Decompression sickness (“the bends”) in sea turtles

Short title: The bends in sea turtles

D. García-Párraga1,2*, J. L. Crespo-Picazo1,2, Y. Bernaldo de Quirós3, V. Cervera4,

L. Martí-Bonmati5, J. Díaz-Delgado3, M. Arbelo3, M. J. Moore6, P. D. Jepson7 and

Antonio Fernández3

1
Oceanografic, Veterinary Services, Parques Reunidos Valencia, Ciudad de las Artes y

las Ciencias, C/ Eduardo Primo Yúfera 1B, 46013 Valencia, Spain.

2
VISAVET Center and Animal Health Department, Veterinary School, Complutense

University of Madrid, Av Puerta del Hierro s/n, 28040 Madrid, Spain.

3
University of Las Palmas de Gran Canaria. Institue of Animal Health. C/Transmotaña

s/n, Arucas, 35416, Las Palmas, Spain.

4
Hospital Veterinario Valencia Sur, Avda. Picassent, 28, 46460 Silla. Valencia, Spain.

5
Grupo de Investigación Biomédica en Imagen GIBI230. Radiology Department,

Hospital Universitario y Politécnico La Fe, Av. Bulevar Sur s/n, 46026 Valencia, Spain.

6
Woods Hole Oceanographic Institution. Department of Biology. 266 Woods Hole

Road, Woods Hole, MA, 02543, United States of America.

7
Institute of Zoology, Zoological Society of London, Regent's Park, London, NW1 4RY,

United Kingdom.

*Correspondence to: [email protected]

1
 ABSTRACT

Decompression sickness (DCS), as diagnosed by reversal of symptoms with

recompression, has never been reported in aquatic breath-hold diving vertebrates despite

the occurrence of tissue gas tensions sufficient for bubble formation and injury in

terrestrial animals. Similarly to diving mammals, sea turtles manage gas exchange and

decompression through anatomical, physiological and behavioral adaptations. In the

former group, DCS-like lesions have been observed on necropsies following behavioral

disturbance such as high-powered acoustic sources (e.g. active sonar) and in bycaught

animals. In sea turtles, in spite of abundant literature on diving physiology and bycatch

interference, this is the first report for DCS-like symptoms and lesions. We diagnose a

clinico-pathological condition consistent with DCS in 29 gas embolized loggerhead sea

turtles (Caretta caretta) from a sample of 67. Fifty-nine were recovered alive and 8

recently dead following bycatch in trawls and gillnets of local fisheries from the east

coast of Spain. Gas embolization and distribution in vital organs, was evaluated through

conventional radiography, computed tomography and ultrasound. Additionally, positive

response following repressurization was clinically observed in 2 live affected turtles.

Gas embolism was also evidenced post-mortem in corpses and tissues as described in

cetaceans and human divers. Compositional gas analysis of intravascular bubbles was

consistent with DCS. Definitive diagnosis of DCS in sea turtles opens a new era for

research in sea turtle diving physiology, conservation and bycatch impact mitigation, as

well as for comparative studies in other air-breathing marine vertebrates and human

diving.

Key words: decompression sickness, the bends, gas bubbles, sea turtles, bycatch,

hyperbaric treatment, gas embolism, breath-hold divers.

2
INTRODUCTION

Decompression sickness (DCS) is a clinical diagnosis encompassing a wide range

of manifestations related to formation of gas bubbles within supersaturated tissues after

decompression (Francis & Mitchell 2003). In human divers, the effects range from

trivial to fatal, and most often involve neurological and musculoskeletal symptoms

(Francis & Simon 2003, Vann et al. 2011), including severe pain. In an analysis of 1,070

central nervous system DCS cases, 77% involved the spinal cord (Francis et al. 1988).

A wide range of symptoms are caused directly or secondarily by the mechanical,

embolic, and biochemical effects of intra- and extravascular bubbles (Vann et al. 2011).

Direct effects include the distortion of tissues and vascular obstructions. Secondary

effects include endothelial damage, capillary leakage, plasma extravasation, and

hemoconcentration (Vann et al. 2011). Definitive diagnosis of DCS is difficult and only

confirmed by successful recompression treatment in a hyperbaric chamber (Ferrigno &

Lundgren 2003).

Breath-hold diving vertebrates, including marine mammals and sea turtles,

classically are considered to be protected against DCS through anatomical,

physiological and behavioral adaptations (Berkson 1967, Rothschild & Martin 1987,

Burggren 1988, Lutcavage & Lutz 1997, Piantadosi & Thalmann 2004, Fossette et al.

2010, Castellini 2012). However, an acute and systemic gas and fat embolic syndrome

similar to DCS in human divers was described in beaked whales that stranded in

temporal and spatial association with military exercises involving high-powered sonar

(Jepson et al. 2003, Fernandez et al. 2005). Since this first report, there has been

accumulating evidence demonstrating the presence of gas bubbles in diving marine

mammals (Jepson et al. 2005, Bernaldo de Quirós et al. 2012, Dennison et al. 2012),

including dysbaric osteonecrosis (Moore & Early 2004) and gas embolism in bycaught

3
animals (Moore et al. 2009). Although these findings have challenged our

understanding of diving physiology in these species, conclusive clinical data (i.e.

diagnosis and therapy) supporting the occurrence of DCS are lacking due to the

complexity of working with wild marine mammals.

Sea turtles are among the longest and deepest marine air-breathing diving

vertebrates (Byles 1988, Sakamoto et al. 1990, Houghton et al. 2008). They may spend

over 90% of time submerged in apnea (Lutcavage & Lutz 1997) and efficiently use

oxygen through cardiovascular adjustments, similar to other air-breathing vertebrates

(Rothschild & Martin 1987, Burggren 1988, Southwood et al. 1999, Southwood 2013).

In addition, osteonecrosis-type lesions, being one of the few long-term lesions

observable after certain episodes of DCS, have been described in monosaurs and sea

turtle fossils from the Cretaceous Age but are very rarely described in animals younger

than the Miocene Age (Rothschild & Martin 1987). This suggests that more recent taxa

have evolved physiological and behavioral adaptations to mitigate hyperbaric conditions

like DCS.

Bycatch is a well-documented, worldwide problem resulting in considerable

mortality of non-targeted species (Lewison et al. 2004a). Over the past decades, there

has been a dramatic global decline in sea turtle populations with six of seven species

currently categorized as vulnerable, endangered, or critically endangered by the IUCN

Red List (IUCN.www.iucnredlist.org (accessed 14 January 2014)). Fishery bycatch is

recognized as the greatest threat to their conservation (Wallace et al. 2010) and is

considered a moderate or high threat for more than three-fourths of all sea turtle

Regional Management Units globally (Wallace et al. 2011, Lewison et al. 2013).

Approximately 85,000 sea turtles were reported incidentally captured worldwide from

1990 through 2008, but true total bycatch is estimated to be at least two orders of

4
magnitude higher (Wallace et al. 2010). Total numbers of global bycaught sea turtles

(Lewison et al. 2004b, Hamann et al. 2010, Wallace et al. 2010) and resulting mortality

(Lutcavage & Lutz 1997, Epperly et al. 2002, Hamann et al. 2010) remain unclear.

Primary limitations in bycatch estimates are the lack of reliable comprehensive

information on total fisheries effort, bycatch in small scale fisheries (Wallace et al. 2010,

Casale 2011), and the rate of survivorship of released animals (Chaloupka et al. 2004,

Mangel et al. 2011). The rate of survivorship following interaction is considered to be

one of the main obstacles to understanding the true impact of fisheries on sea turtle

populations (Lewison et al. 2013). Consideration of causes of sea turtle mortality

resulting from fisheries interaction largely have focused on the effects of drowning and

direct trauma from gear (Poiner & Harris 1996, Gerosa & Casale 1999, Casale 2011,

Lewison et al. 2013). The present work describes a previously undescribed condition

that can compromise post-release survivorship of incidentally captured sea turtles.

In this study, 67 loggerhead turtles (59 alive, 8 dead) bycaught in trawls and

gillnets at depths ranging from 10 to 75m, were evaluated by intensive clinical and

pathological examination. Gas embolism (GE) was a consistent finding in a large

proportion of live and dead animals. Clinical signs, diagnostic imaging, gross and

histological observations and response to recompression and controlled decompression

treatment definitively demonstrate that marine air-breathing vertebrates can suffer from

DCS. These findings offer a new paradigm to consider in many different aspects of sea

turtle research, conservation and management, including basic patho-physiological

aspects of diving adaptations, implications on post-capture survivorship estimates,

bycatch impact mitigation strategies and devices, clinical treatment of affected turtles,

as well as potential additional risks associated with intentional capture of diving turtles.

5
MATERIAL AND METHODS

Animal acquisition

All sea turtles included in this project were under the authority of the

“Consellería de Infraestructuras, Territorio y Medio Ambiente” of Valencia

Community Regional Government in collaborative official agreement with the

Oceanografic Aquarium of the “Ciudad de las Artes y las Ciencias of Valencia” for

animal rehabilitation and posterior release, and for the postmortem examination

of dead individuals.

In 2011, an active campaign involving fishermen from the Valencian coast of Spain

was established to collect all (live and dead) sea turtles incidentally captured by gillnets

and trawling so that bycaught animals could be medically evaluated. During the period

from January 1, 2011 to January 2, 2014, a total of 67 bycaught loggerhead turtles

(Caretta caretta) were received. Eleven turtles arrived dead and 56 arrived alive.

An additional five of 56 live turtles died within 72h. All live animals received

comprehensive clinical examination. Examination of all dead turtles included

necropsy and histopathology.

For all cases, the date of capture, fishing depth, and sea surface temperature at

the originating port were documented (SeaTemperature. www.seatemperature.org

(accessed 14 Jan 2014)). Any comments from fishermen related to the condition and

behavior of turtles upon capture were also noted.

Clinical diagnosis

6
 All live bycaught turtles were examined within the first 24 hrs (average 12 hrs).

Evaluation included routine general veterinary physical and neurological examination,

hematology and biochemistry, followed by imaging studies.

Blood was collected from the dorsal cervical sinus with a 5ml syringe and 21G

40mm hypodermic needle (Henry Schein Inc, Melville NY, USA) and transferred to

2ml lithium heparin tubes (Aquisel®, Barcelona, Spain) for immediate analysis

(maximum elapsed time of one hour). Analysis included automated hematology with an

Abbott Celldyn 3700SL hemocytometer (Abbott Laboratories Illinois, USA), standard

manual hematocrit determination and cytological study including manual differential

count, and complete biochemistry and electrolyte panel using an Olympus AU400

autoanalyzer (Mishima Olympus CO, LTD, Shizuoka-ken, Japan).

Diagnostic imaging studies included the following:

- Plain radiographic evaluation with a Philips Practix 400 unit (Philips Medical

Systems, , Hamburg, Germany,) and a Kodak Direct View Classic CR System

(Carestream Health, INC. Rochester, New York, USA) with 35x43cm Kodak

cassettes (Kodak PQ Storage Phosphor Screen Regular, and 100 Microns,

Carestream Health, INC. Rochester, New York, USA) in dorsal-ventral (DV),

cranial-caudal (CC), and lateral-lateral (LL) projections. Focal distances varied

between 1-1.5m, using average exposure values between 75-120kV and 7,2-20

mAs depending on projections and animal size. Digital images were processed

afterwards through the Kodak Acquisition Software (Onyx-RAD Diagnostic

Viewer, Rochester, New York, USA) for better visualization and image

interpretation. Some dead bycaught turtles were also radiographed.

- Ultrasonographic general examination was conducted using a General Electric

Logiq E Vet ultrasound machine with commercial linear, phase-array, and

7
 microconvex probes (models 12LRS (GE Healthcare, Japan Corporation,

Tokyo, Japan), 3S (GE Medical Systems CO, LTD, Jiangsu, China) and 8CRS

(GE Medical Systems CO, LTD, Jiangsu, China)), respectively.

- Selected individuals with DCS compatible signs were examined by computed

tomography (CT) using a Toshiba Aquilion 16 CT unit (Toshiba Medical

Systems, Nasu, Japan). Acquisition parameters through the whole body

exploration of the turtle were 5mm slice thickness and 5mm slice interval, with

0.5mm retro-recon acquisition under lung and mediastinal algorithms. Images

were post processed with Osirix software version 3.3.1 (Pixmeo, Geneva,

Switzerland) and Philips Brilliance Workspace CT software (Koninklijke

Philips, Netherlands). A 3D air volume was recreated through volumetric

segmented reconstruction (volume rendering).

Based on imaging findings upon arrival at the rehabilitation center and/or post

mortem examinations, the severity of gas embolism was scored based on total amount

of intravascular gas observed and the distribution (Table 1):

-Mild embolism: small amount of gas was only evident at the kidney region on

ultrasound and LL radiographic projection.

-Moderate embolism: larger volume of gas was present in kidney region, being

clearly evident in ultrasound, LL and also even DV radiographic projections.

Other minor vessels in the periphery of the coelom or the liver were also full of

gas (gas angiograms) on DV radiographs. On ultrasound, occasional free gas

bubbles could be observed in the lumens of major vessels and cardiac chambers

(mostly the right atrium).

- Severe embolism: Gas was evident in kidney, liver, major systemic vessels and

even cardiac chambers in DV radiographs. Kidney ultrasound images were often

8
 impeded by the large amount of gas present in the area. Abundant bubbles in the

blood stream: gas accumulations were present in most cardiac chambers and

larger vessels.

Treatment

Individuals without clinical signs and mild embolism detected in imaging studies

did not receive any specific supportive treatment on arrival. Individuals that were

unresponsive or exhibited neurologic signs, such as stuporous behavior, atonic or single

retracted extremities, or reduced sensitivity of the skin as detected by pinching with

forceps, received supportive therapy including normal saline solution (FisioVet® saline

B. Braun Medical SA, Barcelona, Spain) (10-15ml/kg body weight (bw)) intravenously

(IV) and/or subcutaneously (SC). Additional drugs commonly used based on severity of

symptoms included: cardiotonics (atropine 0,1mg/kg bw intramuscularly (IM),

(Atropine Braun 1mg B. Braun Medical SA, Barcelona, Spain)), respiratory stimulants

(doxapram chlorhydrate 5-10mg/kg bw IM (Docatone-V® Fort Dodge Veterinaria SA,

Girona, Spain)), analgesics (meloxicam 0,2 mg/kg IM bw (Metacam® Boehringer

Ingelheim Vetmedica GmbH, Rhein, Germany), tramadol 5-10mg/kg bw IM (Tramadol

Normon, Laboratorios Normon SA, Madrid, Spain)), corticoids (dexamethasone 0,5-

1,2mg/kg bw IM, (Fortecortin® 4mg, Merck SL, Madrid, Spain)) and/or supplemental

oxygen therapy through an endotracheal tube (Rüsch® Uruguay Ltda., Montevideo,

Uruguay), face mask (Kruuse®, Langueskov, Denmark) or commercial critical care unit

(Vetario Intensive Care Unit, Brinsea Products Ltd., Sanford, England).

Recompression with hyperbaric oxygen was applied to two clearly lethargic and

poorly responsive animals with moderate embolism (one of them with evident paresis

and retraction of the hind extremities under the shell). Pressurization was achieved

9
using a power disconnected regular autoclave (Selecta, Presoclave 30, J.P. Selecta SA,

Barcelona, Spain) modified to work as a hyperbaric chamber by means of a connection

of a pressurized oxygen cylinder to the draining tube of the autoclave. Animal breathing

inside the chamber was stimulated with a previous injection of doxapram chlorhydrate

and needle insertion at the acupuncture GV26 point (Litscher 2010). As there were no

previous references for reptiles, the most commonly used human recompression-

decompression table was applied (Vann et al. 2011). Briefly, an initial pressure of

1.8atm (relative pressure) was applied for 1hr, then decreased to 1atm over the next

30min, stabilized at 1atm for another 3hrs and finally progressively decreased to surface

pressure (0atm relative pressure) over 30min. Pure oxygen was used for the entire

procedure. Monitoring of the animals inside the chamber was not possible.

Recompressed-decompressed individuals were reevaluated through simple radiology,

ultrasound and CT (only one case) before and immediately after treatment. Only turtles

smaller than 30cm straight-line carapace width were candidates for decompression due

to the size of the chamber. Larger individuals were followed clinically for outcome

without decompression treatment.

Postmortem examination

Necropsies were performed within 24 hrs after retrieval from fishing gear (except

in one case at 36h) or in less than 12hrs following death at the rehabilitation center.

Systematic sea turtle necropsy procedures were performed (Flint et al. 2009), with extra

caution to minimize artifactual gas infiltration by traction of tissues and during

sectioning blood vessels (especially when removing the plastron). Presence of

intravascular gas was specifically documented. Samples of skin, muscle, pre-femoral

fat, liver, spleen, heart, major vessels, brain, intestine, salt glands, plastron, thyroid

10
gland, both kidneys, both lungs, both gonads and any gross lesions were routinely

collected for histopathology. All tissues were fixed in 10% neutral buffered formalin,

processed routinely into paraffin blocks for histopathology and stained with

hematoxylin and eosin (H&E). Histopathological examination was conducted in all

individuals suspected from DCS. Gas sampling and analysis was performed as

previously described (Bernaldo de Quirós et al. 2011) in 13 different samples collected

from the same individual approximately 36 hours post mortem.

Ethical statements

Animal care was applied within institutional guidelines. In live animals, clinical

information generated for this study was derived from the regular veterinary procedures

provided in order to establish an appropriate diagnosis for the application of the best

feasible treatment. Hyperbaric oxygen treatment was administered with Governmental

and veterinary medical consent and was decided to be necessary based on fatal outcome

of similar cases without hyperbaric treatment.

RESULTS

Sea turtle bycatch was higher during months of the year when the water was

coldest, particularly from November to March. Regional average monthly water surface

temperature ranged from 13.4ºC in February up to 26.3ºC in August (SeaTemperature.

www.seatemperature.org (accessed 14 Jan 2014)) (Table1).

Clinical diagnosis, treatment and outcome

11
 Evidence of gas embolism (GE) was found in 6/18 (33.3%) gillnet and 23/49

(46.9%) bottom trawl net bycatch cases (43.3% of all incidental captures) from a depth

range between 10-50 m and 25-75 m, respectively. Summary information for different

cases is provided in Table 1. The severity of GE was assessed to be mild in 16 cases,

moderate in 9 cases and severe in 4 turtles.

According to the fishermen, clinically abnormal turtles exhibited two clearly

distinct anomalous behaviors when they surfaced within the fishing gear: comatose or

initially hyperactive progressing to stuporous with increasing surface time. Some of the

comatose animals showed aspiration of sea water in the respiratory tract as evidenced

by an alveolar pattern in radiographs and expelled copious fluid after endotracheal

intubation for resuscitation. These animals were diagnosed as drownings and generally

responded well to conventional emergency treatment (Norton 2005).

Twenty-one loggerheads arrived at the rehabilitation center alive and were

clinically evaluated. All individuals presented with good body condition and normal fat

stores. Eight exhibited normal behavior, four were comatose, and nine turtles were

hyperactive or developed progressive neurological symptoms, including limb paresis or

loss of nociception. The latter group was all caught by trawlers and in some cases

terminally displayed rigid pressing of the front flippers against the plastron (Fig. 1a and

1b). These turtles also exhibited an initially increased hematocrit, positive flotation and

erratic swimming when returned to water. Without hyperbaric treatment, neurological

signs gradually progressed to complete unresponsiveness and death within 72 hrs of

capture. Additional animals may have had these signs upon capture and become

comatose or died before arrival at the rehabilitation center.

In radiographs, intravascular gas was observed as radiolucency within or

distending the heart and vessels (Fig. 1c). The lungs were partially collapsed in severely

12
affected individuals as evidenced by reduction in field volume and increased

radiodensity. In mild cases, latero-lateral projections resulted in the most diagnostic

radiographs, providing higher sensitivity than dorso-ventral views for gas visualization

within the renal vessels.

Gas bubbles were detected by ultrasound as hyperechoic spots, typically with

comet tail artifacts. In all affected individuals, renal ultrasound revealed the presence of

gas inside the parenchyma and kidney vessels (Fig. 1d). Cardiac ultrasound

demonstrated a much higher prevalence of bubbles in the right atrium compared with

the left, similar to the pattern observed in scuba divers (Francis & Simon 2003).

CT imaging techniques were used in 11 cases to confirm the presence and

distribution of GE (Fig. 1 and 2a, 2b, 2c). Embolism was confirmed within the kidneys,

liver, heart, spleen, and central nervous system (Fig. 2a and 2b). In simple CT slices,

gas was revealed inside different regional vessels as hypoattenuated (black) compared

to surrounding tissues. As in radiographs, the lungs of severe cases were

hyperattenuated (whiter) and expansion reduced due to partial collapse. Notably,

midline-sagittal multiplanar reconstructed CT images demonstrated gas within the

vertebral canal and central nervous system (Fig. 2c) that was not seen by ultrasound or

in radiographs. Gas within or surrounding the nervous system was apparent even in mild

cases (Fig. 2c). These findings were observed to be compatible with life even without

treatment, although subsequent renal and/or neurological damage or temporal functional

impairment could not be discarded.

Five out of 49 (10.2%) bycatch trawl animals were active while presenting

moderate to severe GE at the arrival to the rehabilitation center. More animals could

have surfaced on board with similar symptoms dying before arrival at the rehab center.

All these cases of GE resulted in death within 48-72 h post-capture if not treated with a

13
hyperbaric protocol while severe cases were generally lethal in the first 6-8 h, thereby

reducing the chances for hyperbaric treatment. Two of these animals survived following

a hyperbaric oxygen treatment (Table 1). After treatment neurological signs resolved

and the sea turtles recovered normal activity. Post-treatment radiographs and CT

confirmed the dissipation of most of the intravascular gas and re-expansion of the lungs

(Fig. 2d and 2e). After two months under observation, both were considered clinically

healthy and were reintroduced into the Mediterranean Sea.

Pathological diagnosis

Complete necropsies were performed on a total of 16 deceased bycaught

loggerheads (8 dead on the gear, 3 dead during transport and 5 dead at the rehabilitation

center). Gas embolism was found in 13 turtles (81%), which included 8 out of the 11

that arrived dead and the 5 that died following admission. In severe cases, gas was found

within the median abdominal, mesenteric, gastric, pancreatic, hepatic and renal veins,

as well as within the post cava and other major vessels (Fig. 3). The atria (especially the

right atrium) and the sinus venosus were distended by gas (Fig. 3). In very severe cases

the spleen was gas dilated. Grossly, the kidneys had multifocally extensive red areas

consistent with marked congestion. Segmental congestion of the intestinal mucosa was

also present. The lungs of some animals were partially collapsed with cranial pulmonary

emphysema. Various amounts of fluid within the respiratory tract were evident in some

individuals. Other gross findings included coelomic transudate in individuals with

severe GE and partially digested contents within the stomach and intestine in most

turtles. In moderate cases, GE were not as obvious as observed by imaging and required

careful examination. Gas was most visible within mesenteric and renal vessels, as well

14
as the postcava and sinus venosuson. In one mild case with concurrent radiographic

evidence of drowning, GE could not be found macroscopically in any explored tissue.

Histopathological findings included moderate to severe multisystemic congestion

with the presence of intravascular gas bubbles in multiple organs including the lung,

liver, kidney, and heart (Fig. 3). In addition, perivascular edema and hemorrhages,

varying in extent and severity were also present in different tissues. Acute, multifocal,

myocardial necrosis with vacuolar degeneration of myocytes, alveolar edema, diffuse

microvacuolar hepatocellular degeneration, sinusoidal edema, and intrahepatocyte

hyaline globules were frequently evident.

Gas composition analysis in one case confirmed that the main component was

nitrogen (75.3 ± 0.9% μmol), followed by carbon dioxide (18.6 ± 2.0% μmol) and

oxygen (6.0 ± 1.3% μmol).

DISCUSSION

Differential diagnoses

Alternative differential diagnoses for GE, including traumatic or artifactual intrusion

and putrefaction, were ruled out based on clear demonstration of antemortem occurrence

in live turtles and absence of any apparent traumatic injuries or surgical procedures.

Pulmonary barotrauma could cause arterial air embolism (Vann et al. 2011); however,

the physical requirements for barotrauma are not met in bycaught turtles. Turtles are

breath hold divers, meaning that the internal pressure in the ediculi (homologous to

mammalian alveoli) at the beginning and at the end of the dive would be the same or

even lower at the end of the dive due to oxygen consumption. Thus, overexpansion of

the lungs is very unlikely. In addition, gas was mainly found in the venous side of the

15
circulation (as in DCS) instead of in the arterial side. In addition, necropsied turtles

were in a good state of preservation and systemic GE was consistent with pathological

findings described in DCS in human divers and in stranded beaked whales (Knight 1996,

Francis & Simon 2003, Jepson et al. 2003, Fernandez et al. 2005). Also, hydrogen, a

putrefaction marker, was not detected in the gas samples collected during necropsy

(Bernaldo de Quirós et al. 2013a). Furthermore, decompression-related GE is the only

process that is reversed by a hyperbaric treatment (Vann et al. 2011). Dissipation of GE

and clinical response fulfill human criteria for medical diagnosis of DCS (Paulev 1965,

Vann et al. 2011).

Key facts for the finding of GE in sea turtles

To the best of the authors’ knowledge, no report of live or dead wild sea turtles

suffering from acute GE has been previously presented. Most of the literature and

research done until present, considers this possibility as highly improbable based on

different anatomo-physiological adaptations, including relatively small and collapsible

lungs (Berkson 1967) and confinement of lung gas to non-respiratory, cartilage-

reinforced airways during deep dives (Kooyman 1973, Lutcavage et al. 1989, Lutcavage

& Lutz 1997). The metabolic adaptations and physiological mechanisms underlying

their diving capacity have been the subject of intense interest for many years, including

early studies on forced submergence response in laboratory settings (Berkson 1966) and

more recent physiological investigations based on sophisticated remote-monitoring

technologies in free-swimming sea turtles (Hochscheid et al. 2007, Southwood 2013).

Berkson (1967) pressurized green turtles to different depths in a hyperbaric

chamber demonstrating tolerance to over 100 minutes of forced submergence at 18-

16
25ºC. Two animals compressed to 18.7atm died several hours after compression (one

fast compression and the other in progressive steps) and then fast decompression with

numerous gas emboli observed in capillaries of the cervical fascia and right atrium.

Death was attributed to gas emboli in the brain after emergence. The study concluded

that equilibrium conditions with full nitrogen solubilization were never attained even

during a prolonged deep dive (at different depths), providing some kind of underlying

protective mechanism, but, in certain extreme circumstances, enough nitrogen could

enter the blood to render the green turtle susceptible to gas emboli in the brain and death

after emergence. Our findings with wild individuals under field conditions are

significantly different. We observed dramatic lesions, with not only bubbles but actually

several milliliters of gas in wild animals entrapped at much shallower depths compared

to Berkson's studies. The explanation of this disparity remains uncertain, but could be

attributed to different factors, including animal species, time of forced submergence,

water temperature, movement capabilities when submerged (Berkson’s animals in the

chamber were fastened to a board with very restricted in movement inside the chamber)

and the previous diving profile of exposed individuals. Situations in which wild sea

turtles are forcibly submerged due to entrapment in fishing gear suggest that behavioral

and physiological responses are drastically different from what has been recorded under

controlled laboratory conditions (Berkson 1966, Lutz & Bentley 1985, Lutz &

Dunbarcooper 1987, Harms et al. 2003, Stabenau & Vietti 2003, Snoddy et al. 2009,

Southwood 2013).

Multiple studies reveal that entanglement in fishing gear has significant effects on

the physiology of sea turtles (Lutz & Dunbarcooper 1987, Harms et al. 2003, Stabenau

& Vietti 2003, Snoddy et al. 2009, Snoddy & Southwood Williard 2010) but have never

described DCS. Various factors may have contributed to the discovery in the current

17
study, including close collaboration with fishermen allowing access to alive and fresh

dead bycaught animals, capacity for intensive medical evaluation following capture,

availability of modern imaging technology and familiarity with diving animals and

pathology related to GE. In addition, local oceanic conditions and type of fisheries could

be unique relative to the circumstances of previous studies.

DCS findings in other marine air-breathing vertebrates: comparative

physiology

Similarly to the present description in sea turtles, DCS had not been suspected

in marine animals until GE consistent with DCS was described in beaked whales that

mass stranded in close temporal and spatial association with military exercises using

high-intensity mid-frequency active sonar, as well as in single stranded cetaceans in the

UK coast (Jepson et al. 2003, Fernandez et al. 2005, Jepson et al. 2005, Fernández et al.

2013). Over the last decade, there has been an increasing body of evidence showing that

marine mammals may suffer from acute and chronic GE, including the description of

gas bubbles forming in tissues of fatally bycaught marine mammals trapped in nets at

depth and rapidly brought to the surface (Jepson et al. 2003, Moore & Early 2004,

Fernandez et al. 2005, Jepson et al. 2005, Moore et al. 2009, Bernaldo de Quirós et al.

2011, Bernaldo de Quirós et al. 2012, Bernaldo de Quirós et al. 2013b). In a recent study

of gas composition of bubbles in bycaught dolphins, the authors concluded that nitrogen

rich bubbles were formed by off gassing of supersaturated tissues (Bernaldo de Quirós

et al. 2013b). These findings provide new evidence of nitrogen accumulation in breath-

hold diving taxa despite anatomical and physiological adaptations. However, all marine

mammal examples were already dead upon discovery, thus a definitive diagnosis of

DCS could not be clinically established. Sea turtles afford a new opportunity for

18
studying this condition due to their amazing capacity for anoxia tolerance (Berkson

1966, Lutz & Bentley 1985, Lutcavage & Lutz 1997, Southwood 2013) and relative

ease of handling, treatment and transport compared to marine mammals.

Hypothetical patho-physiological mechanism

The causal relationship between breath-hold diving in humans and DCS is

increasingly being accepted due to the growing number of cases of DCS-like symptoms

(Schipke et al. 2006). The pathophysiology of this condition in bycaught sea turtles is

unknown.

Turtles have three muscular cardiac chambers, two atria and one ventricle, which

allows some intraventricular mixing of systemic and pulmonary blood flow (Shelton &

Burggren 1976, Hicks & Wang 1996, Wang et al. 2001). All sea turtles also have

vascular adaptations for shunting during diving, including muscular sphincters within

the pulmonary arteries and an anastomosis between the left and right aorta (White 1976,

Wyneken et al. 2013). Cardiac shunting in sea turtles may confer some advantages under

certain physiological conditions, such as diving (Hicks & Wang 1996), but could also

risk bypass of gas bubbles from the pulmonary to systemic circulation (Germonpre et

al. 1998, Harrah et al. 2008, Vann et al. 2011).

Different studies correlate exercise with breathing frequency, pulmonary blood

flow and heart rate in green turtles (Butler et al. 1984, West et al. 1992, Southwood

2013). Exacerbated muscular activity leading to lactic acid built up is induced in free-

swimming bycaught turtles, even under very short forced submersion episodes (Lutz &

Dunbarcooper 1987, Stabenau et al. 1991, Stabenau & Vietti 2003). Additionally, heart

rate and pulmonary blood flow in turtles often increase immediately before breathing

19
starts, which is suggestive of central mechanisms based on elevated sympathetic tone.

This effect could also be induced by catecholamine release during fight-or-flight

response resulting from capture (White & Ross 1966, Shelton & Burggren 1976, West

et al. 1992, Wang & Hicks 1996, Wang et al. 2001).

We hypothesize that entrapped, submerged turtles develop DCS due to increased

activity and catecholamine-induced sympathetic induction/parasympathetic inhibition.

These processes disrupt the normal physiological and protective vagal diving reflex that

minimizes blood flow through air filled pressurized lungs during diving. This

hypothesis is supported by observed disruption of the dive response in struggling green

sea turtles that are forcibly submerged (Berkson 1966).

Although speculative, the shunting ability in diving reptiles may not only represent

a mechanism of regulating metabolism through modulation of oxygen supply to the

tissue (Wang & Hicks 1996, Wang et al. 1997, Wang et al. 2001), but also could

minimize nitrogen solubility in blood and subsequent risk of DCS. Sea turtles and sea

snakes have the highest shunting capabilities (White 1976, Lillywhite & Donald 1989,

Wyneken 2009). If this is the case, the longer the duration of the forced submergence,

the higher the amount of nitrogen absorbed. As breath-hold divers, bycaught turtles

cannot eliminate all absorbed gas at depth nor in ascent while the gear is retrieved.

When the animal is surfaced with the fishing gear, gas bubbles start to form. We also

speculate that the spastic retraction of the limbs (Figure 1a) may in part be comparable

to the bending of limbs in humans. In our experience, it takes several hours or days for

GE to resolve in turtles with even mild embolism.

Potential contributing factors

20
 Environmental conditions, including water temperature and depth and time of

immersion, are known to affect risk of DCS in humans and likely are important in sea

turtles as well (Germonpre et al. 1998, Harrah et al. 2008, Vann et al. 2011). Tolerance

of forced submergence in sea turtles is known to be affected by turtle size, turtle activity,

and water temperature (Lutcavage & Lutz 1991, Stabenau et al. 1991).

In the present study, the highest rate of bycatch occurred between November and

March, when most GE cases were encountered. When considered by proportion of

captured animals with DCS, February, September, and October (average surface

temperatures 13.4ºC; 24.5ºC, and 22.0ºC, respectively) were the months with highest

occurrence. Hochscheid et al (2007) reported that Mediterranean loggerhead sea turtles

increase time of submergence and rest on the bottom during the coldest periods of the

year. This overwintering behavior could explain the higher trawling capture rates

observed during winter in our region. However, the implications of temperature remain

unclear from this study due to limited sample size and bias for presentation of cases

during colder months.

Lower body temperatures in sea turtles compared to mammals, has been considered

a potential protective mechanism against DCS, as body fluids would tolerate a higher

pressure of gas dissolved without forming bubbles (Fossette et al. 2010). However,

decrease in temperature would also increase nitrogen solubility at depth proportionally,

thus increasing the risk of DCS when surfaced compared to mammals. Overwintering

behavior could thereby increase the risk of DCS upon capture, especially if the turtle

warms up out of water.

Regarding the influence of depth, some animals captured by trawlers fishing at over

60 m depth were full of gas after surfacing while others of similar size, coming from the

same waters, same fisheries, same depth and during the same season had no detectable

21
gas. Possible explanations for this disparity are differences in actual depth of capture

(unknown for trawler captures), the length of time submerged, and individual

susceptibility to stress. Large depths do not seem to be required for the development of

the DCS in sea turtles, as animals entrapped in gill nets as shallow as 10-20m deep

presented with moderate or severe GE. One mild case was observed in a turtle bycaught

by a vessel fishing at 30 m, although all severe cases of GE in trawlers occurred in turtles

bycaught by nets fishing at over 60m depth. Based on these findings, even coastal or

shallow fisheries like bottom trawls used to capture shrimp and other coastal fish

resulting in high bycatch (Finkbeiner et al. 2011) could induce DCS in sea turtles.

Duration of submergence is another consideration. Berkson (1967) determined

that submersion time was not a limiting factor to allow nitrogen saturation during diving,

as the nitrogen tensions in blood reached a maximum and then dropped or leveled off

well below saturation level. The author suggested that there might be an underlying

mechanism for compensation. In contrast, our results suggest that time of submersion is

correlated with severity of GE. Animals entrapped in gillnets (generally set at depths as

shallow as 10-15m but for an average of 12hrs) tend to show more dramatic embolism

than similar animals captured in trawlers in the same waters at a significant deeper depth

(25-70m) but with much shorter operating times (2-6hrs).

Potential impacts and future research

The actual contribution of DCS resulting in sea turtle mortality on a global scale is

unknown; however, it is notable that our observations originated from interaction with

two gear types of foremost concern with regard to sea turtle bycatch. Bycaught sea

turtles that are initially active are usually immediately released and are not considered

22
lethal interactions. Our results show that many turtles could have GE and may

subsequently die within hours or days post-release. Mortality following fisheries

interaction could be much higher than previously estimated. Accurate data on both

immediate and post-release mortality data are crucially important for refining the current

mortality estimates used to govern management decisions with far-reaching

conservation, economic and social consequences (Southwood 2013).

The cause of death in comatose and dead net caught turtles should be re-evaluated

to clarify the percentage of animals dying from DCS instead of drowning or dying from

both. Current procedures used aboard fishing vessels to revive comatose turtles, while

useful for drowning, are probably ineffective for DCS. Although GE can be detected in

the field (e.g. with on-board portable ultrasound) any mitigation measures should focus

on prevention and minimization of risk of DCS given that effective treatment is unlikely

to be practical under most at-sea conditions.

CONCLUSIONS

The current study demonstrates that bycaught marine turtles can develop and die

from DCS. Diagnosis was based on clinical signs, detection of intravascular gas by

imaging and necropsy, gas composition analysis, and successful resolution with

hyperbaric treatment. To our knowledge, these findings represent the first example of

DCS in air-breathing marine vertebrates that fulfill all of these medical diagnostic

criteria, providing new clues for the better understanding of the diving response and

DCS avoidance in other breath holding diving vertebrates (Piantadosi & Thalmann

2004).

23
 This discovery has significant implications on sea turtle conservation. It would be

important in light of the present findings to review regional sea turtle bycatch

intervention protocols worldwide after elucidating the real prevalence of the condition

based on different fisheries techniques, geographic areas, oceanic conditions, sea turtle

species and individual characteristics.

ACKNOWLEGEMENTS

We thank all professionals at the Oceanográfic, especially at the ARCA

Rehabilitation Centre, for their many efforts and complete dedication to the best animal

care. In particular, we are grateful to all the fishermen contributing to the project as well

as to the Consellería de Infraestructuras, Territorio y Medio Ambiente of Valencia

Community Regional Government, very especially to Juan Eymar and all the other

technical workers from "Forn del Vidre", "La Granja del Saler" and "Santa Faz" who

made the Project possible. We also thank Roberto Sanz-Requena from Quirón Valencia

Hospital for his assistance in imaging post-processing. We would like to thank the

"Institut Cavanilles de Biodiversitat i Biologia Evolutiva", University of Valencia, for

their collaboration on necropsy procedures. Also thanks to Dr Javier Guayart for his

contribution in information about local fisheries operations and bycatch. We thank the

ULPGC Cetacean Research Unit, Dr. Brian Stacy from NOAA Fisheries, Dr. Andreas

Fahlman, Dr. Micah Brodsky as well as other anonymous reviewers for their comments

and suggestions. This work has been supported with funds from the Pfizer Foundation,

the VISAVET Center of Complutense University of Madrid, the Oceanográfic of the

“Ciudad de las Artes y las Ciencias” of Valencia, and by the research projects: CGL

2009/12663, CGL2012-39681, and SolSub C200801000288.

24
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Table 1. List of bycaught turtles diagnosed with gas embolism, biological, clinical and pathological data.

Depth range CCL range Temperature

Gear type Clinical classification GE Diagnosis Treatment End result
(m) (cm) range (ºC)
1 Mild/Moderate None
2 Comatose
Gillnet 1 Mild/Drowned Medical
10.5 - 50 30.2 – 41.5 13.4 – 24.5 Dead
(n=6) 3 Moderate
4 Dead None
1 Severe
8 Normal 8 Mild Medical Reintroduced
2 Mild Medical Reintroduced
2 Mild/Moderate Medical Reintroduced
Medical (1) Dead
9 Hyperactive Hyperbaric
3 Moderate
/Neurologic & Medical Reintroduced
(2)
Trawl
30 - 75 28.6 - 74 13.8 - 25 1 Moderate/Severe Medical Dead
(n=23)
1 Severe Medical Dead

2 Comatose 2 Mild/Drowned Medical Reintroduced

1 Moderate
4 Dead 1 Moderate/Severe None Dead
2 Severe
Abbreviations: CCL, Curved Carapace Length; Temp, average sea superficial temperature on the month of capture.

34
1 FIGURES AND LEGENDS

3 Figure 1. Sea turtles at reception: signs (a and b) and preliminary detection of

4 clinical gas (c and d). (a), Case CcGE21 at arrival. Moderate systemic GE. Note spastic

5 retraction of the hind limbs under the carapace before recompression therapy. These

6 signs resolved immediately after hyperbaric oxygen treatment. (b), External aspect of

7 case CcGE18 with severe systemic GE after a few hours postmortem. This animal

35
 8 arrived alive and did not respond to emergency medical treatment. Note retraction of all

9 four extremities under the body at rigor mortis. (c), Dorso-ventral digital radiographic

10 image (technique, 90Kv, 10mAs, 1 m focal distance, right side is to the left of the image)

11 of case CcGE15 with severe systemic GE. Note the lumen delimitation of right and left

12 atrium, sinus venosus, and major vessels by the massive presence of intraluminal gas

13 (evidenced as a radiolucent region). Minor vessels are also clearly visualized in the area

14 of projection of the liver and kidneys (gas angiograms). (d), Renal ultrasound of patient

15 CcGE23 with moderate systemic GE. Image obtained with a 12MHz linear probe on the

16 left prefemoral fossa with a ventrolateral-dorsomedial orientation. Note the presence of

17 intraluminal gas in renal major vessels as evidenced by hyperechoic spots and comet

18 tail artifacts (long white arrow). Smaller collections of gas are also clearly visualized

19 disperse inside the kidney parenchyma (short white arrows). Renal margin (yellow

20 arrows). Abbreviations: RP = right precava, LP = left precava, RA = right atrium, SV =

21 sinus venosus, HV = hepatic veins, PC = postcava, H = venous hepatic system , MC =

22 marginocostal vein, and K = kidney.

23

36
24

25 Figure 2. Evidence of GE on computed tomography. (a), Transverse image of mid-

26 cranial coelomic region at the level of the heart of case CcGE15 with severe systemic

27 GE. There is evidence of intraluminal gas (black) inside the heart and major vessels.

28 Gas is also present within the venous hepatic system and vertebral canal. Lungs are

29 hyperattenuated (whiter) due to partial collapse. (b), Dorsal oblique view of 3D volume

30 recreation through volumetric segmented reconstruction (volume rendering) from

31 patient CcGE15 with severe systemic GE. Note the presence of gas within the different

32 peripheral and intracoelomic vessels. Lungs contain less gas than normal. The kidneys

37
33 are clearly visualized due to the massive presence of intravascular gas in this region.

34 (c), Mid-sagittal image of patient CcGE20 with mild systemic GE. Notice the presence

35 of abnormal gas at the central nervous system, spinal cord and renal and minor hepatic

36 vessels. (d) and (e), Dorsal views of 3D air volume rendering view of total gas volume

37 inside the patient CcGE23 with moderate systemic GE before (d) and after (e) oxygen

38 hyperbaric treatment for recompression. Images were obtained 6 hours apart. All gas in

39 brighter color and intravascular gas pointed with stars. (d), Notice the delineation of

40 hepatic veins and renal vessels by the presence of intraluminal gas before treatment.

41 Lungs expansion is also reduced. (e), Most gas contained in the large vessels has almost

42 disappeared after hyperbaric treatment indicating gas reabsorption while pulmonary

43 expansion is back to normal. Few minor vessels still contain gas in the periphery of the

44 hepatic and renal projection areas. Abbreviations: VC = vertebral canal, L = lung, H =

45 venous hepatic system, HV = hepatic veins, SV = sinus venosus, RA = right atrium, MC

46 = marginocostal vein, A = aorta, K = kidney, and SC = spinal cord.

47

38
48

49 Figure 3. Gross (a-c) and histopathological findings (d-f). (a), Caudo-ventral view of

50 the heart, dorsal surface, of case CcGE18 with mild/moderate systemic GE. The right

51 atrium and sinus venosus (amplified) are diffusely distended with a moderate amount of

52 intracameral, gas bubbles. (b), Left dorso-lateral view of the stomach greater curvature

53 (after being reflected cranially) and liver of case CcGE14 with moderate systemic GE.

54 Note that gastric veins from greater curvature and the pyloric vein are diffusely

55 expanded with variably sized gas bubbles. (c), Small intestine and mesentery of case

56 CcGE14 (moderate systemic GE). Note that mesenteric veins are diffusely expanded

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57 with a large amount of variably sized gas bubbles, coalescing at the mesenteric venous

58 root. (d), Right atrium of case CcGE15 (severe systemic GE). Atrial lumen shows

59 multifocal to coalescing, variably sized, round to oval, fat-negative gas emboli,

60 compressing the adjacent myocardium. H&E; 2x. (e), Kidney of case CcGE18

61 (mild/moderate GE) Interrenicular veins are multifocally occupied by round to oval,

62 variably sized, fat-negative gas emboli. H&E; 10x. (f), Lung of case CcGE7 (severe

63 systemic GE). Pulmonary veins show intravascular, variably sized, round to oval, fat-

64 negative gas emboli. H&E; 10x. Abbreviations: V = Ventricle, RA and LA = right and

65 left atriums, LPV = left precaval vein, P = pericardium, PF = pericardial fluid, SV =

66 sinus venosus, LLL = liver left lobe, S = stomach, GA = gastric artery, GV = gastric

67 veins, PY = pylorus, PV = pyloric vein, PA = pancreas, IN = intestines, MA and MV =

68 Mesenteric arteries and veins.

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