Submission 009
Safer Waterways Bill 2018
Submission by Terri Irwin, Director, Australia Zoo 1638 Steve Irwin Way Beerwah Qld 4519
To: Committee Secretary Innovation, Tourism Development and Environment Committee Parliament House George Street Brisbane Qld 4000
Introduction
Like its previous iteration, the Safer Waterways Bill 2018 is a misnomer in name and intent. Rather than making waterways safer for people in Queensland, this Bill will increase the risk of more crocodile attacks on people.
Both the Introductory Speech and Explanatory Notes are high on emotion, anecdotes and misleading statements but short on data and research. Like the previous Bill, the Bill's authors have purported to have consulted widely but have made no contact with Queensland researchers who have carried out the world's longest continuous study of crocodilian behaviour - Australia Zoo and the University of Queensland.
In this submission, I will outline why this Bill increases the risk of crocodile attacks, correct the many inaccuracies in the Introductory Speech and Explanatory Notes and make a number of recommendations.
I would like to appear before the committee if my diary permits or alternatively request that another Australia Zoo representative appear before the committee hearing.
Background
Steve Irwin began crocodile research in the 1980s, and his capture and study techniques remain world's best practice to this day. Australia Zoo, in partnership with the University of Queensland (UQ) and Australia Zoo Wildlife Warriors, now manage the largest and most successful crocodile research project in the world, utilising these very techniques.
Each August, a team of crocodile experts, scientists and conservationists travel to the Steve Irwin Wildlife Reserve Nature Refuge on Queensland's Cape York Peninsula. Through research conducted it has been discovered that crocodiles can spend more than seven hours underwater; we've
Open everyday 9.00am - 5.00pm 1638 Steve Irwin Way Phone; +61 7 5436 2000 [email protected] Ciosed Christmas Day, ANZAC Day open 1.30pm - 5.30pm Beerwah Sunshine Coast Qld 4519 Fax: +61 7 5494 8604 australlazoo.com.au
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unlocked secrets regarding their diet, vital information on their movement patterns, and much more to aid in the conservation of these incredible apex predators. These findings have contributed significantly to the knowledge base of crocodilians, with a large focus of the project also being to educate those that share the crocodiles' habitat.
This world-renowned research involves:
• Tagging and tracking crocodiles in the Wenlock River with Acoustic technology, GPS Satellite transmitters-over 150 animals have been tagged • Monitoring crocodile behaviour, their movements and physiology • Vital research uncovering the distances crocodiles move, their ability to return to their home range after relocation and revolutionary findings on their ability to remain submerged, and their behaviour during flood events
• Australia Zoo's longstanding partnership with UQ dates back more than 15 years. With UQ Professor of Zoology, Professor Craig Franklin, and his team at the helm of the UQ scientific research team, the collaborative annual croc research trip with the Irwin family and Australia Zoo is going from strength to strength.
• Acoustic Telemetry is used to track the estuarine crocodiles in the Wenlock River. Once captured, an acoustic tag is surgically implanted into the crocodile. These acoustic tags send a signal to 50 receiving stations set up on the length of the Wenlock River and some surrounding water bodies. These signals are logged and when analysed enable us to discover how the crocodiles are using the river and interacting with each other.
• Another exciting part of our research on the Steve Irwin Wildlife Reserve Nature Refuge is isotopic analysis. Isotopic analysis identifies markers in bloods/muscle/bone to give us an insight into what makes up the natural diet of the estuarine crocodile and just how important their roles are in their natural environment.
• This is just the tip of the iceberg for us as there are many questions that remain unanswered. All this information is critical in learning how to successfully manage our wild crocodile populations, and most importantly, keep people safe.
• Each research trip to the Steve Irwin Wildlife Reserve Nature Refuge continues to break new ground in crocodile research globally and is central to managing the co-existence of crocodiles and people.
Crocodile Research Focus
As we move forward with our research, we hope will see us capture new crocodiles to provide additional data for the project and recapture crocodiles we've been following for the past 10 years. From recapturing crocodiles that have been tagged and living in the river, we're able to ascertain diet, examine environmental drivers for movement and behavioural patterns of Individual crocodiles with a focus on temperature, and deploy satellite-dlve transmitters to look at long-scale movements and diving behaviour.
We also aim to continue our research with other predatory species living in the river, which involves deploying acoustic tags in animals such as bull sharks, whip-tail rays, golden catfish, spear-tooth sharks and barramundi. We aim to eventually track over 230 animals in the river, and this will allow us to monitor them for the next 7-10 years.
Educating local communities
Each year, the Irwin family not only take part in the conservation and research work on Cape York but they venture into local schools in Weipa to conduct talks with the children, to educate them on how to safely live alongside crocodiles. The Irwin family also conduct community talks to educate Safer Waterways Bill 2018 Submission 009
and inform the local communities, it is the Irwin family and Australia Zoo's belief that individual culling and relocation are not effective ways to manage crocodile/human co-existence; rather, research and educating people are the key.
Misleading claims of Safer Waterways Bill
The Explanatory Notes and Introductory Speech make unsubstantiated claims such as:
• The Bill aims to eliminate from our waterways all crocodiles that pose a threat to human life, while protecting crocodiles from becoming endangered as a species. • Waterways, which people previously regularly swam in, are now infested with crocodiles. • From 1985 to 2015, the Department of Environment and Heritage Protection recorded 25 crocodile attacks in Queensland - seven of them fatal. • The Bill aims to create a significant and sustainable crocodile industry in Queensland through controlled egg harvesting. • The Bill gives power to manage crocodiles on their land and places a value on crocodiles and creates an unprecedented economic opportunity for the landholder. • The Bill allows people to pay to hunt crocodiles on private land with the landholder's consent if the landholder is a permit holder.
The Bill will increase risk of crocodile attacks
Through our research of crocodile movements and the probability of crocodile-human interactions, the removal of crocodiles, either through trapping or culling, will instead increase the likelihood of crocodile attacks as people become more complacent. It certainly will not eliminate them.
White the removal of dangerous crocodiles which have attacked or threatened to attack humans is justified, the removal of crocodiles will not limit the chances of further attacks and will create a false sense of security for residents and tourists.
Our peer-reviewed and published research, some of which is attached to this submission, notes that large male saltwater crocodiles (greater than 2.5m) can travel hundreds of kilometres in the six- month breeding season from September to February in search of unattached females. (Home Range Utilisation and Long-Range M ovement of Estuarine Crocodiles during the Breeding and Nesting Sea so n , Campbell, Dwyer, Irwin & Franklin, 2013) The dominant males tend to remain close to their breeding females which also have localised movements. Therefore, crocodiles removed from the waterways are easily and quickly replaced by roving crocodiles in search of partners.
Translocating crocodiles presents even more danger of crocodile attacks - and not because relocated crocs become more dangerous as stated in the second reading speech. As our ground breaking experiment found in 2004 that when a 4.5 metre male crocodile, was relocated from the west coast of Cape York to the east coast of the Cape, it swam back to its original location - a distance of over 400km in under 20 days.
In further research (Predicting the probability of large carnivore occurrence: a strategy to promote crocodile and human coexistence, Campbell, Dwyer, Wilson, Irwin & Franklin, 2014), the probability of human-crocodile contact increases between September to December, at night and during a high tide.
Crocodiles are highly effective ambush predators and perfected this technique over millions of years of evolution. In estuarine environments, crocodiles are virtually undetectable underwater. Research Safer Waterways Bill 2018 Submission 009
[The intrinsic properties of an in situ perfused crocodile heart, Franklin & Axelsson,1994} has shown crocodiles can slow their resting heartbeat to enable them to stay underwater for periods of up to seven hours to enhance their ambush abilities.
Based on the evidence provided, it is clear that simply removing crocodiles will not mitigate the probability of attacks on humans and the best course of action is for people in crocodiie territory to be "Croc-wise" and take sensible steps to minimise human-crocodile interaction
Accurate, long-term surveys of crocodile numbers needed first
While such highly emotive words as "infested by crocodiles" and "explosion in crocodile numbers" are included in the Bill's supporting documentation, no data is included to justify these claims and therefore justify the need for crocodile relocation, culling or egg harvesting.
A detailed population count of estuarine crocodiles in Queensland has not been carried out in the past 20 years and Australia Zoo welcomes the Queensland Government's action to carry out a survey of crocodile numbers this year. Therefore, it is surprising that the Member for Dalrymple said, in his Introductory Speech, that "people are sick and tired of hearing about more studies or more counts" when such research has not taken place for decades.
Australia Zoo believes, as a minimum, that any action on Queensland crocodile numbers should await more crocodile surveys over several years so an accurate trend in population growth or decline can be ascertained, This is a species that was on the verge of extinction in the 1970's, Changes to legislation to ban hunting was their saving grace. In reality what we are seeing now is a recovery of a critically endangered species. What we don't know is how well it has recovered and how fragile populations may still be. The research is critical before making any changes.
Critical use of statistics
As noted Scottish poet and novelist Andrew Lang wrote in 1910; "Politicians use statistics in the same way that a drunk uses lamp-posts—for support rather than illumination."
While it is true that crocodiles can be very dangerous creatures and there have been 25 crocodile attacks in Queensland over a 30-year period from 1985 to 2015, these figures have to be put into their proper context. In many cases, these attacks could have been avoided through sensible precautions, including a recent one when a teenage boy jumped into a known crocodile habitat to impress a girl and literally jumped on top of a waiting crocodile!
It could be argued that crocodiles are not the most dangerous native animal in Australia if attacks and deaths are used as the only measure. The most dangerous native animal based on human deaths over a 10-year period are kangaroos with 18 deaths from 2000-2010 (National Coronial Information System Factsheet 2011) with 15 of those deaths related to motor vehicle accidents. For deaths based purely on native animat attacks, bees and sharks are the highest with 15 deaths. As far as I'm aware, there have been no calls to cull bees. Crocodiles rate eighth on the NCIS' overall list of dangerous animals in Australia with nine deaths. The top three are horses (77 deaths), cattle (33) and dogs (27), These domesticated animals accounted for 54% of all deaths caused by animals from 2000-2010 in Australia. In fact, the same number of people killed by crocodiles (9) died from tripping over their dogs. We are not trying to belittle these deaths and the tremendous impact they would have had on the affected families but it is a demonstration of how statistics can be used to illustrate a certain point. Safer Waterways Bill 2018 Submission 009
Any increase in crocodile attacks could also be attributed to increased human populations and increased tourism in north Queensland with no increase in education programs, however, more research needs to be undertaken to ascertain the true origins of increased human-crocodile interactions.
As I wrote earlier, if sensible "Croc-wise' precautions are taken - principally, not entering water known to have crocodiles - crocodile attacks can be minimised and most likely, entirely stopped.
Crocodile-egg harvesting
No evidence is given to support the claim that a "a significant and sustainable crocodile industry in Queensland" can be created through croc-egg harvesting. The only reference to evidence is that: "Evidence su g g e s ts egg harvesting can help sustain crocodile populations rather than diminish them
In most arguments in favour of croc-egg harvesting, Northern Territory is held up as an example of sustainable croc-egg harvesting. However, it is misleading to compare Northern Territory crocodiie populations with Queensland populations and the histories associated with egg harvesting.
Through the remoteness of crocodile populations and comparatively less human impact, especially crocodile hunting. Northern Territory would have had a larger, stronger and more diverse crocodile population base when crocodile hunting ceased. In addition, there have been ongoing crocodile population surveys in the Northern Territory to monitor the size and viability of the Northern Territory population,
The same could not be said of the Queensland crocodile populations which have had to start from a lower base and no long-term surveys have taken place. Without long-term population surveys and scientifically robust research Into the effects of egg harvesting, no-one can claim that egg harvesting is "sustainable". The habitats are simply not comparable as they are vastly different In nature. There Is far less suitable habitat for crocodiles In Queensland- there probably always have been, but with increasing pressure around estuaries many of the once prime areas on the east coast are now densely populated by people.
Landholders and shooting safaris
Just because someone owns land in crocodile territory or have lived in north Queensland all their life doesn't necessarily make them an expert in crocodile behaviour, management or egg harvesting.
Over more than a decade, Australia Zoo and UQ researchers have devoted thousands of hours in research into these magnificent animals and we are still discovering amazing facts about them. We have refined the techniques of capturing crocodiles by monitoring their behaviour over many years - yet this Bill proposes that anyone with a permit can capture or shoot crocodiles. It Is a recipe for disaster and will increase the number of attacks and deaths caused by crocodiles by increasing contact with crocodiles by people with limited experience.
Additionally, Injured animals become more dangerous. If they are struggling to catch their natural diet due to being shot, they may become dependent on other food sources which In turn can increase the human-animal conflict. This was demonstrated by a crocodiie called 'Nobby'. Steve Irwin had been asked to capture this animal as it was noted as a potential problem to domestic Safer Waterways Bill 2018 Submission 009
animals on a private property. Upon investigation, it had actually been scrounging from a dump area and when captured, it was missing half of its lower jaw. This injury was clearly the cause for the animal to be moving in and around a homestead area and the type of scenario that would likely increase with any implementation of hunting.
Let's put this statement from the Explanatory Notes - "The Bill legalises... and allows people to pay to hunt crocodile on private land with the landholder's consent" - in its true context. It is a very unsubtle move to legalise crocodile hunting safaris. Not only does this Bill allow for landholders with little or no training to cull crocodiles but allows anyone with enough money to indiscriminately shoot crocodiles and possibly result in long and painful deaths for some of these animals. How can a small Authority which will be funded entirely from within OEM's already meagre budget be expected to regulate and enforce legalised shooting of crocodiles without wholesale slaughter occurring?
This Bill does place a value on each crocodile but for all the wrong reasons which will only hasten the decline in crocodile numbers. Under this Bill, greater value is placed on a dead crocodile or one that will be hatched and spend its life in the brutal conditions within crocodile farms. I would urge Committee members to closely inspect the conditions of these farms when you carry out your consultation.
From my experience, these farms are hideously cruel. The hatchling crocodiles are kept in tiny dark boxes with loud rock music playing, 24/7. They only see daylight for a moment when they are thrown a bit of food. Apart from the "high value" crocodiles whose leather will be used to make expensive handbags for rich people - the other older crocodiles go into an overstocked enclosure with a large number of crocs resulting in jaws, limbs, and parts of tails being torn off.
As far as the money generated, most of the focus is on the high end of the market - not the croc farm or the local Indigenous community. I have always wondered why so much effort is put into 2% of our leather market. We are helping a few rich multinationals, not Australians. This also enables an illegal trade with crocodilians. Short of a DNA test on the leather, there is no way to tell if it is a legally processed crocodile skin or not.
There is only one line in Explanatory Notes with which Australia Zoo agree with - "If managed responsibly, crocodiles have the potential to be a great asset for Queensland, rather than a danger to people". Countries in Africa generate billions of dollars through non-consumptive wildlife tourism. Therefore, more revenue can be generated for local communities through nature-based tourism with a higher price placed on the head of each live crocodile. It directly counters the Member for Dalrymple's argument and this so-called "explosion" in crocodile numbers will only have a positive impact on international tourism.
Legal status of native wildlife
Under federal and state legislation, native wildlife is protected which means they are effectively 'owned' by the state. Further, waterways below the high-water mark are the responsibility of government. Landholders, therefore, do not own any wildlife, especially in waterways, and therefore, have no rights to trade in their products or organise shooting safaris without substantial changes to environmental legislation and the underlying intent of these pieces of environmental legislation.
If human safety is the driving motivation for this Bill, then why not extend this legislation to horses, cows and dogs which also contribute to loss of human life. Safer Waterways Bill 2018 Submission 009
Role of apex predator
Researchers have only recently turned their attention to the role of apex predators, like crocodiles, and the effects they have on biodiversity and the health of ecosystems. It has been found in all case studies of numerous apex predators that their re-introduction into ecosystems have led to substantial improvements in the overall health and biodiversity of these ecosystems.
Predators play a key role in maintaining ecosystem integrity in terms of species and genetic composition, ecosystem functions, and long-term stability. Through a process, known as trophic cascading, apex predators in a food web suppress the abundance or alter the behaviour of their prey, thereby releasing the next lower level from predation.
Studies by the Oregon State University, over 50 years, have shown the re-introduction of the wolf has dramatically improved the biodiversity of Yellowstone National Park. Wolves were able to reduce the populations of elk which in turn gave willows and other trees a chance to take hold along streams, cooling water temperatures for trout and encouraging the return of beaver, whose ponds host long-absent amphibians and songbirds. The Yellowstone Park example proved that damage to a terrestrial food web could be reversed and an ecosystem restored with the return of a single species.
Whiie no definitive research has been carried out on the direct impact of crocodiles and biodiversity, anecdotal evidence has shown that where there are healthy populations of crocodiles, there are an abundance of barramundi and other fish species.
Australia Zoo tagging of other species in the Wenlock River will provide useful insights into the health of one waterway which is "infested” with crocodiles.
Human safety in Northern Territory
It was made patently clear in the rhetoric of the Explanatory Notes and the introductory Speech that human safety is the primary driving force behind this Bill. Supporters of this Bill will point to the Northern Territory as a defining example of why this Bill should be passed as NT crocodile management has many of the same elements of this draft legislation. Apart from allowing croc-egg harvesting (70,000 per annum), NT allows the removal or harvesting of problem crocodiles and has an extensive crocodile farming industry but faiied in its bid to introduce crocodile hunting safaris due to restrictions under the federal government's Environment Protection and Biodiversity Conservation Act 1999. Additionally, NT allows the removal of up to 500 hatchlings, 400 juveniles and 500 adults. (Management Plan for Saltwater Crocodile in the Northern Territory of Australia 2014-15} So potentially, permit holders in NT can legally remove up to 71,675 crocodiles (including a five-year average of 275 problem crocodiles).
With the removal of such an enormous number of potential predators, one would think the number of deaths and attacks caused by crocodiles would have fallen in NT in recent years? The opposite has happened. A study by the National Critical Care and Trauma Response Centre, Royal Darwin Hospital and the Menzies School of Health Research has found 14 people died from croc attacks between 2005 and 2014 in NT, compared with 10 deaths in the 33 years to 2004. Large scale removal of crocodiles in NT has not reduced crocodile-related fatalities, so why repeat it in Queensland and expect a different result? Safer Waterways Bill 2018 Submission 009
Recommendations
1. That the current State Government funded crocodile surveys be continued and enhanced over several years to gain an accurate picture of crocodile populations. 2. That no interference with crocodile populations either through culling or egg-harvesting be undertaken based on our research conducted with UQ. 3. That State Government research into croc-egg harvesting be undertaken to ascertain its true impact. 4. That the State Government explore nature-based tourism ventures for Indigenous communities. 5. That the State Government invest in research to explore the important role crocodiles play in creating healthy waterways. 6. That a comprehensive Croc-wise campaign be funded for Queensland and international markets to ensure crocodile-human interactions are minimised. (Having a few notes on a State Government website and croc warning signs will not work in the long term.) 7. That the Safer Waterways Bill 2018 be rejected in its entirety as it is a piece of poorly researched and poorly drafted legislation which would have devastating consequences for both crocodiles and humans if enacted.
Terri Irwin AM
Director-Australia Zoo Safer Waterways Bill 2018 Submission 009
OPEN Q a c c e ss Freely available online PLOSI
Home Range Utilisation and Long-Range Movement of Estuarine Crocodiles during the Breeding and Nesting Season
Hamish A. Campbell^^ Ross G. D w y e r\ Terri R. Irwin^ Craig E. Franklin^ t The School of Biological Sciences, The University of Queensland, Brisbane, Australia, 2 Australia Zoo, Steve Irwin Way, Beerwah, Australia
Abstract The estuarine crocodile {Oocodylus porosus) is the apex-predator in waterways and coastlines throughout south-east Asia and Australasia. C porosus pose a potential risk to humans, and management strategies are implemented to control their movement and distribution. Here we used GPS-based telemetry to accurately record geographical location of adult C. porosus during the breeding and nesting season. The purpose of the study was to assess how C porosus movement and distribution may be influenced by localised social conditions. During breeding, the females (2.92iQ.013 metres total length ffL), m ean ± S.E., n=4) occupied an area<1 km length of river, but to nest they travelled up to 54 km away from the breeding area. All tagged male C porosus sustained high rates of movement (5.49±0.9 km d~'; n = 8) during the breeding and nesting period. The orientation of the daily movements differed between individuals revealing two discontinuous behavioural strategies. Five tagged male C. porosus (4.17+0.14 m TL) exhibited a 'site-fidelic' strategy and moved within well-defined zones around the female home range areas. In contrast, three mates (3.Bl±0.08 m TU exhibited 'nomadic' behaviour where they travelled continually throughout hundreds of kilometres of waterway. We argue that the 'site-fidelic' males patrolled territories around the female home ranges to maximise reprodurtive success, whilst the 'nomadic' males were subordinate animals that were forced to range over a far greater area in search of unguarded females. We conclude th at C. porosus are highly mobile animals existing within a complex social system, and mate/con-specific interaaions are likely to have a profound effea upon population density and distribution, and an individual's travel potential. We recommend that impacts on socio-spatial behaviour are considered prior to the implementation of management interventions.
C ita tio n : Campbell HA, Dwyer RG, Irwin TR, Franklin CE (2013) Home Range Utilisation and Long-Range Movement of Estuarine Crocodiles during the Breeding and Nesting Season. PLoS ONE 8(5): e62127. doi:lO.1371/journal.pone.0062127
E d ito r: Michael Somers, University of Pretoria, South Africa
R e ce ive d July 11, 2012; A c c e p te d March 19, 2013; P u b lis h e d May 1, 2013
Copyright: 2013 Campbell et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
F u n d in g : This study was supported by the Australian Research Council linkage scheme with Australia Zoo as industrial partners, and funds donated by the golfer Greg Norman. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: [email protected] introduction between river .systems [14], stich a large growth in population i.s likely to be altering the dynamics of the wider community and Animals gcncTalJy confine their movements within discrete ecosystem [3j. 'i'his will occur not only by the consumption of' areas. The size, placement and shape of the activity space has been lower trophic animals but also through die alteration of prey termed die home range, and reflects the animals' behavioural species’ behavioural ecology [3J. repertoire as it searches to procure food, shelter, and mates [l|. Cromfy/u-i /m m iis are generally considered to be highly territorial For m any species .social conditions influence the size of the hom e animals, with dominant males excluding con-specifics from their range, and consequcntlv, the abundance and dislribuiion of the home range [15]. More recently however, telemetiy studies ha\e population. L'ndei'Siandmg homo range dviiamici is essential for recorded large adult male C. fiorotus li\ ing in close proximitv' to the pragniaiic managcmeni of any species [2J, but is particularly each odicr, thereb)’ refuting prciious claims of C. p m s/u as an Important in managing top predators because of their influence exclusively territorial species [B,1G]- Understanding which olThese upon losver trophic le\cls f.'h4,.5]. social conditions is must apparent is of profound importance The estuarine crocodile is the apcx-predaior towards managcmcni because the formin' would result in social in its eiivironment and will feed upon a variety of prey items [6]. condition.s altering population densiiy. dispersal, and distribution The species has a vsidc disiribuliou across norlhem Australia, whilst the latter would not. occurring in coastal areas, estuaries, rivers, inland swamps, Prc\ ioLis estimates of home range upon b'. pomiij. ha\'c relied billabougs. and off-shore islands |’7,8.9]. Unlike a vast majority upon cither visual sightings or the manual collccdoti ul’ location of the world’.s ape’C-predatnrs. the .\ustralian population data via \ ’HF-radio-telomctry [BJfi]. We suggest that the home has undergone significant growth n\ er the last 3d yeans. Once in range e.stimates of these, studies may hax'c been biased by serial risk uf imminent extinction the eurreni Australian population is autocorrelation because temporal irregularities occui'red in the estimated to be greater than 75 000 non-luitchling individuals period between location Irxing |17|. Furthermore, these .studies 110.! 1.12.13|. .\lthough the population density varies considerably
PLOS ONE I www.plosone.org May 2013 | Volume S [ Issue S | e62127 Safer Waterways Bill 2018 Submission 009
Movement and Home-Range in Estuarine Crocodiles
and others upon crocodilians have defined tlie home range using l3xTG M 410, Tclonics, .Vrizona, U.S.A.f The OPvS-units were mid-stream linear distance or the minimum cr>nvex polygon secured onto the dorsal surface of the crocodiie with aluminium method [18,19]; whilst these techniques provide a measure of the crimps threaded onto the stainless steel wire (Fig. 2). The process full extent of the area visited by an individual they ignore patterns of remo^'ing the crocodile from the trap to eventual release took of selection within the home range. This is important if we are to appro.ximately 60 min. The crocodiles were released at the point assess the difference between an individual’s daily usage of an area of capture. To a\ oid any bias in crocodile behaviour occurring compared to an. area that is merely passed through or only from the baited traps or Increased boat traffic during the trapping frequented occasionally. In oi’der to make this assessment, kernel period, only GPS-based location data obtained after 01 September utilisation distributions fKL'Ds) arc convenient analytical tools, were used In the final analysis. because tiiey calculate density based upon the entire sample set of relocations during the period of interest rather than the emphasis Data analysis being on the most outward location points f20|. ft was the aim of The devices utilised the global-positionlng-sysiem of satellites to this study to use KUDs to as.sess the relationship het\veen daily determine geographical location twice daily (0800 h and 1800 h). movements and area utilisation distribution in male and female C. The location data were stored on board the unit and parsed to the porouu. We selected to moiiitar the crocodiles during the breeding ARGOS satellite system betvveen 1000 h-1600 h every' other day. and nesting .season (September - February) as the effects of social For each of the GPS-based location llxes. the accompanying conditions upon movement and space-use were expected to be satellite dilution of precision (SDOP) \'alue was used to define the most apparent during these periods. positional resolution and precision. Stationaiy logging tests (7 d) To apply kernel utilisation distribution plots it is important tn prior to the study tvere used to pre-determine the a^•erage de.gree collect accurate location data at a sufficiently high freciuency and of error lor each GPS-unic. .\il units performed eqmilly and an regularity [‘21]. To achiese this, tve utilised high preci.sion global SDOP of^3 had a mean accuracy of error 12.1 ±1.1 m. All positioning system-(GPS) based telemetry data-loggers. which had location ILxes with an SDOP^3 were excluded from the final an inbuilt capacity to parse the collected location data through the analysis. .-kRCIOS satellite system. In the light of previous telemetry studies Fo assess h o m e ran g e size, we a d o p te d the ILxed kernel (FK.) u p o n C. fjoroiUi, [0,16], we hypothesised that there would be method [21]. Kernel density estimators are known to be sensitive profound diiferenccs in space-use between molc.s and females and to their choice of the .smoorhing parameter 7z) [2.3]. The least- the home ranges of individuals would overlap within and between squares cross validation (LSCV3 method has been suggested as the the sexes. Furthermore, due to the high temporal resolution and most accurate way of estimating the appropriate .smoothing spatial accuracy of the GPS-based location data, we suspected that parameter [25], it was not however suitable for the present study new insights into crocodile movement, interaction, and space-use because it resulted in the delineation of numerous .small di.sjunct would also i^e revealed. coniQurs, excluding connecting stretches of river. A second cnmmoniy used smoothing estimator, the reference bandwidth Materials and Methods method [26]. resuited In large areas beyond the outermost locations being included in the utilisation divSlrihutions. To em ure Study site and animals a contiguous home range boundaiy extending throughout the Trapping was conducted on the \VenIock River, Cape York length of the ri\'cv and accurately represent the outermost Peninsula. Australia during August 2010 (Fig. 1). A field camp wa.s locations, we selected a smoothing parameter o ( k—lbO m. For run from the Steve Irwin Wildlife Reseiwe (142.18''N, — 12.38^E). each individual, the 95% and .30% v^olume contour of the KUD The trapping occurred from the freshwater tidal reaches of the diereafter the KL'D Oo*!'© and KUD 50%. respectively) were rh'er down to the macro-tidal brackish water, between 20 and determined using the ’adchabitatHR’ package [27] implemented 60 km from the river mouth. The bank vegetation in the lower in the statistical software R [28]. To examine temporal variation in reaches of the crapping zone was mangrove palm {\ypa finticaiis) home range use volume contours vvcre constructed for si.x time changing to MetaUiica dominated forests. It has been suggested chat periods (01 September—30 September, 01 September—31 October. out of all the rh'er systems along the we.sterii side of Cape York 01 Septcmbcr-30 November, 01 September—31 December, 01 Peninsula the Wenlock system pro\'idcs the most suitable nesting September-31 Januaiy and 01 September-28 Febrnaiyh Croco habitat for estuarine crocodiles [22]. dile movcmeni was constrained within the river channel, and A d u lt CrocodyliLi poroMis (males = 3.91 ±0.14 m total length. therefore, rhe area produced by the FK-mcthod was considered meaniS.E. n~8; females - 2.93±0.13 m total length. n = 4' over-represeniuthe of the actual area utilised by C. puronts. wci‘e captured bets\ecn the non-ddal freshwater reaches of the Stretches of river intersecting the volume contours were con.sc- Wenlock River through to the macro-tidal brackish (Fig. 1). Fhc C|uently extracted to ensure diat habitat inacccs.sible to C. porfl.){c> traps were Qoatcd on the water surface or placed at the water edge were noi included in the final home range esliinalcs. A high along the river bank. Each trap was baited with wild pig [StLS scrofa] resolution spatial polygon of the Wenlock and Duric Rh'cr and the trap door was sprung by the crocodile when pressure was catchment was constructed using sa tellite Imageiy data (Fig. 1) and applied to the bait, via a trigger mechanism [23]. Gncc captured, conwncd to a .30x30 m raster object using /XRCGIS 10 (ESRI, crocodiles were removed from the trap and mamiaily restrained. Redlands. California. UhS.Af Areas of riv'er contained ^rithin the Total length (TL) and snoiit-veiu length (S\T, mcastiremcnls were KUD 95% and KUD 5U%, and the corresponding centroid within taken and a local anaesthetic (5 ml of lagnocaiue. Troy the KUD .30%. \vere obtained using functions contained within laboratories. Sniitlifield. Au.stralia) was injecied under rhe nuchal the 'sp' [29]. 'rgdaf [30] ancl 'rgcus' [31] R packages. This river rnscTte. O n ce the an aesth esia had taken effect, a .singie hole wa.s intersection method reduced the KUD 93% by 90.7±4.1% and drilled in each of the tour raised osteoderms of llie nuchal rosette the KUD 50^0 by 71.4±3.2%. j24| Stainless steel mulii-strand, plastic coated wire i80 kg To explore the liner-scalc niovenicnts in taggcfl purusns^ two breaking strain! was inserted through the drilled holes and laced measures of directional movement were inve.stigated. The first into attachment pomes on the GPS-ba.sed satellite transmitter (in measure, the distance moved fi'om the KUD 50% centroid rluring 2009 5xGPS units Sirtrack, Hamilton. New Zealand: in 2010. the j^eriod Oi September—30 September, would reveal rxplorntory
PLOS ONE I www.plosone.org May 2013 I Volume 8 | Issue 5 | e62127 Safer Waterways Bill 2018 Submission 009
Movement and HomS’Range in Estuarine Crocodiles
t4 5 ’0'E
H 6 ] F4 20'^0'S M1
M 3 i F3 M8 M4
M2 25"0'S M7 QLD
30=0'S
NSW Sydney / ' iKm
Figure 1. The Wenlock and Ducie River, Cape York, QLD, Australia.The capture locations of each Crocodylus porosus tagged for the study are displayed. doi:10.1371/journa!.pone.0062127.g001
movements from the centre of the home range. The second included in our modcL Analysis was undertaken ittt dtatistica 10 measure, the minimum, distance bemveen two locations in series, (StatSoft Tnc, Tulsa, USA) and P<{).05 was con.sidered significant. would reveal periods of activity. As crocodile movements were limited by the trajecturv' of the river, the minimum distance mo\ cd Results between two locations was calculated along the trajecLoiy uI the river using the ‘raster’ [32] and ‘gdisiance' packages [33] in R. d’he majority of the crocodiles tagged in this study remained within the Wenlock River for the duration of the study, but one A general linear mixed model iCrLMM^ was used lu assess Lhe male travelled to the adjacent Ducie River system, and some males induencc of body size and sex on movement patterns in C. porosus. and females moved into seasonal creeks located far upvher. Daily rale of inovcineiu (ROM) was included as the respunsc [.ocation data were collected ivvice daily for eight male and four variable, with days from 01 SepLcmber (date) and body mass fem ale ('. porosus from the 01 September 2010 until the 28 (extrapolated from SV’L using the conversion lactors in [3+] as February 2011 (Table I). 7.11:0.4% of location fixes did not have covariates, sex as a factor, and crocodile TD as random cfTcct. A a sufTiciently low SDOP for inclusions in the analysis and were second model assessed the relationship benveen the daily distance therefore removed from the analysis. each individual wa.s located from the centroid of it.s KL'D .>0%. witJi date and body mass as cov'ariates, sex as a factor, and Male Movements crocodile ID as random effect, Dttc to Ihc correlation between I'he application of kernel density estimators to the location data bodv mass and sex the interaction betw'ccn these variables was and calculation of the cuinulath’e home range illustrated that the movement patterns of the eight males could be gi'ouped Into two discrete categories. The ‘nomadic’ males (n = 3) were clclined by the fact they did not denion.strate a stable KUD 95% during the 6- m
PLOS ONE I www.plosooe.org May 2013 | Voiume 8 | Issue 5 j e62127 Safer Waterways Bill 2018 Submission 009
Movement and Home-Range in Estuarine Crocodiles
Tabfe 1. Summary statistics for four female and eight male Crocodylus porosus tracked by GPS-based telemetry between 01 September 2010 and 28 February 2011.
Total Body Length Total distance Max distance from C ro c ID (m) Day ROM (m/h) Night ROM (m/h) moved {km} KUD 95% {km^l KUO 50% (km^) c e n tro id
M l 3.2 301 352 S34 34.2* 6,6* 73.2 i
M 2 3.7 153 428 1054 42.1* 6.4* 69.2 M 3 3.9 24S 373 1269 72.5* 4.7* ___J M 4 4 3 290 589 1179 9.0 4.2 19.2
MS 3.9 67 Its T73 7.3 3 5 12.2
M 6 3.7 84 243 197 8.7 3.6 27.9
M 7 4.1 166 447 954 1 0 5.1 9.6 ]
M S 4.5 200 270 324 7,1 4.2 8.07
FI 3,0 123 S6 25S 12.3 3.9 54.3
F2 2.9 17 39 127 7.2 2.2 54.8
F3 3.2 34 92 165 4.9 0.8 33.!
F4 2.6 27 23 154 1.1 0.5 22,5
^indicates that the monthly kernel utilisation distrrbutlon (KUO) had not stabilised by the end of the study.
hundreds of kilometres und on a\-eruoe triivelled l02.6± lr0.B kni located away from the KUD 50% centroid (Fig. 5b). FI iravtdled Irorn the KUO 30% centroid Fig. 3bj. The mean total-lcngth of upriver whilst F2, F3 and F4 travelled dov\ nriver. and within a 21- the 'nomadic' mules was 3.6±0.2 m (mean ± S.E.h h period all females were located a considerable distance from the As the name implies the 'site-fidelic' males (n = 5) e^thibited a KUD 50% centroid, ft seemed logical due to the timing that these stable RL'D 50% in which they confined their inovemenLs during long-range movements exJiibited by the females were towards the 6 months of study IFig. 4aj. The KUD 95% and KUD 50% nesting areas. F2, F3 and F4 remained at the new location lur less were comparable across tlio group (Table and the KUD 50% than 48 h before travelling back to the KUD 50% centroid within comprised a large cnmponent of the total KUD 9^3% a 24-h period. They remained within their original KUD 50% for (48.1 ±2.8%). There was o\criap in the KUD 50^^4i between U2 weeks bd'orc undertaking the same journey back to the males, but this was never greater than 47.1% (35.1 ±6.1% nesting location. Once at the nesting location for the second time, inean±S.E., n = 8). The 'site-fidclic’ male.s moved a niiniinum they remained there until the end of the .study '28 Febniaty). Fl river disUince of 334.4±o3.7 m h ” ' during darkness, decreasing to did nut show this repetitive movement and undertook a single 16l-4±4l.2 in h~' during daylight hours (Table 1). .Although the long-distance mo\'ement in January, remaining at the new^ location average hourly rate of movement for the 'site-fidelic’ males was less until the end of the study. than exhibited hy the 'nomadic' males, there wa.s no significant difference in the daily distance tras elled between the two groups GLMM tliroughout the study 'ffable 2). 4’hc 'site-fidelic' males moved back The general linear ntixcd ellecis model showed that body mass and forward within their home range and therefore the daily had no significant effect upon the daiK rate of movement (ROAF; distance they were located away from the KUD 30% centroid or the river distance an individual was located away from it.s KUD closely matched the daily rate of movement (Fig. 4b). The 50% centroid (Table 2). Sex did ha\'e a siguiiicant effect upon maximum river distance the 'site-lidelic'' males were located away ROM but not dUlaiice from the KUD 50% centroid and date had from the KUD 50% centroid averaged 15.4±3T km for the group a significant elFcct upon both ROM and distance from the KUD (Table 1). The mean-total length of the site fidelic males was 50'% centroid. C rocodile ID ex erted a significant efTecl w ithin the f. I ±0.18 m. model upon both ROM and distance from die KUD 50% centroid, but classifying males into either hiomadic’ or Ahe-fidelic’ Female movements groups accounted for the signifit:ant elfect of crocudilc ID The four tagged female (•'. were of a similar size range .F| .j = 67/U F<0,01). and were smaller than liie lagged males (Table I). .All fem ales occupietl the main trunk of the river and exhibited a defined KUD Discussion 93% that was stable between Ul September and 01 December (Fig. 5a). i'he KUD .50% of two females o\erlapped at 324 anrl Male movements 34.4% area, whilst the otiier two lemales heid di.screte KUD 50% U'e recorded two distinct behavioural tacdc.s exhibited by m close proximity. The daily rate of movement for females was tagged male pn m ti\ throughout the si.x month study. The flaily much lower than recorded for the males (nighr = 52.5± 13.4 m rate of movement was not significantly dilTcrcm between groups h”\ daylight = 50.3±22.2 m h~' tiiey did not exhibit the exhibiting either beha\ioural tactic, hut the temporal directionality male preference Ibr nocturnal activity (4’ablc f). of movcmern defined each group. Males exhibiting a nom .idic’ During Dcccmijcr and January, each female showed .m lactic ranged throughoiU die \\'enlock and Ducic Ri\ er calch- approximate 30'% oxpan.sion of their KU4) 95'h>. This increase naents; their movement along the river were t\pically unidirec in the KUD 95^n and KUD 50% was due to a sharp increase in tional upon cnnscruiivc day.s and coniined only by rhcr daily activitv and a lengdiening of rhe distance rlie female was geogniphv. In contrast, male.s exhibiting a ‘.site-fidelic’ lactic
PLOS ONE I www.plosone.org May 2013 | Volume 8 j Issue 5 j e62127 Safer Waterways Bill 2018 Submission 009
Movement and Home-Range in Estuarine Crocodiles
B
60 r Ml
TJ P 60 - Crocodlla ID E o cc 142* P 20
O 0) CC ^ Q -n ll
Figure 3. Movement patterns of 'nomadic' male Crocodylus porosus.{a) GPS-location 1^xes obtained twice daily between 01 September and 28 February (n = 3). Inset Ime graph shows the me^ " cumulative KUD 95% for each individual, (b) The relationship between daily rate of movement (ROM) and daily distance from the KUD 50% centroid {grey = primary y-axis; black = sec o n d a ^ y-axis). doi:10.1371/journal.pone.0062127 g003 confined their movements within a discrete stretch of river. Fach strongly reflective of the Tighting’ or 'sneaking' alternative individual male maintained its selected behavioural tactic reproductive tactics often displayed within polygamous mating throughout iht: breeding and nesting sctison. systems [38,39]. That is, dominant males maximise their The patterns of movement recorded by CrPS-bascd location reproductive succe.ss by defending mating rights with co-habiting fixing and defined by RUD home range analysis strongly reflected females, whilst subordinate males maximise their rhance by territorial patrolling beha\'iour and mate-defence [35,3b]. fagged biieaking’ copulations with unguarfled females. Further support eon-.spccilies were located imidc the honie range of the ‘site-lidelie* for this theorV'' in C. porosus populations comes from the genetic male.s. but the rate of movement of these individuals would have analysis of eggs collected from nests in the: wild, which showed resulted in them passing through the home range quickly, and the muitiple-patcniity is widespread with some clutches luuing mure lack of total exclusion may simply be a function of the large home than two sires |40|. range area and the high mobility of the eon-spcciiics. It is likely A surprising obsenation that contradicts much ol the lilLTaiure that the 'nomadic' males passed through the territories of many jfi.1.5] was the sustained high daily rates nfm o\‘emenr exhibited by other uiuagged ‘sitt;-fidelic’ males during this period. all the tagged jmiosfis. Even the sitc-fidclic males travelled The prrsnu study was undertake’!i during the breeding and hundreds of kilometres during the study, albeit w ithin a discrete lie s ling season and all tagged males would ha\e been of area, 'frauslocated male porona hat’c been pret iously reported reproducliie age. Body-size is a good surrogate of social status CO ha\‘c travelled over hundred.^ of kilometres In a quest to return in C. fm om s [37]. and although beha\ioural strategy- was not home [16,H.12J. These were however, considered extreme rates .significantly .segi'egated by size in this .study. \se argue that it is the of movement, undertaken hy the individual only becau.‘:e of the most likely detei'ininate between a 'nomadic' or a 'site-lidelic’ manipulated conditions and a sti’ong homing instinct. On the lifestyle. Cenainly. the dichotomy of inoveniciit patterns ^vere eoiurary. high [reqLU'nc\ CrPS-baicd location sampling revealed
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Movement and Home-Range in Estuatine Crocodiles
AB
Crocodile ID 1 ■25S CD — 1 5: 30 ••■ -I ■D ^ ' £ O 20
O a :
I M 8
30 ! 30
20 20
10 10
o Q. O c Figure 4. Movement patterns of 'site-fidelic' male Crocodylus porosus.(a> KUD 50% and KUD 95% (dotted boundary) calculated from GPS- location fixes recorded between 01 September and 28 February (n = 5). inset graph shows the monthly cumulative KUD 95% for each individual, (b) The relationship between daily rate of movement (ROM) and daily distance from the KUD 50% centroid (grey = primary y-axis; black = secondary y- axis), doi:l 0.1371/journal.pone.0062127.g004 that adult male C. poronis are extremely active animals rottlinely ierriioi 7 that lower rates of location sampling or anecdotal moving many kilometres per day. Presumably, it is because observations have given the impression of far lower potential for dominant males move back and ioith vvithin the coniines of a movcmeni in ptiivsus. Female movements Table 2. The results from two general linear mixed-effects III u o rth c n i A ustralia, C. pnrosus iiest from Novcmhf'r through models to examine the covariates and factors influencing until March [13]. The time bet^veeii copulation ancl the laying of daily rate of movement (ROM) and site-fidelity for Oocodylus eggs in captive C. porostvi is betaceen + to fi weeks f6j, and therefoi'C. porosus (male = 8; female = 4). coiutsliip and mating ntay occur an^'vvhere between the end of yepiombcr and early December. During this period our lagged female C‘, coniined iheir movements wiihin a few kilomclrcs Daily distance from KUD D a ily R O M 50% centroid of the main trunk of the river, ft has been suggested previously that fem ale C. poro.s}L\ remain close to the nesting locatinii throughout DF F P F P the year [I-3]. This \s-as not the case in the present study ho\ve\er. S ex L9 19.67 0-001 0,99 0.76 and all our tagged females travelled con.siderable dLstauces lup to B o d y m ass ],9 0.29 0.6 1.3 0.27 .34 km) to a location where we presume they nested (.based upon mo\eincnts that were represeuiathc uf ailenlivc iicsi-guardiug). D a te 1.2153 4.3 0.02 629 0.0001 btich large movement between the breeding and nesting site has doidO.l 371/journal.pone.0062l27.t002 PLOS ONE I www.plosone.org May 2013 | Volume 8 | Issue 5 | e62127 Safer Waterways Bill 2018 Submission 009 Movement and Home-Range in Estuarine Crocodiles B ^ fO V/- PI * ! I n ♦ im Figure 5. Movement patterns of female Crocodylus porosus.(a) The KUD 50% and KUD 95% {dotted boundary} calculated from GPS-location fixes recorded between 01 September and 28 February (n = 4). Inset graph shows the monthly cumulative KUD 95% for each individual, (b) The relationship between daily rate of movement (ROM) and daily distance from the KUD 50% centroid (grey = primary y-axis: black = secondary y-axis}. doiilO.l 371/journal.pone.0062127.g005 noi been reported previously lor female C. and may be resources and the males defend territories around these areas to reflective of the local environment. maximise their mating opportunities [43,46]. Further investigation The females that were captured and lagged in this study is required to confirm this social structure, which would have inhabited the tidal freshwater reaches of the river. In this area, the profound influence upon population densiry and di.stribution. river is relatively narrow and bordcri'd by steep sandy banks A novel observation of this .study was that three out of the four ■S]iarsely covered w ith Afel(detica trees. The river vvotild be fast- tagged females travelled to the locality ol' the nest site a few weeks ilowmg through this section in the wet season, and this lucauon prior to the at:iual nesting movemeni. These journeys would have does not contain good nesting habitat for C. poroML-i. P rio r to required considerable energetic expenditure, and therelbre are nesting, three out of the four tagged females travelled dow nstream likely to have offered some advantage to the offspring. We can to a much wider, .saline-brackish, section of the ri\’er. [n this only speculate on what this may have been, and the motivation for stretch, the river is bordered by thick stands of n'iangro\'e\ M ’pa this repeated-mm'ement so close to nesting retriains an avenue of palms and sak-marsh; vegetation and habitat that is much more future investigation. suited lor C. porosus nesting [4T]. Moreover, this section of the river contains a disproportiorially high densilv^ of hatchling C. porosus Effects upon the ecosystem compared to other stretches of the river [10]. This suggests that The movcnienls of the 'nomadic’ and the 'site-fidelic’ males female migration into this area may be a common behavioural would ha\e resulted in very different feeding opportunitie.s and stratcgv- vvithin the local population. O ne of uttr tagged females did likely required disparate foraging strategies. The 'nomadic’ C. how ever: migrate over 40 km upstream from the breeding area to porosus would need to select a variety of prey items from freshwater the nesting location. This area did not appear to be ideal C. porosixs and saline-brackish ecosy.stems, whilst 'site-fidelic' C. porosus w ould nesting habitat [44]. but there was a large pennanent freshwater need to take prey whenever it wa.s ai ailable within the limits of swamp in close proximity. their home range. Gonsequendy, C. porosits are likely to vaiy in It seems reasonable to assume that the tagged female C. porosus their degree of individual specialisation across spatial scales. Stable travelled long distances to a nesting location because ol better nest isocopic studies upon the tissues of American alligators (Alligator building materials, access to freshwater, and a reduced likelihood fnississippiensis} in the Florida F.vcrgiades revealed a poptilation of the nest being flooded during the wet season [44]. W hat is less composed of both generalist and specialist feeders [48]. There \vas clear is w^hy the females did not breed in the locality of the nesting a strong con-clation betvveen ingested prey item.s and broad-scale areas and save ihemselves from these cncM'gctically expensive mo\emenis, and we argue that aliernath'e behavioural laclics journeys. A possible reason is that the breeding area had better driven b>' social status may ha\'C undcipinned the obsenx'd diet resources than at the nesting areas. Over a four-year period we selection by intlividuals. have laid numerous traps throughout a 60 km stretch of the When highly mobile predators move rapidly betvveen habitats V\'enloek Ri\ er but onh' caught females of breeding size within a and feed on a s'arier.^ of prey specie.s, they create habitat linkages fe\v discrete locations tCampbell. personal obsei'varinn:, The GPS which transport nutrients and energy between systems [.3]. A location data revealed that during the breeding period the females predator that ra|hdlv moves between habitats and switrhe.s prev cxhil.)ii high siie-lideliiy lo these areas. We argue that these will stabilise the ecosyscetn by increa.sing pressure upon oue hi'eeding areas are located '\ ithin productive sections of the ri\'t;r. channci of cncrg\- whilst freeing up a depleting energy channel and the female.'; select these areas in order to build up fat-stores for from strung predaLoiy pressure [3]. In coiUra.st. a sesdlc predator egg gestation and nesting. If this is trite then it suggests that may take foocl whenever available, resulting in negligible transport poro<:us have a social sy^aem ba.sed upon resource-based mate uf energy or nutrients. The diL hotumy of rnovenieni strategies choice. Thai is. the females select areas containing the best observed in this studv for adult C. poroms would result in ven PLOS ONE I www.plosone.org May 2013 | Volume 8 | Issue 5 j e62l27 Safer Waterways Bill 2018 Submission 009 Movement and Home-Range in Estuarine CrocodiJes dilFcrcm lop-dovvn regulation upon trophic intcraoUons ancl the porosus captured in permanently set-traps are males between 2 and coupling of ecosystems and habitacs. Consequencly, understanding 3 m total length (Yusuke Fukuda, Scott Sullh'an, personal the relationship between C. porosxis density, spatial mo\ ement. and communicationj. and the high rates of niovement exhibited by home range dynamics are important in defining the wider the subordinate males in this study explains this capture bias. coirm'iunily and ecosystem effects of a g ro w in g C. poroius Although implemented less frequendy, the removal of dominant population. m ale fil pvwsus U also considered as a viable management inierveniion to reduce crocodih* demity in particular areas. We Implications for management recommend that the impact of this management interv’ention is Since the legislated protection of C. porosus tliere has been a thoroughly evaluated because, as has beeii shown for other general increase in population abundance across northern vertebrate species, dominant male remo\al can camse social Australia. Within some rivers, crocodile density has remained perturbations and can increase movement and immigration from stable tbr the last 10 to 20 years whilst total crocodile biomass has neighbouring areas [49,50,51]. Only by thorough evaluation of continued to increase, whereas other rhers are increasing in each management intervention, Caking into account any conse crocodile densirv but with no matching increase in total bioma.'is quences of social perturbation, can the desired outcome be [14|. The social dynamics of the C. porosus in this study may aid to achieved in the management of C. porosiLs. explain some of these obsen ed trends. For example, the theory of female rcsoui’ce-bascd mate choice [4-7,4-y] in C. pov(i'>iis w'oukl Acknowledgments serve lo stabilise popiilaLioii density in areas of good crocodile habitat, and because displacement is unlikely to be achieved by a W e thank .\,ti.strr\iia Zoo staff for aid in the ra p tu re and tagging proee.ss, smaller rhal. total crocodiie biomass of the area would increase Yu.suke Fukuda .uui the anniiym ous jo u rn a l relen*f*.s f{>r a References I. Kivijs JR, Dasies NB :[‘tQ7'^ Behavioiir.il Frolug^': es’oliuicinniy approach. IB. Kay VVR 21)945 Movements and home ranges ol’radio-lraeketl L'rotodyiuj p'jmus OxtorcL Blackscdl. in the Cambridge Gulf Region of 5\’estetn Australia. Wildlife Rescaich 3 I: 49-5- ■J, Dntiovaii TM. Freeinan ^[. .Vhouolezz ft. Royar K. I-rov>ar PLOS ONE 1 www.plosone,org May 2013 I Volume 8 | Is5ue 5 | e62127 Safer Waterways Bill 2018 Submission 009 Movement and Home-Range in Estuarine Crocodiles !il. Bis’micl R R , Cl (2012} rtfi-os; IiiTt-rfLito to niKim- upen 3our PLOS ONE I www.plo5one.org May 2013 | Volume 8 | Issue 5 | e62127 Safer Waterways Bill 2018 Submission 009 J. exp. Biol. 186. 269-2SS (1994) 2 6 9 Primed in Great Britain © The Company o f Biologists Limited 1994 THE INTRINSIC PROPERTIES OF AN IN SITU PERFUSED CROCODILE HEART CRAIG E. FRANKLIN* Department o f Zoology, University o f Queensland, Brisbane 4072, Australia AND MICHAEL AXELSSON Department o f Zoophysiology, University o f Gotehorg, Goteborg, Sweden Accepted 1 October 1993 Summary An in situ perfused crocodile {Crocodylus porosus) heart preparation was developed to investigate the effects of input and output pressure on cardiac dynamics and to detennine the conditions that lead to a right-to-left cardiac shunt. The pericardium was kept intact, both the left and right atria were perfused and all three outflow tracts (right aortic, left aortic and pulmonary) were cannuiated. enabling pressures and flows to be monitored. The perfused heart preparation had an intrinsic heart rate of 34 beats min“ ' and generated a physiological pow er output. Both the left and right sides of the heart were sensitive to filling pressure. Increasing the filling pressure to both atria resulted in an increase in stroke volume and cardiac otitput (Frank-Starling effect). Increasing the filling pressure to the right atrium also had a positive chronotropic effect. Large right ventricular stroke volumes initiated a right-to-left shunt, despite the left aorta having a pressure 1.5 kPa higher than the pulmonary output pressure. The letf ventricle was able to maintain its output and stroke volume up to an output pressure of approximately 8kPa. However, the right ventricle was significantly weaker. Right ventricular output and stroke volume showed a marked decrease when the output pressure was increased above 5 kPa. A right-to-left shunt occuiTed when pulmonary output pressure was increased. Surprisingly, a shunt occurred into the left aorta before the pressure in the pulmonary artery became greater than that in the left aorta. Once the pressure in the pulmonary artery exceeded the left aortic pressure, pulmonary artery flow ceased and right ventricular output was solely via the left aorta. A right-to-left shunt could also be initiated by increasing the filling pressure to the left atrium. Introduction The architecture of the crocodilian heart and associated outflow tracts is complex. In contrast to the rest of the reptiles, the crocodilian heart has anatomically separated right and left ventricles but, compared with the birds and mammals, where only one systemic ’’'Present address; Gatty Marine Laboratory. University of St Andrews. St .Andrews, Fife KY16 8LB. Scotland. Key words; crocodiie, Crocodylus porosus, heart, cardiac output, shunting, heart perfusion, heart rate. Safer Waterways Bill 2018 Submission 009 270 C , E. Franklin and M. A x e l s s o n arch has been retained, the left in mammals and the right in birds, both systemic arches are present in the crocodilians. The right systemic arch arises from the left ventricle and the left systemic arch and the pulmonary arterial trunk arise as separate distinct vessels from the right ventricle. Moreover, the systemic arches are connected via the foramen of Panizza, a small aperture found in the inter-aortic septum at the base of the outflow tract, a feature which is uniquely crocodilian. Another connection between the two aortic arches can be found further downstream; it is usually called the ‘anastomosis’. This complex anatomical arrangement of vessels allows for a diverse array of flow patterns. In the undisturbed crocodile, blood flow in the left aorta (LAo) is derived entirely from the right aorta (RAo) via the foramen of Panizza. The circulation pattern is thus essentially the same as in mammals and there is a complete separation of systemic and pulmonary blood flow. However, if resistance in the pulmonary outflow tract is increased and/or systemic blood pressure is reduced, blood is ejected into the LAo from the right ventricle (right-to-left shunt) (Axelsson et al. 1989; Shelton and Jones, 1991). To date, our understanding of the physiology/flmctioning of the crocodilian heart is based on in vivo measurements and on speculations drawn from anatomical studies (White, 1956,1969; Webb, 1979; Grigg and Johansen, 1987; Axelsson e/a/. 1989, 1991; Grigg, 1989; Shelton and Jones, 1991). In this study, a completely different approach was taken: a perfused heart preparation was developed and used to examine the intrinsic mechanical properties of a crocodilian heart. Perfused heart preparations have been effectively used to study cardiac function in a range of vertebrates, and many different types of preparations have been developed to meet the specific aims of the investigation. However, if the flow and pressure dynamics of the heart (cardiac dynamics) are to be studied, the integrity of the perfused heart preparation is crucial; that is, all chambers of the heart should be perfused, all outflow tracts cannulated and monitored and the pericardium kept intact (see Perry and Farrell, 1989). Furthermore, the cardiac outputs generated by the preparation and filling and output pressures should be similar to those found in vivo. In reptilian studies, the perfused heart preparations that have been used have had only one side of the heart perfused and one outflow tract cannulated (Reeves, 1963fl,6; Wasser et al. 19906; Jackson et a l 1991). These preparations are useful for studying cardiac metabolism but are of little value in understanding cardiac dynamics. The aim of this study was to examine the effects of filling and output pressures on cardiac dynamics in the crocodile heart. To accomplish this, crocodile hearts were perfused in situ, via the left and right atria, and output cannulae were positioned in the RAo, LAo and the left pulmonary artery (LPA). This preparation enabled us to investigate the sensitivity of the heart to fi lling pressure (Starling effect), the maintenance of cardiac output/stroke volume with increasing output pressure and the conditions that result in a right-to-left shunt. Materials and methods Experimental animals Crocodylus porosus Schneider (body mass 1.09±0.07kg, mean ± s .e . m ., /V=7) were obtained from the Edward River crocodile farm. Cape York. Australia. The crocodiles Safer Waterways Bill 2018 Submission 009 Perfused crocodile hearts 271 were transported by light aircraft to the University of Queensland, where they were housed outdoors in a large 4 m diameter fibreglass tank. They were kept in fresh water heated to 30 “C and had access to platforms on which they could bask. The animals were fed once a week. Perfusate composition The physiological Ringer used to perfuse the crocodile hearts was based on plasma ionic concentrations and blood pH values measured in C. porosus (see Grigg, 1981; Seymour et al. 1985). The Ringer had the following composition (gl“ ’): 6.4 NaCl, 0.3 KCl, 0.41 CaCb.2H20, 0.34 MgS04.7H:0, 0.43 NaH:P04.2H20, 2 NaHCOs and I glucose. The Ringer was bubbled with 3 % CO 2 and 97% O 2 and had a final pH of 7.4-7.5 at 23 °C. Adrenaline (adrenaline bitartrate, Sigma Chemicals) was added to the Ringer to a final concentration of 5 nmol 1 “ '. Just prior to perfusing the heart, superfine Sephadex (0.2gl“ Q was added to the Ringer. The Ringer was stirred and gassed continuously, preventing the Sephadex from settling out and resulting in a homogeneous suspension. The Sephadex particles permitted the perfusate to be monitored by a Doppler flow system. In initial experiments, we found no difference in the performance of the heart, with or without the Sephadex. The Sephadex beads were 10^0/j.m in diameter, which compares favourably with the size of the blood cells in C. porosus (appro.ximately 40/xm in diameter, see Grigg and Caimcross, 1980). Surgical protocol Crocodiles were killed instantaneously by a sharp blow to the head and then weighed. The trachea was intubated with a 3.5 mm rubber tube and the lungs were immediately ventilated with medical grade oxygen. The body cavity was opened with a mid-ventral incision and the vessels going to and from the heart were exposed and cleared of connective tissue. Heparin (2000 i.u. sodium heparinate. Sigma Chemicals) was injected into the right anterior vena cava and allowed to circulate. The left and right anterior vena cavas and the right pulmonary, carotid and subclavian arteries were tied off with silk thread (Ethicon, 2-0). Stainless-steel cannulae (12 gauge, 2.2 mm) (see Fig. 1 for design of cannula), connected via silastic tubing to a Marriot bottle filled with Ringer, were inserted into the right and left pulmonary veins and tied firmly in place with silk thread. The tips of the cannulae were positioned so that they were in close proximity to the opening of the left atrium. Perfusion was started immediately, care being taken to prevent air bubbles going into the heart. The right aorta was cannulated with an 8 or 10 gauge (3 or 2.5 mm) stainless-steel cannula that provided an outlet for the Ringer being pumped by the left side of the heart. To perfuse the right side of the heart, the right hepatic vein was exposed by slicing through the liver and a stainless-steel cannula (8 gauge, 3 mm) supplied with Ringer from a Marriot bottle, was inserted and tied in place. The tip of the cannula was positioned inside the sinus venosus, close to the opening of the right atrium. The left pulmonary artery and left aorta were catmulated with stainless-steel cannulae (8 gauge, 3 mm), providing outlets for the perfusate being pumped by the right ventricle. The three output cannulae were inserted in so that their tips were positioned within the outflow tract. Safer Waterways Bill 2018 Submission 009 272 C. E. F r a n k l in a n d M . A x e l s s o n The crocodile was then decapitated and the tail cut off, and the body region with the in situ perfused heart transferred and submerged in a preparation bath filled with physiological saline at 23 °C. The input cannulae (right and left pulmonary veins and hepatic vein) were connected to constant-pressure devices (see Farrell et al. 1982) that could be raised or lowered to effect a change in the filling pressures to either the left or right atrium. The filling pressures to the left and right atria could be adjusted independently. The three output cannulae (right and left aortas and pulmonary artery) were then coimected to separate pressure heads that were adjustable and which set the diastolic output pressure for each outflow tract (see Fig. 2). There vvas no noticeable leakage of perfusate into the saline bath from the heart preparation. The coronary system in the crocodile heart starts at the base of the right aortic outflow tract, so, as a consequence of perfusing the heart, the coronary system was also perfused. Instrumentation The stainless-steel cannulae used in the preparation had 23 gauge (0.6 mm) needle tubing running along the inside wall and opening at the tip of the cannula (Fig. 1). Pressures were recorded via the 23 gauge (0.6 mm) needle using saline-filled polyethylene tubing connected to Statham pressure transducers (P23XL). Thus, the pressures recorded were a direct measure of the pressure within the vessel or chamber and did not require correction for cannula resistance. Pressures were referenced to the level of the saline in the preparation bath in which the heart preparation was fully immersed. Five pressures were recorded; right and left atrial pressures, right and left aortic pressures and pulmonary artery pressure (see Fig. 2). Flows in the left and right aortas and pulmonary artery were recorded using a Doppler flow system (University of Iowa, Bioengineering, model 545C-4). Extracorporeal flow probes (Titronics Medical Instruments) were inserted into the output tubing approximately 10 cm from the stainless-steel cannulae. The pressure transducers and flow probes were calibrated daily, and the zero for the pressures was checked several times during each experiment. Fig. 2 shows the experimental set-up we used to perfuse and monitor the in situ crocodile heart preparation. Pressure and flow signals were amplified and displayed on a chart recorder (Grass Polygraph model 7). The flow signals were electrically dampened in the Doppler flow system and the various pressure signals were recorded undampened on the chart recorder; further averaging was performed on-line in the computer. Heart rate was derived from the right aortic pressure signals via a Grass 7P44 tachograph unit. In addition to the Grass 23 gauiie needle 8-12 gauge cannula Fig. 1. Diagram of a double-bore staiuless-steel cannula used to cannulate the vessels of the crocodile heart (23 gauge=0.6mm. 8-12 gauge=3—2.2 mm diameter). Safer Waterways Bill 2018 Submission 009 Perfused crocodile hearts 273 polygraph recordings, a data-acquisition software package (AD/DATA; P. Thoren, Hassle AB, Sweden) was used and all data were fed into a Toshiba computer (model T5200), The sampling frequency was set to 10 samples s“ ' and on-line mean values were calculated over 5 s periods. The digital display provided by the computer allowed us to set Doppler flow system Grass polygraph - Perfused heart Perfusion bath Constant pressure device Periusate Fig. 2. A schematic diagram of the experimental set-up used to perfuse and monitor an in situ crocodile heart. F. How signals from output vessels; P, pressure signals from vessels; LAo, left aorta; PA. pulmonary artery; PVs. pulmonary veins; RAo, right aorta: RHV, right hepatic vein; LP.A, left pulmonary artery. Safer Waterways Bill 2018 Submission 009 2 7 4 C . E . pRANKLrN AND M. A xelsson input and output pressures accurately and to set flows to predetermined values dictated by our experimental protocol. Data were stored on disk for later analysis. Terminology Cardiac output is traditionally defined in mammalian hearts as the volume of blood pumped by the left ventricle into the systemic circulation. However, the anatomy of the crocodilian heart, with the left aorta arising from the right ventricle and connected to the right aorta via the foramen of Panizza, makes defining cardiac output more difficult as right ventricular blood can be shunted into the systemic circulation via the foramen. For the purpose of this paper, the flow in the right aortic cannula will constitute left ventricular output (the carotid and subclavian arteries were ligated; hence, flow that would have been in these vessels is directed into the right aortic cannula), and the combined flow in the left aortic and pulmonary cannulae will constitute right ventricular output. We have made the assumption that foramen flow into the LAo from the RAo was nil, which was the case under control conditions. Total cardiac output is defined as the sum of the flow in the LAo, RAo and PA; this represents the total volume of perfusate being pumped by the heart. Experimental protocol Control conditions The left and right ventricles were each set to deliver a flow of approximately 20mlmin“'kg“ ' body mass (i.e. a total cardiac output for the heart of 40mlm in“ ' kg“ ' body mass) against a mean output pressure of 3.5kPa for the RAo and the LAo and 2.0 kPa for the pulmonary artery (PA). The ventricular outputs were set by raising or lowering the filling (input) pressures to the left and right atria, which subsequently increased or decreased the stroke volumes in the respective ventricle. Heart rate was determined by the intrinsic rate of the sino-atrial pacemaker and by the adrenergic stimulus provided by the SnmolH* adrenaline present in the perfusate. The preparation was allowed to settle under these conditions until stable values for the recorded variables were reached (usually about 5 min) before the following responses were tested. Response to filling pressures: Starling curves With the output pressures kept constant at control levels, the filling pressures applied to both atria were dropped to zero or slightly subambient pressures. As soon as flow in all output vessels had stopped, the filling pressures were increased by approximately 0.05 kPa and the new ventricular outputs were allowed to stabilise. The filling pressures were then increased in further steps of 0.08-0.1 kPa to a maximum of 0.8 kPa (8-10 steps). After each pressure increment, a stable reading on the computer of ventricular output was required before the filling pressure vvas increased further. The end point was reached when an increase in filling pressure no longer augmented ventricular output. This point was deemed the maximum ventricular output. Filling pressure had a noticeable effect on heart rate, so in one preparation a further test Safer Waterways Bill 2018 Submission 009 Perfused crocodile hearts 275 was performed. The filling pressure was increased first in the right atrium while that in the left atrium was kept constant, and then the filling pressure was increased in the left atrium while the right atrium’s filling pressure was kept constant. Response to output pressure After testing the response of the heart to filling pressure, the heart was returned to work under the control settings for 2-3 min or until stable values for the various variables had been obtained before its response to output pressure was tested. Changing the pressures against which the heart pumped was performed to meet two major aims; (1) to investigate the ability of the left and right ventricles to maintain flow with increasing output pressure and (2) to investigate the conditions that lead to the right-to-lefl shunt (i.e. the shunting of perfusate from the PA to the LAo). The left ventricle was challenged first by increasing the pressure in both the RAo and LAo in steps of 0,5-1.0kPa. At each pressure increment, the flow was allowed to stabilise to its new rate before the pressure was increased further. The left side of the heart was tested to a maximum of 10.5 kPa. During this challenge, the right ventricle was exposed to the control output pressure of 2 kPa (this corresponds to the pressure in the PA, as all the output from the right ventricle was via the PA; there was no shunting to the LAo). To test the right side of the heart, the PA pressure was increased in increments of 0.5-1 kPa until the flow had been completely shunted into the LAo. At this point, the LAo and RAo pressures were increased in similar steps to a maximum of 8.5 kPa. During this phase, the pulmonary pressure was kept above the RAo/LAo pressure to prevent the perfusate from being redirected back into the pulmonary cannula. Flow through the foramen of Panizza was investigated in one animal by first clamping the PA, thus shunting the perfusate into the LAo, and then clamping the LAo. Histology o f the ventricle Upon the completion of the protocols, the heart was removed from the body of the crocodile, excess water was blotted off and the various chambers of the heart were weighed. Some hearts were fixed in 3% formaldehyde in 0.9% NaCl. Three of these hearts had their ventricular compact and spongy myocardial layers separated from each other and weighed to determine their percentages. The rest of the fixed hearts were dehydrated through an alcohol series and embedded in paraffin wax, 8 /i.m sections were cut and stained with Mallory’s triple stain. The coronary circulation to the myocardium was studied, in particular noting the differences in supply to the left and right sides of the heart. Data analysis and calculations Heart rate, pressure and flow data collected and stored on computer disk by AD-DATA were transferred into a spreadsheet and other cardiac variables were calculated. Stroke volumes ( Ls) were calculated for each ventricle (flow//H, where/ii is heart rate). In the left ventricle, Fs was calculated fi'om RAo flow, and in the right ventricle, f's was calculated from the combined flow of the LAo and PA. Flows and Fs were normalised per kilogram Safer Waterways Bill 2018 Submission 009 2 7 6 C. E. Frank.l[n and M. A x e l s s o n body mass. Flows from the left and right sides of the heart were combined to give total cardiac output. Cardiac power output was also calculated for each side of the heart and for the whole heart. Cardiac power outputs were calculated using the following equations: Left ventricular power output (mW) = [(/’rao ~ ELungv) X F rao ] X 0.0167, (A) Right ventricular power output (mW) = [(7’p a “ Evcava) X F r a ] X 0.0167 + [(7*1X0 ~ E’tungv) X A lao ] X 0.0167, (B) Total cardiac power output (mW) = A + B , where Frao is flow in the RAo (mlmin^^), Alao is flow in the LAo (ml m in"'), Fp,\ is flow in the PA (ml m in"'), Prao is pressure in the RAo (kPa), P lao is pressure in the LAo (kPa), PpA is pressure in the PA (kPa), Pvcava is pressure in the vena cava (kPa), PLungV is pressure in the lung veins (kPa) and 0.0167 is the conversion factor to calculate power in milliwatts. Cardiac power output for the whole heart was normalised per gram heart mass. Flowever, as the mass for each side of the heart could not he measured accurately, the power outputs for the left and right ventricles were normalised per kilogram body mass. For each preparation, / h , F s , flow and power output for each ventricle were plotted against filling pressure (preload graphs) and output pressure (afterload graphs). Curves were fitted to the data using a third-order polynomial. The correlation coefficient was always greater than 0.97, which meant a highly significant curve fit was obtained (P<0.01, A-6-10 data points). The graphs from the individual preparations could not be directly combined as input and output pressures were never completely the same between preparations. To overcome this problem, we used the polynomial equations to generate data at set pressure intervals. For the preload curves, data points were calculated in 0.05 kPa steps and for the afterload curves in 0.5 kPa steps. We were then able to combine the data from the different preparations to produce composite graphs. Data are presented as means ± S.E.M. and statistical comparisons were made using the Wilcoxon matched-pairs signed-rank test. Differences where P<0.05 were regarded as statistically significant. Results Cardiac morphology Heart morphometric data obtained from sub-adult C. porosus are presented in Table 1. The heart mass of C.porosus was 0.279±0.012 % of body mass and the combined mass of the right and left ventricles represented 0.232±0.009% of body mass. The compact myocardial layer made up 42.3±5.9% of the total ventricular mass, the rest constituting spongy myocardium. The left ventricle had a thicker layer of compact myocardium than the right ventricle. The left ventricle also had a greater supply of coronary vessels than the right. When the coronary system was examined in the paraffin sections it was found to be completely devoid of red blood cells, indicating that the coronary system had been perfused by the Ringer. Safer Waterways Bill 2018 Submission 009 Perfused crocodile hearts 277 Table 1. Heart morphometric data for Crocodylus porosus Body mass (kg) 1.09±0.07(7) Heart mass (g) j.05±0.21 (7) Relative heart mass (%) 0.279±0.0I2 (7) Relative ventricle mass (%) 0.232±0.009 (7) Relative atrial mass (%) 0.048±0.004 (7) Compact myocardium (%) 42.3±5.9 (3) Spongy myocardium (%) 57.7±5.9 (3) Data are presented as means ± s .e .m . (/V). The relative masses are expressed as a percentage of body mass. The outflow tract was removed from the heart before weighing. Intrinsic properties o f the heart preparation Table 2 summarises the physiological performance data obtained from the heart preparation. Heart rate varied between preparations (28.5^2.5 beatsmin“ '). The heart preparation had an intrinsic heart rate of 33.8±2.0 beatsmin“ ' when working at ZOmlmin^'kg^’body mass and at an adrenaline concentration of 5 nmol 1“ ' in the perfusate. The heart rate remained stable throughout the duration of the experiments, with only small variations due to changes in the right atrium filling pressure. Response to filling pressure Increasing the filling pressure to the left atria, via the pulmonary veins, resulted in a significant increase in RAo flow, which was due chiefly to an increase in Fs (Figs 3A, 4B,C). Left ventricular Fs increased sixfold as input pressure was increased from 0.1 to 0.5 kPa. This range of input pressures represented the most sensitive range for altering Fs (Fig. 4B). Increases in filling pressure above 0.7 kPa had no significant effect on Fs and RAo flow. The maximum values of left ventricular output and Fs were 39.5±2.3 m lm in"'kg“ 'body mass and 1.18±0.09mlkg“ 'bodymass, respectively. As left ventricular output increased against a constant output pressure in the RAo (3.5 kPa), Table 2. Cardiovascular measurements from Crocodylus porosus Heart rate (beats min"') 33.8±2.0 Maximal cardiac output (ml min"' kg"* body mass) 79.9±3.5 Maximal right ventricular output (ml min“* kg'* body mass) 40.5±2.3 Maximal left ventricular output (ml min~* kg'* body mass) 39.5±2.3 Maximal total stroke volume (ml kg'* body mass) 2.58±0.12 Maximal right ventricular stroke volume (ml kg'* body mass) 1.39±0.09 Maximal left ventricular stroke volume (ml kg'* body mass) 1. 18±0.09 Maxittial cardiac power output (mW g'* ventricular mass) 1.46±0.20 Ma.ximal cardiac power output (mW kg'* body mass) 3.99±0,22 Ma.ximal right ventricular power output (mW kg'* body mass) 1.73±0.25 Ma.ximal left ventricular power output (mW kg'* body rnass) 2.26±0.15 Data arc presented as means ± .s.e..vi. (.V=7). Safer Waterways Bill 2018 Submission 009 278 C . E , F r a n k l in a n d M , A x e l s s o n A > 1 m ill 50- • a - 0 - Fig. 3. Recordings of the effect of (A) increasing the filling pressure in the pulinonar>- veins (Rpv) on the flow in the right aorta (^Ao) and (B) increasing the filling pressure in the right hepatic vein ( P r h v ) on the flow in the pulmonary artery ( ^ a ) a n d left aorta ( ^ ao ). the power output of the left ventricle increased to a maximum of I.56±0.19 m W kg^' body mass (Fig. 4D). Increasing the filling pressure to the right atria, via the hepatic vein, significantly increased right ventricular output and Fs (Figs 3B, 4B,C). The right side of the heart was significantly more sensitive to filling pressure than the left side of the heart, For example, at an equivalent filling pressure of 0.2 kPa, the right ventricular output and Fs were almost double those of the left ventricle (Fig. 4B). The increase in right ventricular output was a result of both PA and LAo flow. Cardiac outputs below about 20mlmin“ ' kg“ ' body mass were solely due to PA flow. It was at higher cardiac outputs that flow occurred in the LAo (Figs 3B, 4C); this despite the LAo output pressure being 1.5 kPa greater than the PA pressure. The maximum total cardiac output for the heart preparation was 79.9±3.5 ml min^' kg^' and left and right ventricles contributed equally to this output Safer Waterways Bill 2018 Submission 009 Perfused crocodile hearts 279 40 35 A 30 T 1 2.5 ^ D 2.0 - 1.5 - 1 0 _ o 0.5 Q o o n 0.0 0.2 0.4 0.6 0.8 Input pressure (kPa) Fig. 4. The effects of increasing liiiing pressure in the pulmonary veins (i.e. left atrium) and right hepatic vein (right atrium) on cardiac performance. Output pressures were 3.5 kPa for the left and nght aorta and l.OkPa for the pulmonary artery. (A) Heart rate(/i-i); (B) stroke volume (Fs) o f the left and right ventricles; (C) flovv of perfusate from the right and left ventricles and From the left aorta; (D) power output from the left and right ventricles {O right ventricle; • left ventricle; ■ left aorta). * indicates a significant difference between the left and right ventricles (P<0.05, N=l). (Table 2). The maximurn right ventricular output was 40.5±2.5mlmin“ ' kg^' body rnass, which was not significantly different from the maximum left ventricular output (39.5±2.3 ml tnin“ 'kg“ Table 2). Likewise, the maximum right and left ventricular Fs values were not significantly different from each other. The power outputs for the two sides of the heart were different, however, as a result of the left ventricle working against a higher output pressure (Fig. 4D). The pressure in the PA was only 2 kPa compared with 3.5 kPa in the RAo. Safer Waterways Bill 2018 Submission 009 280 C. E. Franklin and M. A x e l s s o n Increasing filling pressure from 0 to 0.5 kPa caused an increase in stroke volume and also an increase in heart rate in six of the seven preparations (Fig. 5A-C), The average increa.se in heart rate was 4.5% (from 34.5 to 36.1 beats min“ '). If the filling pressure was simultaneously increased to both sides of the heart, then an elevation in heart rate occurred (Fig. 5A). An increase in filling pressure to the right atrium resulted in an increase in heart rate (Fig. 5B); however, there was no increase in heart rate when filling pressure was increased in the left atrium (Fig. 5C). Response to output pressure Increasing RAo pressure only slightly reduced left ventricular output and Vs over the output pressure range tested. It was only when the RAo pressure was increased above 8 kPa that a more marked decrease in left ventricular output and Fs occurred (Fig. 6B,C). Heart rate was not affected by RAo pressure (Fig. 6A). As RAo pressure increased, left ventricular power output also increased to a maximum of 2.26±0.15 mW kg” ' body mass at an output pressure of 9.5 kPa and remained at about this level as the RAo pressure was 0 T 50 30- Increase in F rhv and Ppv Increase in P ruv Increase in Ppv 45 1 L 1 1 1 1 _ 4 5 1 1 1 . 1 c ■3 40 p 40' t/3 « , - 1 k 35 - 1 35- I t 1 I t 1 1 t 1 1 30 ^ " 3 0 0 0.1 0.2 0.3 0.4 0.5 0.6 O.I 0.2 0.3 0.4 0.5 F rhv (kPa) Fpv(kPa) Fig. 5. The effect of filling pressure in the pulrnonaiy veins (i.e. left atrium) and right hepatic vein (i.e. right atrium) on heart rate. (A) Recordings of right hepatic vein pressure (F rhv), pulmonary vein pressure (f*p\) and heart rate (/it). Heart rate increased when both pressures were increased together and when the right hepatic pressure was increased and the pulmonary vein pressure remained at control levels (B). There was no increase in heart rate when the pressure in the pulmonary veins was increased and the pressure in right hepatic vein remained at control levels (C). Safer Waterways Bill 2018 Submission 009 Perfused crocodile hearts 281 elevated to 10.5kPa (Fig. 6D). The right ventricle was subjected to an output pressure challenge by first raising the PA pressure and then the LAo pressure. Heart rate was not affected by an increase in output pressure to the right ventricle. Right ventricular stroke volume and flow did not change significantly until the output pressure was increased above 5 kPa, after which they both significantly decreased (Fig. 6B,C). With regard to maintaining ventricular output and Fs against an output pressure challenge, the right 40 ■n 35 25 l.O - B 0.8 0.6 0.4 0.2 0.0 30 25 20 15 10 5 0 - L.U 0 * - 0 5 1 0.0 Output pressure (kPa) Fig. 6. The effect of increasing the output pressure to the left ventricle (increase in right aortic pressure) and to the right ventricle (increase in pulmonary artery pressure followed by an increase in left aortic pressure) on cardiac performance. (A) Heart rate (fw). (B) Stroke volume (Fs) for the left ventricle {•) and right ventricle (O). (C) Flow of perfusate from the left ventricle ( • ) and right ventricle (O). (nset: the flow in the pulmonary artery (▼) and left aorta (V) (ordinate and abscissa labels as in C). (D) Power output in the left ventricle (•) and right ventricle (O). * indicates a significant difference between the left and right ventricles (P<0.05, ,V=7). Safer Waterways Bill 2018 Submission 009 282 C . E . F r a n k l i n a n d M . A x e l s s o n ventricle was significantly weaker than the left ventricle (Fig. 6B,C; Table 2). The output pressure which could be maintained without a significant decrease in cardiac output for the left ventricle was SkPa, whereas it was only 5kPa for the right ventricle. Right ventricular power output Increased to a maximum of 1.73±0.25 mWkg“ 'body mass, which is significantly lower than that of the left ventricle (Table 2). The right ventricular output and Fs dynamics were determined by flows in the PA and LAo (see Fig. 6C inset). Increasing PA pressure to 3.0 kPa caused a significant shunting of perfusate into the LAo. Shunting occurred even though the LAo pressure (3.5 kPa) was greater than the PA pressure. Once the pressure in PA exceeded LAo pressure, PA flow r/Forauieii 4.a 22.2 (inlmin^') RForamen 2.6 7 (Paminml-') LAo clamped LAo released PA clamped H S I i i Fig. 7. Effect of occlusion of the pulmonary artery followed by occlusion of the left aorta on How in the right aorta (^ya). left aotla (rjr.Ao) and pulmonary artery ((JFa ) and on the pressure in the right aorta {P ra o ) and pulmonary artery {Pi>.y). The increase iti flow in the right aorta after the pulmonary artety and left aorta had been clamped is due to How through the foramen of Panizza. Foramen flow (r/Forameu) and the resistance of the foramen (/fForamen) are presented helow the right aortic How trace. Safer Waterways Bill 2018 Submission 009 Perfused crocodile hearts 283 ceased and right ventricular output was attributed solely to LAo flow. Shunting of perfusate from the PA to the LAo did not significantly decrease the right ventricular output. That is, the initial flow in the PA was not significantly different from the flow in the LAo once PA flow had ceased. Fig. 7 shows recordings of the effect of clamping the PA and LAo on the flow dynamics in the RAo, LAo and PA. Clamping the PA shunted the blood into the LAo. A small foramen flow of 4.8mlmin“ ' from the LAo to the RAo resulted from the PA occlusion. Clamping both the PA and LAo resulted in the output from the right ventricle being shunted through the foramen of Panizza and into the RAo. A foramen flow of 22.2mlmin~^ was recorded; this is appro.ximately the same as the flow recorded in the LAo. The foramen had a resistance of 88.7Paminml” ' at this flow rate compared with a Discussion The heart mass relative to body mass of C. porosus was 3.05 g kg” ' body mass This is similar to the relative heart masses recorded for snakes (3.1 gkg” '; Poupa and Lindstrom, 1983) and the lizard Amphibolunis vitticeps (2.85 gkg” '; Else and Hulbert, 1983), but considerably greater than the heart mass of the tortoise Emydura signata (1.6gkg” '; C. E. Franklin, unpublished observations). Among the fishes, the relative ventricle mass is greater in the more active species, such as yellowfin and skipjack tuna, than in sluggish fish, such as the sea raven (Fan'ell, 1991). A similar relationship with respect to athleticism may also occur among the reptiles, Poupa and Lindstrom (1983) found that a large variation in relative heart mass occurred among the snakes, with the tree snake Dispholidus typiis having a very high heart mass (4.5gkg” '). They proposed that this may be related to the active and fighting prey they consume, which would reflect their athleticism. In situ perfused heart preparation This is the firstin situ perfused crocodile heart preparation to be developed and also the first of its type for the reptiles. The surgical procedures used to perfuse the crocodile heart subjected the cardiac tissue p er se to minimal interference. The heart remained in situ, and the pericardium was kept intact, both of which maintained the structural integrity of the heart with its vessels and surrounding tissue. Furthermore, both the left and right atria were perfused and all three outflow tracts (RAo, LAo and PA) were cannulated and their outputs monitored. All previous reptile heart perfusions (turtles) have involved removing the heart from the body cavity, perfusing only one side of the heart and having only one outflow tract cannulated (Reeves, 1963a,h; Wasser et al. 1990n,h; Jackson et al. 1991). Isolated heart preparations, such that as described above, involve suspending the heart in an organ bath, which results in considerable mechanical and physical manipulation of the cardiac tissue and this would have a detrimental affect on cardiac performance. Altliough this cannot be substantiated among the reptiles, Houlihan et al. (1988) found that the isolated trout {Salma gairdneri) heart preparation they used had an inferior perfonnance to the in situ trout heart preparation used by Farrell et al. (1986). Removal or puncturing Safer Waterways Bill 2018 Submission 009 284 C. E. Franklfn and M. A x e l s s o n the pericardium has been shown to have a detrimental effect on cardiac performance in a range of vertebrates (Janicki and Weber, 1980; Farrell et al. 1988; Franklin and Davie 1991, 1993). The main disadvantage with previous isolated reptile heart preparations is that there was only one input and one output; therefore, cardiac outputs are very unlikely to reach physiological levels. A key feature of the in situ crocodile heart preparation is that it produced cardiac outputs (determined from the three outflow tracts) that were similar to those recorded in vivo (Shelton and Jones, 1991) and these flows were generated at physiologieal output pressures. Perry and Farrell (1989) stress that perfused heart preparations must work at physiological pressures and cardiac outputs if the results are to be meaningful, especially if the flow and pressure dynamics of the heart are being investigated. The coronaiy circulation, which arises from the base of the RAo outflow tract, was perfused as a result of heart perfusion. Transverse sections of heart tissue revealed a coronary circulation that was devoid of red blood cells. The extent of perfusion and the flow rate in the coronary vessels were, however, not established. The high Po^ o f the Ringer would have assisted in oxygenating the compact myocardium directly via the lumen of the heart, thus helping to offset the possibility that the compact myocardium was not effectively oxygenated by the coronary system. The perfused heart preparation had a stable and intrinsic heart rate of 34 beats m in^’. W right et al. (1992) recorded in vivo a mean heart rate of 23.5 beats min~' from resting C. porosu.'i at 25 °C. The lack of an Inhibitory vagal lone in the perfused heart preparation, as a result of the vagus being cut, could account for the higher heart rate. The effect o f filling pressure The perfused crocodile heart relied on positive filling pressures to generate physiological cardiac outputs {vis-a-tergo filling). If the filling pressures to the left and right atria were reduced to below ambient, flow in all vessels ceased. Vis-ci-fronte filling (suctional filling) of the atria does not appear to function in the crocodile heart, unlike the hearts of some fishes, such as dogfish, trout and tuna (see Farrell, 1991). The heart preparation was exposed to filling pressures ranging from approximately 0 to 0.8 kPa. The perfused crocodile heart responded to an increase in filling pressure by increasing stroke volume, as predicted by the Frank-Starling mechanism. This positive relationship between filling pressure and stroke volume has also been found in the hearts of fishes, turtles and mammals (Reeves, 1963£i,^i; Farrell, 1991; Strobeck and Sonnenblick, 1986). Only low filling pressures were needed to stimulate the heart to produce a physiological cardiac output. Furthennore, small changes in filling pressure resulted in large changes in stroke volume; i.e. the heart was very sensitive to filling pressure. The right side of the heart was more sensitive to filling pressure than the left. In I'ri'o measurements of venous pressures in crocodiles have not been made, although Johansen and Burggren (1984) recorded central venous systemic pressures in varanid lizards that ranged from about 2 to lOcmFhO (approx. 0.2-1.0kPa). It is reasonable to assume that crocodile central venous pressures would fall within this range (as in indeed the central venous pressures of most vertebrates do, the exception being some of the fishes, which have lower pressures). Thus, the filling pressures used in our heart Safer Waterways Bill 2018 Submission 009 Perfused crocodile hearts 285 preparations are apparently physiological and the marked Starling response operated within this physiological range. However, the role venous pressure plays In determining stroke volume in vivo remains to be established, although the intrinsic mechanism exists in the crocodile heart to augment stroke volume several-fold via the Starling response. Increasing filling pressure not only elevated stroke volume but also initiated a right-to- left shunt. A significant flow was recorded in the LAo, despite this outflow tract having an output pressure 1.5 kPa greater than the PA outflow tract. The functional basis of the Starling response is that the force of contraction increases with the length of the muscle fibre (although beyond a certain sarcomere length, force of contraction decreases, see Lakatta, 1986). Presumably, as stroke volume increased with greater filling pressure, the force of contraction also increased until the right intra-ventricular pressure exceeded the pressure in the LAo and a shunt resulted. Shelton and Jones (1991) proposed that shunting could result from increasing the right ventricular fibre length by increased filling pressure. Our results support this hypothesis. Filling pressure also had a chronotropic effect. Increasing filling pressure to the right atrium increased heart rate, whereas an increase in left atrial pressure failed to increase heart rate. It appears that increases in right atrial filling pressure are stimulating the sinoatrial pacemaker to cycle (discharge) at a higher frequency. Heart rate also increases with filling pressure in mammals, but not in trout heart preparations in situ (see Perry and Farrell, 1988). Effect o f output pressures The left ventricle was relatively insensitive to output pressure (in the RAo) and was intrinsically capable of maintaining left ventricular stroke volume up to an output pressure of approximately SkPa. The right ventricle was significantly weaker, only maintaining stroke volume up to an output pressure (in PA and LAo) of 5 kPa. This marked difference between the systemic and pulmonary circulations is characteristic of mammalian hearts also. Ventricular wall thickness is considerably greater in the left than in the right ventricle and would account for the difference in maintaining stroke volume. Right-to-left shunts Right-to-left shunts have been recorded in vivo in crocodiles during diving and after intravenous injections of acetylcholine (Grigg and Johansen, 1987; Axelsson et al. 1989). After acetylcholine injections, Axelsson et al. (1989) found that pulmonary resistance increased and blood was shunted into the LAo. White (1969) noticed that during forced dives there was an increase in the resistance of the pulmonary circuit. The shunting of blood into the LAo is generally associated with an increase in pulmonary resistance or, as proposed by Shelton and Jones (1991), a decrease in systemic blood pressure. In both cases, it is assumed that the blood travels along the path of least resistance (lower pressure). A bradycardia also occurs during forced dives and as a consequence of acetyleholine injections and may be an important component of shunting. It is conceivable that a bradycardia could result in an increase in right ventricular stroke volume as a consequence of an increased filling time; the larger stroke volume generating intra- Safer Waterways Bill 2018 Submission 009 2 8 6 C. E. Franklin and M. A x e l s s o n ventricular pressures greater than systemic (LAo) pressure. With a shunt operating, the systemic circulation would receive a larger contribution of blood which, in turn, would increase the central venous pressure. Thus, it would be possible to have a shunt without a change in systemic or pulmonary pressure, the shunt being mediated by changes in systemic venous pressure initiating a Frank-Starling response. Flow through the foramen of Panizza Under our control conditions in the in situ perfused heart, no flow occurred in the LAo. This is contrary to resting conditions in vivo in the crocodilians, where LAo flow accounts for approximately 1—3 % of total cardiac output and is due to flow through the foramen of Panizza; i.e. from the right aorta to the left aorta (Axelsson etal. 1989; Shelton and Jones, 1991). LAo pressure was the same as RAo pressure; thus, the additional resistance the foramen of Panizza incurs would inhibit flow in the LAo. However, flow through the foramen of Panizza from the RjAo to the LAo did occur when LAo pressure was marginally lower than RAo pressure. Grigg (1989) proposed that a reverse foramen flow (Bow from the LAo to RAo) might be possible if the pulmonary perfusion ceased altogether. In this scenario, if the pulmonary perfusion was stopped, no blood would retum to the left ventricle and the systemic circulation would be driven by the right ventricle, cardiac output occurring in the LAo and RAo. As shown in Fig. 7, this hypothetical pathway suggested by Grigg can occur, although its relevance or whether it actually occurs in vivo is yet to be determined. In summary, the cardiac dynamics of the crocodile heart is perhaps the most complex of all vertebrates. Distribution of the cardiac output between the three outflow tracts shows that extreme flexibility and a range of compieteiy different flow patterns and regulatory mechanisms exist. The in situ perfused crocodile heart preparation gives an insight into the cardiovascular possibilities which could occur in vivo and which remain to be substantiated or disproved in the future. This work was performed in Gordon Grigg’s Physiological Ecology Laboratory at the University of Queensland. We offer our sincere thanks to Gordon for providing us with the facilities and encouragement to do this work. Crocodiles were obtained from the Edward River Crocodile Farm, Pty Ltd, and thanks go to Victor Onions and Don Morris. C.E.F. was a University of Queensland Postdoctoral Fellow at the time of this study. M.A. received a travel grant from Wennergren Centre Foundation for Scientific Research. References .Axelsson, M., Fritsche, R., H olm gren. S., G rove, D. J. .a.nd N ilsson. S. (1991). Gut blood How in the estuarine crocodile, Crocodylus porosus. Acta physiol, scand. 142. 509-516. A.xelsson, M.. H o lm , S. .vnd N ilsson, S. (1989). Flow dynamics of the crocodilian heart. Am. J. 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