Attachment X

AUSTRALIAN CENTRE FOR APPLIED MARINE MEGAFAUNA SCIENCE

RESEARCH APPLICATION FORM
2014-15

1. Project title

The influence of diet preference on intra-population segregation of whale shark (Rhincodon typus) in aggregation
site


2. Proponent(s) details

(a) Chief Investigator

Title Miss
Name Lau Ieng Hou
Institutional
Affiliation
University Of Queensland
Address School of Biological Science
University of Queensland
St Lucia, QLD 4072
Phone Number +61 (07) 33651111
Email Ieng.lau@uqconnect.edu.au


3. Project summary
Describe what you are doing and why (maximum of 100 words). In particular explain how your work will contribute towards
advancement in our management and conservation of marine megafauna in Australasian waters.

In this project, plankton samples and skin tissue of whale shark will be collected during the seasonal aggregation
around Christmas Island for diet preference analysis between different size and gender group. Additionally, selected
individual will be satellite-tagged for movement detection through out the season. Overall, this project will allow detail
investigation on diet of whale shark at aggregation site so as to identify habitat in relation to food source. More
importantly, knowledge gain may contribute to explain sexual segregation in aggregation site around the world, which
can be intergrade into management or conservation plan to achieve more effective management.

4. Project priority areas
(a) Select below which ACAMMS priority / priorities the project addresses?

[ x ] Quantify the status, dynamics and forcing factors (physical, biological) of marine megafauna population structure,
distribution and abundance.
[ x ] Characterise and quantify threats to marine megafauna populations, with a focus on understanding the nature and
extent of interactions with humans.
[ ] Develop risk management and mitigation tools/strategies for marine megafauna and human interactions and test their
efficacy.
[ ] Develop powerful, new, non-lethal technologies and methodologies (e.g. molecular, bio-logging, remote sensing) that
best address the above objectives.







Attachment X
(b) Briefly detail how this project will meet this priority / priorities

Genetic study and majority of the population structure study in different aggregation suggest clear evidence on
sexual segregation behaviour on whale shark, however, the drivers of this sexual segregation remain a mystery,
largely due to the lack of female observation. Yet, study on the stable isotope profiles on the living tissue and
foraging ecology insight that the segregation might be result from diet and habitat preference (Borrell et al. 2011;
Ketchum et al. 2013).

In Australia, information on the diet and distribution of whale shark were predominantly come from seasonal
aggregation at Ningaloo Reef, Western Australia (DEHP 2005), Where the aggregation is mainly composed of
males, especially juvenile males (Meekan et al., 2006; Norman & Stevens 2007). However, interestingly, a study
observed female base sex ratio of 1:3.3 at Christmas Island (Hobbs et al.2009).

By addressing the movement and diet preference differences between sex and size group around Christmas
Island, the outcome this project may contribute to locate potential habitat that might be utilize by different groups
that can contribute to explain and quantify population dynamic of whale shark base on the appearance of prey
item.

And therefore the priority Quantify the status, dynamics and forcing factors (physical, biological) of
marine megafauna population structure, distribution and abundance can be met.

Furthermore, By knowing the composition of diet and habitat, potential human activity that might disrupt or
threats to whale shark can be characterised and thus meet the second priority Characterise and quantify
threats to marine megafauna populations, with a focus on understanding the nature and extent of
interactions with humans.
































Attachment X

5. Project objectives (maximum of 1 page)


1. Survey population structure of whale shark (Rhincodon typus) during seasonal aggregation around
Christmas Island.

During the survey, trained snorkelers will be direct to each individual whale shark observed around
Christmas Island to collect individual data on size, sex, maturity and skin tissue sample. Meanwhile, photograph
identification will be taken and environmental data including water temperature, GPS location, weather condition
will also be collected close to where whale shark is observed. Collectively, the data should build a picture of the
population demographic structure and distribution of whale shark around Christmas Island during the season
when the survey is conducted. This data will then be used to categorize whale shark observed for further
analysis. Additionally, when the project is finish, the data can be hand on to resident research group in Christmas
Island that conduct yearly whale shark monitoring programs to study the variation of population dynamic between
years.

2. Identify organism that whale shark (Rhincodon typus) prey on around Christmas Island via plankton sampling
and observation.

Plankton samples will be use to in identification of potential prey item. It will be collected in two types of site:
firstly, random generated GPS location where whale shark absent and secondly, location where foraging
behaviours of whale shark is observed during the survey, additionally, if whale shark is observed consuming
other visible prey item, it will also be record. This data would allow classification of potential prey item that whale
shark consume by comparing plankton composition and density record where whale shark present to the control
(when whale shark absent). Under the same principal, preference on prey item among different sex or size group
can be investigate by adjusting the base line.


3. Identify potential feeding habitat via stable isotopes profile analysis from skin biopsies.

Sampled skin tissue will be used for stable isotopes analysis as an alternative method to examine diet
compositions use in this project. Although not providing species-specific dietary information, stable isotope
values provide information on previous location of subject base on food consumed previously. Thus it is a
powerful tool to study migratory organism. This project will use stable isotope (
15
N) and (
13
C) as an indicator to
identify potential feeding habitat utilize by different groups of whale shark pervious to their arrival in Christmas
Island.

4. Examine intra-population segregation and differences in movement patterns during seasonal aggregation in
relation to mature stage, sex and size groups.

The Main objective of this project is to determinate the probability of diet preference as a diver to intro-
population segregation in whale shark observed in aggregation site worldwide. So as to identify potential
habitats use by female or juvenile shark that are currently unknown but is critical to international conservation
and management of whale shark. Potential habitat will be identified by building a simple occurrence model utilize
data of significant finding (if any) from other part of the project and overlap it with the potential prey and location
of food -net identified








Attachment X



6. Project methodology (maximum of 2 pages)

Background:

Whale shark (Rhincodon typus) is an iconic shark species well known as the largest living fish species, with
maximum body length up to 20 m descripted previously (Stevens 2007). Despite its large body size, knowledge
on whale shark remain very limited due to the difficulty in conducting research with organism of this size and its
wide distribution (Rowat & Brooks 2012). Although highly migratory, currently whale shark are know to
aggregate in predictable location on a yearly and seasonally base (Rowat & Brooks 2012). The predictable
seasonal aggregation of whale shark had enabled the development of ecotourism industry, which allow direct
economic value, generate from the living organism. This also led to the increase funding in research
programmes (Martin 2007).

The K-adapted lifestyle of whale shark make it very vulnerable to threats such as over exploitation, due to the
low reproductive rate and long life span (Jarman $ Wilson 2004). In addition, evidence given in recent genetic
study also confirm its vulnerability state, in which the study indicated that genetic variation contain in the global
population is not sufficient to overcome genetic drift (Vignaud et. al. 2014). Furthermore, study at Ningaloo Reef
indicated a trend of decline in total length and number of individual observed in recent year (Bradshaw et
al.2008). Nevertheless, other argued that this observation was potentially a sight of population recovery
(Holmberg et al. 2009). No matter which is the truth, this question highline the need to obtain information on the
population dynamic of whale shark so as to develop proper international management tool that suit this unique
organism.

Whale shark are filter - feeder organism that feed on plankton as well as larger prey such as small fish ad squid
(Stevens 2007). The seasonal aggregation of whale shark in certain location were through to be due to the
temporal abundance of prey item, and this were confirm by several genetic study on small amount of faecal
samples (Meekan et al. 2009; Jarman & Wilson 2004). The unbalance in sex ratio and maturity state observed in
these feeding aggregation clearly suggest a intra-population segregations but the reason for this segregation is
not known (Ketchum et al. 2013) While some aggregation are known to be feeding ground, it is possible that the
segregation is due to the differences of diet preference in different sex, size and age groups (Ketchum et al.
2013). By studying the diet of different whale shark on a Christmas Island, this research wish to identify new
habitat that may be utilize by female whale shark but yet being ignored as well as create simple model of whale
shark occurrence base of prey appearance.

Study Site:

This project will focus on one seasonal aggregation site around Christmas Island where aggregation of whale
shark occurs from December to March each year. This site was less studied due to its remote location and the
oceanography feature of the island (Meekan et al. 2009). Nevertheless, the site was chosen because: in contract
to many aggregation sites, female base sex ratio was reported here in pervious study (Hobbs et al.2009).

Aerial survey and boat survey

In this project, Whale sharks will be located fundamentally base on boat survey using the network of local tourist
operators daily. Once a whale shark is located, the vessel will approach at a low speed and stop at
approximately ~60 from the organism (a distance that is greater then the minimum distance to be kept according
to the current management code of conduct for whale shark tourism operation (DEHP 2005)). If no whale shark
is observed on the sea surface until early afternoon (1 pm), a quick aerial survey will be conducted to locate
whale shark on the aircraft and if successfully located, vessel will be directed to the specific location. The aerial
survey will be preform according to the advise of local expert.


Attachment X


Prey identifying

Differences in the Plankton composition with and without the present of different groups of whale shark will be
use as indicator for identifying potential prey item. Similar method had been use by pervious study: for example,
Nelson & Eckert (2007) identify copepods as the primarily prey item for whale shark in Bahia de Los Angeles.
Further, Ketchum et al. (2013) identify different prey item consume by different group in regard to their location.
This project will continue to use similar method to detect the differences in prey preference amount whale shark
with different size and gender. Detail of the sample collection and analysis is discussed in Question 7.

Stable Isotope profile

Stable Isotope is another powerful tool that hand been increasingly used in migratory species. Which provide
information on the potential habitat that a migratory species use during the journey using the stable isotope
characteristic as a reference value (Hobson & Wassenaar 2008). Isotopic carbon ratios of primary producers in
marine ecosystem (i.e. Plankton) have distinct spatial gradients increasing from offshore to coastal habitat, in
addition, generally the isotopes value increase with trophic level, therefore stable isotope contain valuable
information such as trophic level and distance from land of the habitat used by the organism previously.

Value of stable isotope on whale shark will be obtained from analysing skin tissue, which will be collected by skin
biopsy technique, the tissue will then be analysed using isotope ratio mass spectrometer. Detail of the stable
Isotope analysis and sample collection is descripted again in Question 7.

Satellite tracking

Although expensive, Satellite tracking had been a powerful tool to study the movement in migratory species. In
this project, 3 male and 3 female in average size (~6 m) occur at Christmas Island will be tracked during the
survey periods. The main propose of this tracking is to test the accuracy of the model generated in term of how
much of the movement can be explain by the occurrence model.

The type of satellite tag that will be use for this study would be the Smart Position or Temperature transmit tags
(SPOT tag), which are capable to record the location of tracked individual every time it come to the water
surface. Satellite tag will be attach to the dorsal fin of the whale shark, the actual tagging techniques will follow
the once developed by CSIRO guidelines to minimize tag loss.

Occurrence model

After all, an occurrence model will be build to visualize the significant finding from other part of the project. The
basic concept of this model is to map the distribution of whale shark base on the abundance and composition of
their prey item through time and space.

The effectiveness of this model will be determinate by comparing to the actual shark occurrence data obtain from
Satellite tracking during the survey, similar to what was done by the CIS and remote sensing group (Sleeman et
al. 2013)









7. Data collection and analysis (maximum of 1 page)
Attachment X
Data collection
All data will be continually collected within a single season of whale shark aggregation around Christmas Island
in Indian Ocean. The survey will be conduct continually through out the season from December to March in the
following year if the weather condition is suitable for the survey.

Methodology to locate individual whale shark is descripted in Question 6. Once located, two experienced, trained
snorkelers will be sent for data collection. Data to be collected include:
- Total length and standard length, measured by laser photogrammetry to obtain acute length measurement
as descried by Jeffreys et al. (2013);
- Sex (and maturity state);
- Skin tissue, obtained using a biopsy spear on the skin behind dorsal fins (Meekan et al. 2009);
- Water temperature
- GPS location, data and time
- Water depth (if known)
- Distance from shore
- Photograph for identification

- 6 individual will be satellite tagged for more detail analysis of the movement of whale shark during the
season. The satellite will pick up remote sensing data automatically every time when tagged individual come to
the surface.
- 3 Plankton sample will be taken as close as 30 meter in front of the whale shake on the vessel If whale shark
foraging behaviours observed, in addition, if whale shark is observed concentrate in a particular area plankton
sample will be taken daily at the location until whale shark not present. The plankton sample is obtained using
conical plankton net; Extra plankton sample is also taken from random GPS location daily as a reference control
of plankton community (Ketchum 2013).

Data analysis
All statistic tests will be preform in R program (R 3.1.1)
Stable isotopes profile
Stable isotopes analysis is base on method as descripted in Borrell et al. (2011). To begin with, skin tissue will
be dried for 3 days and ground. Then, Lipids content will be removed by rinsing with 2:1 chloroform: methanol. At
the end, grounded sample will then be analysed in a continuous flow isotope ratio mass spectrometer. Result is
presented in delta which is calculated as *(

) + , where RS is the ratio of heavy and light

isotope in the sample and RR is the reference. After checking for normality, ANCOVA will be used to preform the
hypothesis test for difference in stable isotopes between Sexes and standard length.

Plankton composition
Plankton sample will be analysed under compound microscope for species identification and composition count
(Nelson 2007). Data from this experiment will first be test for normality. Then ANCOVA will be used as statistic
tool to test for significant difference in Plankton composition and abundance that can be explained by other
variables includes sex, size, appearance and maturity stage of whale shark observed.

Remote sensing data
Remote sensing data will be analysed simply by mapping the location where individual occur and look for
difference in movement and habitat use pattern between different genders.

Model synthesis
A simple occurrence model will be generated by mapping the probability of occurrence of different group of
whale shark in relation to diet reflect by the stable isotope (if available) and the plankton abundance and
composition using significant finding (if any) from prey identification. The model will then be test using the remote
sensing data from tagged individual in term of how much of the movement can be explained. Similar as the
testing performed in Sleeman et al. (2013).

8. Project work plan
Attachment X

Activity to be undertaken Est. start date
(mm/yy)
Est. finish
date (mm/yy)
Satellite-tagging, plankton Sample, environmental data, observation and skin
tissue collection
December
2014
March 2015
Stable isotopes analysis for skin tissue April 2015 April 2015
Plankton composition analysis April 2015 May 2015
Remote sensing data analysis May 2015 June 2015
Statistic analysis on correlation and model synthesis June 2015 August 2015
Preparation of final report August 2015 October 2015
Peer review and justification October 2015 November
2015
Submission December
2015


9. Project outputs

Expected outputs Date of completion (mm/yy)
One seasonal population structure survey in Christmas Island March 2015
Collection ~100 skin tissue sample from whale shark in Christmas Island March 2015
Characterisation of correlation between diet and intra-population segregation August 2015
Research publication December 2015

10. Technical and management capability
How will the proponent manage the project to ensure it will be successful in achieving the objectives listed in Question 5
and any outputs listed in Question 9? Include risk analysis and management.

Fieldwork will be conducted by a collection of experimented researchers, volunteers and tourist operators. All who
participate in the research will be trained properly pervious to the survey in terms of both technical and safety training.
Also, any risk management or safety requirement will be double check in advance pervious to the survey. Finally, during
the survey, there must be member with fist aid certification and proper safety equipment such as satellite phone on board

Lab work will be preformed in different laboratories: Plankton samples analysis will be conducted in laboratories own by
resident research group in Christmas Island using compound microscope. Stable isotopes analysis will be conducted back
to the University of Queensland due to the need of isotope ratio mass spectrometer for the analysis processes. All
laboratories will be conducted after safety induction under the occupational health and safety guideline and specific
laboratories guideline.

11. Project budget

(a) Provide all project costs taking into consideration increases in operation costs over the life of the project (e.g. petrol)
Note: All budgetary items are to be costed at GST INCLUSIVE prices. If the project is successful this work plan will form
the basis for the contract.

Item
(please specify each
item)
Australian Government
funding sought
Other contributions (provide
name of contributor)
Total project budget
Labour costs
- Salaried staff
- Volunteer labour (total
hrs x $15/hr)

$ 13,656
$ NA

$
$

$
$
Operating costs $ 15,800 $ $
Equipment costs $ 24,700 $ $
Other $16,000 $ $
TOTAL $70,156 $NA $70,156


Attachment X
(c) Justification for budget

Labour costs
We are asking for salary for a three-month full time employment of local expert on whale shark to allow effective
searching of whale shark. Considering the level of expertise, the salary level is consider equivalent to a new UQ
professional research staff (i.e.: $1707.04 fortnightly), the total amount will be for three month would be ~$13656.
Volunteer will not be paid in this project, instant, they will be provide accommodations, food and transport free,
the cost will be discuss below.

Operating costs

Aerial survey
Aerial searching of whale shark is one of the most effective ways to locate individual whale shark in open sea;
therefore, we are seeking funding for aircraft rent during the survey, the estimate cost will be $400/hr.
Estimate budget: $ 800 (2 hr / week) x 16 = 12,800

Boat trip will be conducted in a way cooperating with local tourist operators. Therefore, no budget is allocated on
covering the operational cost of boat trip.

Laboratory
In term of laboratory work, we are seeking SCAMMS funding to cover the cost on consumables such as
containers for skin samples and Plankton samples storing. It is estimate with a total of $3000.

Equipment
A range of basic equipment will be provided by UQ, however there are two types of equipment we ask for
ACAMMS funding, which is: 1. Laser photogrammetry that will be use for underwater measurement with estimate
cost $ 700. 2. 12 SPOT tags for Satellite-tracking, estimate to be $4000 each, therefore $4000x6 = $24000.

Other
Travel, accommodation, food
We are seeking SCAMMS funding to cover basic travel, accommodation and food cost for volunteers and
researchers during the field survey on Christmas Island, the budgeting would be $1000 for 5 people, therefore
$1000 x16 = 16000

University Overheads
The universitys policy on the Recovery of Indirect costs from Research Funding and Consultancy Contracts
(PPL4.10.12) indicates that the indirect cost recovery component on all research projects will be 60% of total
employment costs; plus 10% of all other costs





12. Permits/approvals

Does the project require approval from an animal ethics committee?

[ x ] yes
[ ] no


Note: All relevant ethics committee approvals and permits/approvals must be obtained by the proponent and provided
before work can commence.


13. References
Attachment X

Borrel, A., Aguilar,A., Gazo, M., Kumarran, R.P. & Cardona. (2011) Stable isotope profiles in whale shark
(Rhincodon typus) suggest segregation and dissimilarities in the diet depending on sex and size. Environmental
biology of Fish 92, 559-567.

Badshaw, C.JA., Fitzpatrick, B.M., Steinberg, C.C., Brook, B.W & Meekan, M.G. (2008) Biological conservation.
141, 1894-1905.

Department of the Environment (DEHP). (2005) Whale Shark (Rhincodon typus) Issues paper.

Hobbs, J-P-A., Frish, A.J., Hammanaka, T., McDonald, C.A., Giligan, J.J. & Neilson, J (2009) Seasonal
aggregation of juvenile whale sharks (Rhincodon typus) at Christmas Island, Indian Ocean. Coral Reefs 28, 577.

Hobson, K.A. & Wassenaar L.L. (2008) Tracking Animal Migration with stable isotopes, Terrestrial Ecology.
Elsevier Science Pulishing Co Lnc.

Holmberg, J., Norman, B. & Arzoumanian, Z. (2009). Estimating population size, structure and residency time for
whale sharks Rhincodon typus through collaborative photo-identification. Endangered Species Research, 7, 39-
53.

Jarman, S.N & Wilson, S.G. (2004) DNA-based species identification of krill consumed by whale shark. Journal
of fish Biology. 65, 586-591.

Jeffreys, G.L., Dorat, D., Marshall, H. & Broos, K. (2013) The development of robust morphometric indices from
accurate and precise measurement of free-swimming whale sharks using laser photogrammetry. Journal of
Marine Biological Association of the United Kingdom. 93, 309-320.

Ketchum, J.T. Magana, F.G. Klimley, A.P. (2013) Segregation and foraging ecology of whale sharks, Rhincodon
typus, in the southwestern Gulf of California. Environmental biology of Fish. 96, 779-795.

Martin, R.A. (2007) A review of behaveral ecology of whale sharks (Rhincodon typus). Fisheries Research. 84,
10-16.

Meekan, M. G., Bradshaw, C.J.A., Press, M., McLean. C., Richards, A., Quasnichka. S. & Taylor, J.A. (2006)
Population size and structure of whale sharks (Rhincodon typus) at Ningaloo Reef, Western Australia. Marine
Ecology Progress Series. 319, 275-285.

Meekan, M.G., Jarman, S.N., McLean, C & Schultz, M.B. (2009) DNA evidence of whale sharks (Rhincodon
typus) feeding on red crab (Gecarcoidea natalisI) larvae at hristmas Island, Australia. Marine and Freshwater
Research. 60, 607-609.

Nelson, J.D. & Eckert S.A. (2007) Foraging ecology of whale sharks (Rhincodon typus) within Bahia de Los
Angeles, Baja California Norte, Mexico. Fisheries Research. 84, 47-64.

Norman, B. M. & Stevens, J.D. (2007) Size and maturity status of the whale shark (Rhincodon typus) at
Ningaloo Reef in Western Australia. Fisheries Research 84, 81-86

Rowat, D. & Brooks, K.S. (2012) A review of the biology, flheries and conserveation of the whale shark
Rhincodon typus. Journal of Fish Biology. 80 1019-1056.

Sleeman, J.C., Meekan, M.G., Wilson, S.G., Polovina, J.J., Stevens, J.D., Boggs, G.S & Bradshaw, C.JA. (2010)
To go or not to go with the flow: Environmental influences on whale shark movement patterns. Journal of
Experimental Marine Biology and Ecology. 390, 84-98.

Attachment X
Stevens, J.D. (2007) Whale shark (Rhincodon typus) biology and A review of the primary literature. Fisheries
Research. 84 4-9.

Vignaud, T.M., Maynard, J.A., Leblois, R., Meekan, M.G., Vazquez-Juarez, R., Ramirez-Macias, D., Pierce, S.,
Rowat, D., Berumen, M.L., Beeravolu, C., Baksay, S. & Planes, S. (2014) Genetic structure of populations of
whale sharks among ocean basins and evidence fro their historic rise and recent decline. Molecular Ecology. 23,
2590-2601.


Please print this proposal and submit it with appropriate assessment cover page, by Wed 8 October 2014 to:

Help Desk
Goddard Building (Building #8)
School of Biological Sciences, University of Queensland.