (local extinctions)

in the network.

In the proposed study we aim to assess the plant – pollinator network

along an agricultural intensification gradient. The agricultural intensification

gradient provides an array of possible drivers, including pesticide stress and

habitat loss, to any impairment of the network.

Survey of Literatures:

Beginning from basic ecology to possible economic consequences due to

pollinator decline has been reflected in the works of national and international

pollination biologists. The following literatures were surveyed to design the

proposed study:

The study by Kearns & Inouye, 1998 provides the pioneering backdrop to

the present thrust in pollination biology research. The incidents of decline in

pollinators due to agricultural intensification and land use change are reflected

in the works done by Brittain and Potts, 2010; Steffan-Dewenter and Westphal,

2008; Kremen, 2004 etc.

. Devoto et al, 2012 and Baldok et al, 2011 has worked on the temporal

and seasonal shifts of plant-pollinator networks.

Rafferty and Ives, 2012 has worked on effect of altered floral phonologies

on pollinator assemblage.

Gibson et al, 2011 and Memmott, 1999 provides the basic knowledge

about plant-pollinator networks and also the sampling strategies for the same.

Among others, Dordo et al, 2010 (studied trap nests); Ramos-Jiliberto et

al, 2009 (studied in rain forest); Fontaine et al, 2006 etc are also important

studies done on plant-pollinator networks. Ghajoul, 2004 provides methodology

for nocturnal sampling.

Bartomeus et al, 2010; Padron et al, 2009; Vila et al, 2009; Brtomeus et

al, 2008 etc have worked on effect of invasive plants on the native plant-

pollinator networks. Gibson et al, 2006 have worked on pollinators of arable

weeds.

Brosi et al, 2007 studied the effects of distance from forest, tree

management and floral resources in pastures in southern Costa Rica.

Taki and Kevan, 2007 presented study on whether plants and pollinators

become more generalized with increasing habitat loss.

The work done by Alder and Irwin, 2006 provides procedure for pollen

load counting and pollen staining. Balmford, 2006 provides procedure for day

and night exclusion studies.

The concept of ecological networks and attack tolerance is well discussed

in Crucitti et al, 2004.

In India, there is almost no prominent work on plant-pollinator networks.

Notable among Indian studies include Abrol, 2007 and Davider, 2003.

2. Objectives

(i) To identify local pollinator groups of the study areas and determine the

effect of agricultural intensification on the plant-pollinator networks.

(ii) To quantify the effectiveness of each pollinator species.

(iii) To determine the temporal and seasonal variations in pollinator assemblage.

(iv) To determine the role of non crops in vicinity of the crop, in sustaining the

pollinators.

3. Materials and Methods

Study Site

The study will be based on the north-eastern Indian state – Tripura. The

state is surrounded by Bangladesh on three sides. The selected region is

Teliamura subdivision in Khowai district and Baramura hills of West Tripura

district. Natural and planted teak bamboo and rubber vegetation is prevalent.

The study region falls in the basins of two rivers: Khowai and Howrah. Average

temperature 25-30 degree C. Rainfall ranges from 100-200 mm. Traditional

‘jhum’(a local name for shifting cultivation) is practised in hilly areas.

Three nodes of agricultural intensification have been identified:

Low Intensification – Brinjal is produced along with other vegetables and

fruits. Very little or no pesticides are used and surrounded by moderate to thick

natural vegetation.

Medium Intensification – Small vegetable fields of gourds and brinjal, where

moderate amount of pesticides are used. Natural vegetations are located within

100 m.

High Intensification – Large monocultures are maintained for both gourds and

brinjal. Pesticide usage is highest of the three. No natural vegetation within 100

m.

Three replicates of each node of the gradient (each having three sites) will

be surveyed per season.

Study Systems

In our study we will study the pollinators of specific plants along a

gradient of agricultural intensification at landscape scale. The focal vegetable

crops would include solanaceaous crops like brinjal (Solanum melongena),

tomato (S. tuberosum) etc; cucurbits like pointed gourd (Trichosanthes dioica),

spine gourd (Momordica sp.), bitter gourd (M. charantia), bottle gourd

(Cucurbita spp.), cucumber (Cucumis sativas) etc; legumes like cowpea (Vigia

sinensis), broad bean (Vicia sp.) and cruciferous crops like mustard (Brassica

spp.) cultivated in the study sites. The non crops in the vicinity of the crops will

also to be surveyed.

Objective (i): To identify local pollinator fauna in the study areas and to

determine the effect of agricultural intensification on the

plant-

pollinator network

Gradient of Agricultural Intensification

Agricultural intensification is characterised by both the management

practice which dertermines the degree of usage of chemical pesticides and

fertilizers and and, to some extent the landscape structured by influencing the

proportion of natural vegetation in the locality. Three levels of agricultural

intensification have been detrmined:



natural vegetation.



500 m.



m.

Pollinator Sampling

a. Day sampling:

Transect Walk- Belt transects and point transects (whenever applicable) will

be laid along the vegetation, in and around vegetable fields. The length of belt

transects will be 10 m. Two transects per crop per hour will be made, from

7.00am to 4.00 pm. Observations will be made on the flowers on either side, 2

m each side.

The physical parameters like GPS location, elevation, temperature, RH

and time should be noted at the start and end of each transect walk.

Rainy and foggy days are excluded as in those days, insects are less

active.

Focal Observations- The behavior of the insects is also noted in a randomly

chosen 1 square meter area for 10 min. Four focals will be done per hour, two

for crops and another two for non-crops, from 7.00 am to 4.00pm. The key

behaviors to be recorded are:

i. Time spent on each flower.

ii. No. of flowers visited.

iii. Whether it touches the reproductive parts of the flower or not.

b. Night Sampling:

Construction of a pollen library- Pollen from various flowers will be collected

manually and then slides will be prepared. A collection of pollen images will

make the digital pollen library.

Light Trap: light traps will be set near the vegetation. The insects will be

captured. The pollen attached on their body will be stained in the same

procedure (discussed below). Those will be matched with the pollen library

images (Ghajoul, 2004).

c. Sampling irrespective the time

Pan trapping- White, blue, yellow pan traps will be set (Westphal et al, 2008;

Brosi et al, 2007). For day and night exclusions, those will be checked every 12

hours.

Collection and Identification of Insects and Plants

Insects will be captured by sweep net or hand picking and killed in a jar

containing ethyl acetate. Then, for dry preservation, pinning will be done using

a macropin. Then the wings are spread and the legs, antennae are held in semi-

natural condition. Larger ones or gravid females are eviscerated. Plant

specimens will be dry preserved in herbarium sheets. Liquid preservation will

be done in 70% ethyl alcohol. Insects will be identified with the help of

standard couplet keys.

Objective (ii): To quantify the effectiveness of Pollinators belonging to

different taxa

Pollen Load Count - Pollen load from insect body is to be stained as the

following procedure (Alder, 2006):

At first, a slice of basic fuchsin gelatin is cut and the insect body is wiped

with it. It is then placed on a clean slide and a cover slip is placed on it. Then,

the slide is heated gently. The jelly will melt and a semi permanent slide will be

made.

Objective (iii): To determine the temporal and seasonal variations of the

plant

-pollinator network

Day-Night Exclusion – To determine presence and effectiveness of diurnal and

nocturnal pollinators, following experimental sets will be prepared (Balmford,

2006):

i. Flowers bagged only in night

ii. Flowers bagged only in day.

iii. Flowers bagged both day and night

iv. Flowers left open

After that seed set in the following flowers will be counted and collected

to assess effectiveness of diurnal and nocturnal pollinators.

Hand Pollination – To assess stigma receptivity in day or night, flowers will be

hand pollinated and after that, seed set will be measured.

Seasonal Variations – The same sampling procedure mentioned in objective (i)

& (ii) will be carried out in different seasons.

Objective 4: To determine the role of non crops in vicinity of the crop, in

sustaining or competing for the pollinators (in terms of display

and rewards).

Surveys - The above mentioned surveys will be carried out in weeds throughout

the year, in different seasons.

Analysis of floral rewards - analysis of nectar (both quantitative and

qualitative) will be done in lab, to determine whether the non crops (chiefly the

alien weeds) compete with the crops for pollinators or not.

Floral display – The floral characters of crops and non crops will be studied

like colour, contrast, fragrance etc.

References

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floral visitors: experiments with pollen and fluorescent dye. Annals of Botany.

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invasive species. Biotropica. 36 (2): 156- 164.

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networks. Oikos. 120: 822-831.

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biology. Bioscience. 47 (5): 297-306.

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PhD Programme: To forecast the extent to which pollination limits seed set in crop plants along a gradient of agricultural intensification.

Arnob Chatrerjee

1) Identify pollen requirements of three crop species

Objective: Establishing a dose response relationship between the number of

pollen grains on a flower’s stigma and the seed set in the resulting fruit.

Protocol: To eliminate sources of contamination the experiment should be

conducted in an insect proof cage or by placing a mesh bag over each flower

once it opens. If the plant is self compatible, then the flower stamens are

removed with a forceps before the pollens are released. Test of self

compatibility is done by hand pollinating some flower with their own pollens.

A set of flowers are marked with collars made from 6-8 mm of plastic drinking

straw with an opening slit which are conveniently placed around each pedicle

and are numbered. Randomly allocating the flowers a dose using a random

number table.

For hand pollination process first the anthers are collected from at least five

different plants and placed in a small vial.( This will reduce possible variation

arising from variation in pollen vigour among plants) Using a wooden cocktail

stick the pollens are mixed , pieces of anthers are removed and applied to the

stigma at the required dose.

To calibrate the required dose, microscopic preparation of hand pollinated

stigmas are made by squash preparation and pollens are counted.(

experiments should be done with various technique to standardize the amount

of pollen dose. E.g. using one dab, two dabs vs. using a insect pin or by

covering a part of stigmatic surface.) After the flower has senesced the stigma

is retrieve to check the pollen dose. Stigma may be stored individually in

Eppendorfs in a freezer. Finally mature enough fruits are collected to measure

no of seeds, which indicates fertilization has occurred.

From this experiment few things observed and recorded are:

i. Pollen ovule ratio of each species.

ii. Number of pollen required to initiate seed set.

iii. Number of seed developed per flower at a specific amount of stigmatic

load.

iv. Total number of pollen required to produce complete seed set of each

species.

2) Quantifying pollinator effectiveness.

Objective: Establish the effectiveness of flower visits by pollinators.

protocol: the method requires to allow a single pollinator visit to a virgin

flower. To obtain a virgin flower mesh bags or cages are used to cover a late

bud. once the flower is open the mesh bag is removed and wait for pollinater

to visit the target flower. If pollinator visit is not sufficiently frequent the

mobile bouquet technique can be used.

After the insect has visited the flower pistil is carefully removed and placed

individually in an eppendorf vial on top of a moisture tissue that has been

stuck in the end of the vial. The cap is closed and allowed to rest for a few

hours to enable the pollen to germinate. The squash preparation of the stigma

is done to count the number of pollen grains attached on the surface of the

stigma. Specimen can also be freeze for later analysis.

Mobile Bouquet Technique: If insect visitors are scarce it is best to find an

active insect in field and bring the target flower to it. The experimental insect

should be in the process of visiting flowers of the same species as the target

flower, so it should be carrying the right pollen. A mobile bouquet can be

constructed by using a 1m cane and attaching a retainer to the end of the

cane. The flower is placed in the retainer and is offered to the insect.

From this experiment few things observed and recorded are:

i. Number of pollen grains delivered by a single visit of each bees

ii. Number of visits required from each type of insects to satisfy each plant

species requirements.

3) The third target is to quantify flower visitation rates by insects

to each plant species at location along the intensification

gradient.

Gradient of Agricultural Intensification

Agricultural intensification is meant by both the management practice

(degree of usage of chemical pesticides and fertilizers) and the landscape

(distance from natural vegetation). The gradients of agricultural intensification

are as follows:



natural vegetation.



500 m.



m.

Protocol: To quantify flower visitation rate focal observation will be done. This

will be done in a focal sampling 1m2 plots randomly chosen.

The key parameters to be observed are:

i. F = density of flowers per m2

ii. H = handling + travel time for one flower.

iii. B = density of bees per m2.

4) The ultimate target is to combine the observed visitation

pattern with pollen delivery to forecast how pollen limitation

varies along the intensification gradient.

From the data collected we can find out bee visits per flower per hour

FH

1 = visit per flower per hour

F = density of flowers per m2

H = handling + travel time for one flower.

Considering life span of a open flower and density of bees per m2.

FH

LB = bee visit per flower life time.

B = density of bees per m2.

L = life time of flowers in hours.

Proportion of flowers receiving a bee visit =

1FH

LBR

Pollen accumulation rates:

Flowers receive FH

B bee visits flower

-1 h

-1

If a specific bee delivers G pollen grains:

Bees will deliver FH

BGPbee pollen grains flower

-1 H

-1

Therefore importance of a specific bee (for example bumblebee)

%XP

PI

total

bumblebeebumblebee

Therefore flowers of a particular area should require a minimum no. of visits of

one or more pollinators, which can satisfy their stigmatic load and hence

commence to successful seed set. Thereby we can see if the no of visitors

(pollinators) saturate the pollen grains required for successful seed set,

pollination of flower in that area will not be limited; if the no. of visitors fails to

reach the threshold limit then the seed set will be limited. Considering this fact

in mind different factors are calculated which regulates the seed set of flowers

that will reflect the fate of pollen limitation along the intensification gradient.

PhD Programme: The role of Native Plants on Ecosystem Service Deliveryin an Indian Agro-ecosystem

Supratim Laha

Aim-

The native flora of a region supports native faunathat may play a role in ecosystem

service delivery. If the native vegetative cover of a particular region becomes

threatened (e.g. though modern agricultural practices, habitat fragmentation,

urbanization etc.), we can assume that in near future those faunal communities,

dependent on those threatened native plants could also be under threat. So, in order

to conserve native faunal communities of a region we need to maintain those

particular native plant communities of that region; and we have to be very careful

about the characteristics, habit, interaction type, reward type and quality, dispersal

etc. of those native plants, as these features can influence various ecosystem

functions. The aim of this project is to understand the role of native plants in

delivering ecosystem services. The project primarily focusses on two services:

pollination and natural pest control. Ultimately the aim is to develop methods to

conserve the ecosystem service providers (pollinators and other beneficial insects)

through management and re-establishment/re-introduction of some native plant

populations in the agricultural landscape.

Introduction-

Agricultural landscape complexity varies across regions. The agricultural landscape

is primarily divided into crop land and natural vegetation; the latter mainly consists

of native plants. On the basis of the proportion of these two types of vegetation,

agricultural landscape can be of simple, moderately and highly complex type. In

simple type of landscape there are vast stretches of crop land with very little

percentage of native vegetation; while the complex type landscape shows totally

opposite scenario. But in case of moderately complex type, there are good

proportion of both crop area and native vegetation cover.

Agricultural landscapes are totally man made. It has developed by removal of

natural vegetation cover and other natural features of particular region.

Consequently large continuous natural habitat becomes fragmented into several

patches of non native plants. Hence, the native animals, surviving and getting

resources from those native plants now have become less abundant and vulnerable

to local extinction. Habitat fragmentation is also a major problem of dispersion of

those animals. Modern agricultural practices are so intense that it hardly includes

any native plants along the margin of the fields; naturally the native animals become

unable to survive in that situation as the native plants are almost lacking. This can be

assured by studying the diversity and abundance of native insects in intensive

farming areas. In case of highly complex type of landscape, crop fields are present

but, native plant populations are high in proportions. There, thus, one can find

diversity rich composition of native flora and fauna. Interestingly in medium

category, both crop fields and native plants are good in proportion. Here, in this

case, re-introduction of more native plants may support more number of native

insects, thus increase their populations and diversity. In the former case, the area has

already a decreased insect pool; so re-introduction of native plants may not readily

increase native insect diversity to that much as compared to that of the last case-

medium/moderately complex type. On the other hand, highly complex type of

landscape already has rich number and diversity of native insects; so, here also, re-

introduction of native plants may not increase the local insect population and

diversity to a great level (Landscape- Ecosystem Service Hypothesis, Tscharntke et

al., 2005).

Native plant species have major roles for the survival of native insect communities,

as well as they are important to enhance some ecosystem services (like pollination,

pest-control etc.) and after all, conservation of these native plants leads to habitat

restoration. There are various native plant species which provide an array of

resources to many native insects. Some insects take floral reward (pollen and nectar),

some chooses these plants as their larval food material, some makes nest to live or,

some uses these plants in some other ways. These native plants, thus, attract lots of

native beneficial insects and thus helping in enhancing ecosystem services in

agricultural fields if they are present in good number along with the crops.

In this project, moderately complex type of habitat will be chosen. Then, native plant

species of that region will be observed by planting them at experimental plots. After

that, they will be re-introduced into the field to see the realized benefit.

Objectives-

To study the effect of native plants on pollinators and beneficial insect

communities in an agro-ecosystem

To assess the efficiency of some native plants in providing pollination services

by supporting crop pollinators

To assess the efficiency of some native plants in biological pest control

through supporting natural predator population

How these native plants attract pollinator and other flower visitors

What will be the effect if a combination of native plants is present along with

a crop type in a field

Experimental Design & Methodology-

Vegetable fields will be chosen in a moderately complex landscape type.

Hedgerows and weedy strips will be identified in replicate fields

Plants of hedgerows / weedy strips, will be identified and collected to be

identified later if necessary. Similarly, all the insects (pollinators, predators

and pests) visiting and/or inhabiting those plants, will be noted or collected

to be identified later if necessary.

Species will be selected to be grown in experimental plots.

Among all the identified plants, only the native perennial plants will be

grown in experimental plot at our field station.

Native plants either will be uprooted from the fields or, seeds will be collected

for planting in experimental plot. 2m X 2m area will be selected for each plant

species. Inter-plant distance will be one foot. Plants will be established

according to their blooming season. Each plot will be replicated at least 3

times.

Each plant will be observed thrice in their two successive blooming period.

Details of observation are as follows-

Plant traits- Most of the available plant traits will be noted; once for

each plant. These are:

1. Type (herb/shrub)

2. Height

3. Foliage pattern

4. Inflorescence pattern and size

5. Flower size, shape and colour

6. Flower odour

7. Pattern of nectar guide under UV- light

8. Reflectance of fluorescent colour of petal

9. Anthesis time

10. Duration of the opened flower

11. Peak nectar production time

12. Nectar quantity and quality

Visitation rate- 12 flowers of each plant will be observed for 5 min

for 20 days per blooming period.

Focal observation- 20 min observation for each plant for any five

days per blooming period. All the insects (pollinators, beneficial

ones and pests) visiting (eg. feeding/ resting/ nesting) the plants

will be noted along with their behaviour.

You could sweep and set pitfall traps for the pest predators.

Each plant will also be established in different combinations with other

selected plants to find the most efficient combination(s) to attract pollinators

and other beneficial insects.

Lastly, the most efficient combination(s) will be established along the margin

of a certain vegetable crop field. Then, visitation rate and pest population will

be studied on that vegetable.

For control, same vegetable plant field will be studied where these native

plants are not manipulated experimentally.

Master’s Project Protocol 1

Subject: - Determine the pests, predators, visitors and pollinators of members of the

Solanaceae family & whether they share its pollinators with other non-crop plants.

Introduction: - Globally, it has been estimated that more than 100,000 species of wild plants

depend on insects for pollination and reproduction.

Brinjal (eggplant), Solanum melongena belongs to the family Solanaceae. The eggplant

(Solanum melongena) has a perfect flower with purple petals and green or purple sepals. The

anthers are yellow and sit on short filaments surrounding the style. Each anther bears two

chambers that open in a round hole at the upper end. The style may be longer, equal, or

shorter, than the cone of stamens surrounding it. The eggplant is a self-fertile and self-

pollinating plant. However, cross-pollination through insects is possible and in many cases

desirable. The carpenter bee is capable of shaking the flower using the “buzz-pollination”

mechanism in which it specializes. Hence carpenter bees are commonly used for pollination.

In this project our aim will be to determine which specific insects and animals play the role of

pollinators in brinjal plant & also the visitors which take the advantage of the brinjal plants

but do not help in pollination. In second part of the project our aim will be to determine the

pollination network between the brinjal plant individuals & other members of the Solanaceae

family and the non-crop plants. We will also try to identify plant traits (e.g. plant height, stem

and foliage distinct features, distinct fruit characters, crop duration and 1000 seed weight),

that help in the process of pollination.

Protocol: -

1) For the 1st Part of The Project (i.e., identification of pests,predators,pollinators

& distinguishing the pollinators & visitors): -

Flower pollinators & visitors will have to be monitored by direct observation in the field from

6.00 am to 03:30 pm or whatever time suitable throughout the period of study and insect

specimens have to be collected for identification by proper taxonomic study.

A) For Determining visitation rate & Identification Of The Pollinators

Experimental site:- Crop fields of East Kolkata wetlands. Red loamy soil of medium

fertility, treated with agrochemicals.

Period of study:- November,2012-Present

Methodology: -

I. With the help of stopwatch the visitation rates of the pollinators/visitors are

measured & noted in the data sheets.

II. Mark the brinjal plant of which the visitation rate has been measured with the

help of a marking tape or flag.

III. The ambient temperature of the environment is measured with the help of

thermometer.

IV. Repeat the process in different times of the day so that we can compare the

number of pollinators with ambient temperature.

V. Number and diversity of pests, predators and pollinators of the brinjal plants,

are characterised as-Flower, Foliage and Flying, by “line-transect” and “focal

observation” methods. This procedure is repeated for various plots, and the

data is collected.

VI. Insects are collected with the help of insect nets.

B) Distinguishing the Visitors as Pollinators and Non-pollinators :-

Methodology:-

I. Categorise visitors as Pollinators ( those that touch reproductive parts of the

flower ),and Non-pollinators.

II. Collect as many visitors to flowers as we can with an insect net.

III. Use ethyl acetate to kill or stun insects.

IV. Use a dissecting needle to cut a 0.5 x 0.5 cm cube of glycerine fuchsin jelly.

Holding the cube of jelly on the dissecting needle, wipe pollen from the body

of the visitor. Make separate slides for jelly wiped on mouthparts, abdomen,

and legs of the insect (or other body parts as observed). If no pollen is found

on the body of the insect then it is identified as visitor & not a pollinator.

V. Place the cube on a glass slide, put on a coverslip, and gently melt over a

candle or lighter, or place on dark surface in the sun.

VI. Examine the slides under a microscope at 100X & identify and count the

pollens of cowpea. If the pollens of some other non-target plants are recovered

then calculate & mark it in data sheets. Later ratio of target (i.e. brinjal) &

non-target plant pollens are calculated.

For the 2nd

Part of The Project ( Pollination Network Between brinjal plants & other

Non-crop Plants) : -

Methodology:-

We can have two plots having the same area around 100×100m. In the first plot only Brinjal

plants are evenly distributed. In this plot our main aim will be to determine the pollination

network between the individual plants of the Brinjal. As because we estimate species linkage

level daily, i.e., the number of pollinator species visiting a plant species and vice versa (the

number of plant species visited by a pollinator), we will not use rarefaction curves to show

how the number of pollinator species changed with sampling effort. Instead we can use the

Jaccard similarity index (J) to show how the two individuals of a species differed in the

composition of their pollinator fauna. For this purpose in each observation we have to

observe two plant individuals & watch whether they share any pollinator species.

To see whether the brinjal plants share their pollinators with other non-crop plants we will

use the second plot in which the brinjal plants will be planted haphazardly (i.e. in non-

specific manner). To see the effect of introduction of new non-crop plant species on the

pollination network, new non-crop plants are planted in between the brinjal plants.

Regardless of their specific nature, all networks consist of nodes interconnected by links. An

important characteristic of a node is its Linkage Level. To determine the linkage level we

have to observe which pollinator insects are more predominant in pollinating which plants.

Here we can use our previously collected data of most common pollinator species of the

brinjal plant. We have to observe in which non-crop plants these pollinators visit by

observing & collecting the insects. From the collected insects the pollens are separated by the

previously said way. Now by the help of basic fuchsin gelatine the slides of the pollen are

prepared & it is observed under the microscope. From the observation a linkage level graph is

to be prepared by plotting the Brinjal plant in one axis, different non-crop plants in other axis

by the value of linkage level. Network regions are called modules, whereas link-sparse

regions demarcate their boundaries. For each pollination network, we can run the modularity-

detecting algorithm Simulated Annealing (SA). It will produce a modularity index M, which

is a measure of the degree to which the network is organized into clearly delimited modules.

The algorithm will also provide (i) the significance level of M of the real network by

comparing its value to that of similar-sized random networks, (ii) the number of modules per

network, and (iii) the content of species of each module.

Literature Available on The Question :-

1. R. B. Thapa, Institute of Agriculture and Animal Sciences (IAAS), Rampur, Chitwan,

Nepal, A Review, Honey Bees and Other Insect Pollinators of Cultivated Plants.

2. Jens M. Olesen, Jordi Bascompte, Yoko L. Dupont, and Pedro Jordano, The

modularity of pollination networks, PNAS, December 11, 2007, vol. 104,no. 50

,19895.

3. Jens M. Olesen, Jordi Bascompte, Heidi Elberling,And Pedro Jordano, Temporal

Dynamics In A Pollination Network, by the Ecological Society of America,

Ecology, 89(6), 2008, pp. 1573–1582.

Master’s Project Protocol 2

Subject- Determine the effect of at least two commonly used agrochemicals on

stigma receptivity and pollen viability of brinjal (Solanum melongena)

Project lead: Pamela

Introduction

Brinjal, Solanum melongena L. is a very common and favourite vegetable among the Indians.

It is estimated that there are more than hundred varieties of brinjal in our country. Brinjal is

cultivated in almost every part of our country since last 4,000 years (Kumar, 2009). [1]

Pest like brinjal fruit and shoot borer is a serious threat to brinjal cultivation. Pesticides are

commonly used to kill the brinjal fruit and shoot borer larvae. Application of pesticides to

this crop was found to be more than many other crops cultivated in India. The minimum

range of pesticide-use intensity was estimated at 3-4 kg of a.i/ha and 31.43 per cent of the

farms were within this range.[3]

Pesticides are often applied on the foliage as well as flowers using a spray which includes the

stigma and anther. Through killing of pests the pesticides could play an important role in

reducing the economic loss, however, if these chemicals also affect the floral reproductive

parts they could have a negative effect on the yield. Little is known about the effect of such

pesticides on pollen germination and stigma receptivity in the Eastern part of India. The aim

of this study is to quantify the effect of a few selected pesticides on stigma and pollen.

Research: [3]

Percentage of farmers using various types of plant protection chemicals

Pesticide class

Insecticides 100.00

Fungicides 8.57

Biopesticides 0.00

Botanical pesticides 0.00

Types of pesticides used by vegetable growers Category

Organophosphates

Profenofos 50% EC II

Methyl Parathion 50%EC & 2% EC I

Monocrotophos 36% SL I

Quinalphos 25% EC II

Acephate 75% SP III

Carbamates

Carbofuran 3%G I

Indoxacarb14.5% SL II

Carbosulfan 25% EC II

Pyrithroids Cypermethrin 25% EC & 10% EC II

Fenvalerate 20% EC II

Organochlorines

Endosulfan 35% EC II

Others

Fipronil 5% SL II

Imidacloprid 17.8% SL II

Spinosad 2.5% SL III

(**Most of the pesticides used on the sample farms belonged to the moderate risk (category II),

followed by high risk (category I) and low risk (category III) groups as classified based on acute dermal LD50 for

Rabbits/Rat.)

Brand name Chemical composition Class[1]

Thiodine Endosulphan active 35% w/w Organochlorines

Melathion 0,0-Dimethyl dithiophosphate Organophosphates

of diethyl mercapto succinate

Tricel Chloropyriphos Tech. 21%

Solvent Aromax 72%, Emulsifier, Organophosphates

Ustad Cypermethrin 10% Pyrethroid/Pyrethrin

Tata fen Fenvalerate tech (90%) 22.5% Pyrethroid/Pyrethrin Emulsifier (Polyethylenepolyp-

-henolderivative 14%+Alkylanyl

sulphonate 6%)

BSE-50 Lindane 50% Organochlorines

Experiment:

We will have two experimental sets – (a) Application set up

(agrochemicals used crop plants)

(b) control set up(no agrochemicals used)

Determination of Pollen Structure change and viability[4]

Materials required

Carnoy’s solution

Glycerin-gelatin-liquid safranin mixture

ocular micrometer.

2,3,5-triphenyl tetrazolium chloride solution.

Scanning Electron Microscope

Methodology

1. Flower bud specimens are randomly collected from both the control and application set

up(s) and the following methods are applied separately and simultaneously.

2. The flower buds are fixed in Carnoy’s solution.

3. The flowers will be removed from Carnoy’s solution and then the anthers were mounted

in a glycerin-gelatin-liquid safranin mixture.

4. Pollen grains from each group are used for measurements using an ocular micrometer.

5. Measurement of the length of pollen’s polar axis in equatorial and polar view is taken

from equatorial diameter of the pollen.

6. Pollen viability is tested by TTC test using 2,3,5-triphenyl tetrazolium chloride

solution. See TTC preparation for information on this assay.

7. One drop of this solution is placed on a slide.

8. Pollen grains are spread with a brush on the slide, and a cover slip is placed on top.

9. Counting is performed after TTC application.

10. It is divided into 3 groups based on staining density.

o Pollen grains that –

stained dark red will be referred to as viable,

light red as semi-viable,

unstained as non-viable.

11. Viable and non viable pollens of both control and application group are photographed.

12. If possible, scanning electron micrographs will be taken for the pollen in the treated and

untreated set.

TTC preparation: The solution prepared with this chemical, which is normally colorless, turned into Triphenyl

formazan, which is insoluble and appears red in living tissue to which it is applied. This

reaction is formed by some reductase enzymes in living tissue. The activity of redox-enzyme

occurs via the same mechanism in other tetrazolium salts, such 2,3,5 triphenyl tetrazolium

chloride; therefore, according to the level of liveliness in tissue, tissues stain red in

accordance to the density of enzyme activity. When the enzyme activity increases a dark

color is observed. If the same activity decreases, a light color is observed.)

Determination of effect of agrochemicals on stigma receptivity[2]

To test stigma receptivity any one of the four tests can be conducted.

1. Baker’s procedure(Dafni 1992)

Test solution contains 10 ml of 1M phosphate buffer (ph 7.3-7.5), diluted (1

part buffer to 2 parts distilled water) 5-10mg nitro blue tetrazolium to give a

slight yellow color, 6mg of NADP and 1 ml of ethanol(95%).

The fresh stigmas are cut and removed in the field directly into a large droplet

of the test solution on a slide and incubated at room temperature in a closed

petridish containing a moist filter paper in the bottom.

Stigma inspected after 20 to 40 minutes under microscope (x200) to locate

stained areas.

2. Perex Test (Firmage and Dafni 1997)

The test solution ( Merck Chemical 16206) tests for the presence of hydrogen

peroxide.

The tissue turns deep orange within 1-2 minutes confirming peroxide is

present.

The kit has a color chart indicating the concentration range of 10 -500 ppm

peroxide.

A droplet of solution was placed directly on the stigma and inspected after

several minutes for yellow to orange colouration.

3. Hydrogen Peroxide( Zeisler 1933)

A 6% solution was placed on the stigma and appearance of bubbles observed.

4. Macherey-Nagel Peroxtesmo Ko Peroxidase test paper (Motten 1982, Sullivan

1984)

The paper is applied directly on the stigma.

The appearance of a blue color indicates the presence of peroxidases

The paper does not work on dry stigmas.

(Tests 2 or 3 or 4 will be performed)

Quantification to be done:

1. Calculate the length of the pollen tube in germination medium

2. Changes occurred in the pollen structure

3. Stigma receptivity test

Results of control and application set up will be compared.

Expected Results: 1. Distorted structure of pollen grains in application set up.

2. Length of pollen tube may be small in case of pollens from application set up.

3. Pollens from application set up even may not stay viable for long hours.

4. Stigma receptivity may decrease in case of application set up.

References

1. Assam University Journal of Science & Technology :

Biological and Environmental Sciences Vol. 7 Number I pp 84-88, 2011

2. Sex plant Reprod(1998) 11:177-180

3. Agricultural Economics Research Review, Vol. 18 July-December 2005 pp 209-221

4. Turk J Biol 34 (2010) 281-286

MSc. theses and Internship opportunities

Project 1 Aim: To investigate the effect of radio tags on bee behaviour.

Status: To be taken up

Tracking devices like radio tags have been used to track the movement of various insects like

butterflies, bees, dragonflies etc. Such studies provide valuable information on the foraging distance

of the species as well as the habitat requirement for the species. However, such studies are very few. In most the studies what remains untested is whether the tags themselves could have an effect on the

animal under investigation. Tags could affect behaviour because the tags have certain weight which

might limit the insect from performing certain behaviour or affect the distance to which it is capable of flying in absence of the tags on it. A recent study has investigated the effect of these devices on

behaviour of bumblebees. It was observed that the bees with tags would rest more often than the

untagged bees. Also, a difference in flower handling time was noticed.

We aim to investigate the effect of radio tags on carpenter bee behaviour. The suitability of such tags

will be assessed. This will enable us to use the tags for experiments aimed at estimating the foraging

distance as well as habitat requirements of these bees.

Protocol:

1. Two individuals will be collected from the same nest using a net/box at the time of their exit from the nest. (This is done to reduce variation arising due to difference in nest).

2. Both the individuals will be made inactive by freezing/ ethyl acetate.

3. A tag will be fixed with eyelash glue on thorax of one of the individual. 4. Wing will be measured for body size estimation.

5. These inactive bees will be placed inside a polyhouse (See Polyhouse for details).

6. Once the bee becomes active, the observer will start data collection on bee behaviour.

Polyhouse

1. The polyhouse will have floral resources for pollen collection.

2. Brinjal pots at flowering stage could be installed inside the polyhouse.

3. The behaviour of the released bees will be observed.

Observation schedule:

1. Observations will be performed during the peak activity of Xylocopa:

2. Observation time will depend on the time when the individuals emerge from their nest for

foraging. 3. Tentatively, observation time will be between 9:00-13:00 hrs

4. Ad libitum scan sampling will be done on focal animal.

5. Each observation period will last for 25 min.

Tagged bee: 9:00-9:25

Untagged bee: 9:25-9:55

6. Following behaviours will be noted during scan sampling:

Flying inside the tunnel, sitting/holding flower, Sitting on places other than flower,

grooming, walking, buzzing, pollen collection

7. From the scan samples, the most frequent behaviour will be quantified for each bee. The total time spent on each behaviour can also be calculated.

8. Comparisons will be performed between tagged and untagged bees, controlling for identity

and size.

9. If possible, the behaviour of the bees will be recorder using a high definition digital camera. This will be analysed in the lab to obtain the behaviours and collect quantitative data from the

same.

Project 2 Aim: To evaluate the best active sampling method for insects.


To get an estimate of diversity and abundance of insects in the fields, often, transects are laid. Usually, on each transect an observer walks recording all the species observed as well as the number

of individuals encountered during the walk. The length of transect could vary from 10 m to more than

a kilometre.

However, it is also possible that the quality of data obtained from the long walk does not

change even if many small transects are walked upon instead of a continuous long transect. A further

splitting of the design could be many points along the transect line on which an observer could stand note the species encountered.

We aim to quantify which of the three methods i.e. long transect, short transect and point counts is the best method for estimating the insect diversity and abundance.

Protocol:

1. An observation site will be selected.

2. All three sampling methods will be tested at the same site.

3. The sampling methods are as follows:

Long transect - 200 m

Short transect - many 10 m transects

Point transect - many point observations

4. The order of the sampling method tested will be randomly decided for each day.

5. Observations could be done on a single day by different observers; each observer will

quantify all the methods. 6. In absence of many observers, a single observer will perform sampling for at least 10 days.

Based on the results, the period of sampling will be increased. Therefore, for each day data

will be available for each method. 7. Results across methods will be compared to arrive at the best possible way of sampling insect

diversity in an area using the transect method.

8. The time required to finish each sampling will also be estimated, and the surveyor’s ease of

sampling will also be discussed.

Project 3

Aim: To evaluate the use of “bindis” as a method of marking bees for behavioural

observations.


Behavioural observations are often impossible without tagging or marking an individual. Many such

tags are known to facilitate the observations with accuracy. These includes, non-toxic paints markers, florescent dyes, number tags etc. Also, very often these tags have their own limitation of size. Many

of these tags are expensive and the experimenter might be limited in choice of colour. The same holds

true for marking of bees for behavioural observations.

We aim to find an alternative to the usual marking methods. “Bindi” as an alternate marking

tool will be evaluated. Bindis are sticky plastic structures used by women as a cosmetic in India. These come in various shape, sizes and are friendly to the human skin.

Protocol:

1. Tests will be performed on Apis cerana in closed chambers.

2. Bees will be caught and a bindi will be placed on the thorax.

3. The retention time of the bindis on the bees will be quantified. Later the retention time will be compared with other conventional tags.

4. The ease of observation, i.e. how easy it is to locate a bee with bindi will also be looked at.

Spotting frequency of a tagged bee will be recorded. This will be compared with bees tagged with bee tags.

5. Effect, if any of these binids on the bee bahviour will be quantified. Comparison will the

drawn between bees tagged with bindi, bee tags and unmarked bees. 6. Since the bindi has a glue, after a few days any kind of fungal/bacterial/mite growth which

could affect the bee health will be checked.

Project 4 Aim: Can we introduce bee boxes? An investigation of species interaction between

wild bees and domesticated bees from boxes.


Introduction of bee boxes have been found to be important in increasing the yield of crops dependent

on pollinators for fruit set, especially, apples and almonds. Such boxes usually contain a large force of

worker bees who forage for nectar and pollen from the plants and in turn cause pollination. Recent

studies are showing that wild species of pollinators could act as an insurance against loss of domesticated bees.

The study sites at Tripura have been observed to have naturally occurring/ wild bees. The abundance status and also their services towards pollination are not yet completely known. In such an

area, for crops dependent on pollinators, introduction of bee boxes could be useful. Before the

introduction it is important to understand whether domesticated bees could have a negative interaction with the wild bees. This project aims at investigating the interaction between the domesticated and

wild bees.

Protocol:

1. On pilot crop plots survey of the wild bee will be conducted.

2. Survey will be followed by introduction of bee boxes.

3. The diversity and abundance of wild bees will be compared before and after the introduction of bee boxes.

4. Behavioural observations like aggression/chasing etc will also be monitored between

domesticated and wild bees.

5. Flower visitation time in presence and absence of domesticated will be quantified. 6. These results will facilitate decisions on introduction of domesticated bee boxes in crop

fields.

Project 5 Aim: Identification of mite species and quantification of mite load on wild bees in

Eastern India.

Status: To be taken up by Pamela.

Presence of Verroa mites have been a serious threat to the domesticated bees in Western countries. In

India, these mites have so far not been found to be a threat. However, whether mites are present on

wild bees is not very well known. We aim to investigate the presence of mites on wild bees. This preliminary study aims at identification of mite species and quantification of mite load on the wild

bees.

Protocol:

1. Bees collected in pan traps will be inspected for the presence of mites. 2. The mites on the body as well as tracheal mites will be looked at.

3. If present, the mite load on the bees will be quantified, along with identification of mite

species. The function of such mites, whether phoretic or infectious will be deciphered based

on the available literature. 4. Digital photos of the mites will be taken. Also, Scanning Electron Micrographs will be taken.

5. This investigation will be done on the bees collected from two states of Eastern India, Orissa

and Tripura.

Project 6 Aim: To classify the vegetation for selected sites in Tripura.

Status: To be taken up.

We need to understand the elements of landscape to know whether a habitat is suitable for an insect. Many insects are known to forage over longer distances and may need suitable sites for resting. Also,

habitat classification is important for an understanding of intensification of agriculture, for example,

how much of the land is used for crop cultivation in an area. GIS would be used for classifications. All the chosen sites for long-term monitoring will be classified. The following questions will be

asked:

1. What is the proportion of land under crop cultivation?

2. What is the ratio of crop vs non-crop region?

3. Is the landscape simple or complex?

Project 7 Aim: To investigate methods for observing pollinator behaviour in field.


Focal observations are aimed at observing the behaviour of insects. Pollination behaviour, that is transfer of pollen to stigma by a insects could help in separating pollinators from pollen feeders as

well as visitors. Behavioural observations can be done on a dedicated patch of flowers where the

interaction between the insect and flower is observed for a dedicated period of time. Often times it’s

not easy to observe such interactions because there are many species as well as many individuals of the same species which simultaneously interact with the flowers. Also, most often only an expert can

follow the interactions. Under such a circumstance, if the observations are recorder digitally, it is

possible to look at individual interaction and such interactions can be quantified by new observers also.

We aim to compare benefits and drawbacks of focal observation by an observer with a digitally recorded observation period.

Protocol:

1. 1 X 1 m observation plot will be chosen.

2. An observer will be present on one side of the focal plot.

3. The observer will collect data on all interactions for 10 minutes. 4. Next to the observer will be placed a camera on a tripod.

5. The timer of the camera will be set for 10 minutes,

6. At the end of 10 minutes, we will obtain a data matched for time from the observer and camera.

7. The recorded observations will be decoded in the lab.

8. Observations recorded from the digital partner will be compared with the human observer.

9. At least 10 such observations will be taken. 10. Three or more observers could repeat the same; therefore at the end we have 10 data points

from each observer.

Project 8 Status: To be taken up.

Aim: Quantification of relationship between floral phenotype and yield in brinjal


Brinjal has been reported to have three types flowers based on the style length. This might indicate

difference in mode of fertilisation (self vs cross). Also, the pollination mode can affect the seed set

and hence yield in brinjal. The following aspects will be addressed:

1. Quantify the different floral phenotypes in brinjal

2. Establish the pollination requirement of these three type of flowers (self or cross pollination).

3. Is there a relationship between floral type and yield?


Aim: Determine the visitors and pollinators of cowpea and quantify the pollinator dependence of

cowpea.



Aim: Does cowpea share its pollinator with the neighbouring non-crop plants?



Aim: To determine the pollination dependence of a few vegetable crops and horticultural crops in

West Bengal, Tripura and Orissa.


Aim: To determine the pollen viability and stigma receptivity of selected vegetable crops and horticultural crops in West Bengal, Tripura and Orissa.

entre for pollination studies: project protocols long term...

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