amphibian biology 2014

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Amphibians

Biology, ecology and conservation topics and their applications in ecotoxicology

Ecotoxicology of amphibians and reptiles: from theory to practice. Aveiro, April 2014

Manuel Ortiz Santaliestra

Institute for Environmental Sciences

University of Koblenz-Landau (Germany)

[email protected]

Outline

INTRODUCTION

Origin and evolution

Diversity

LIFE HISTORY

Breeding migrations

Mating, fecundation and oviposition

Eggs and embryonic development

Larval development

Metamorphosis

Sexual determination

Maturation and adulthood

ENVIRONMENTAL PHYSIOLOGY

Thermoregulation and water balance

Excretory physiology

Overwintering and aestivation

Gaseous exchange

ECOLOGY

Habitat

Feeding ecology

Antipredatory strategies

CONSERVATION

Global decline

Threats

Outline

INTRODUCTION


Diversity

LIFE HISTORY

Breeding migrations



Larval development

Metamorphosis







Gaseous exchange

ECOLOGY

Habitat

Feeding ecology


CONSERVATION

Global decline

Threats


©Geoff Simpson

©Dan Nedrelo

© Heidi & Hans-Jürgen Koch

© Dan L. Perman

An uncompleted step towards the independence from water

Amphibians = two lives Reptiles

Origin and evolution Current diversity of amphibians

Order Gymnophiona (Caecilians)

200 species (all from Neotropic)

Order Caudata (newts and salamanders)

660 species (10 in the Iberian Peninsula)

© Chris Harrison

Order Anura (frogs and toads)

6396 species (19 in the Iberian Peninsula)

http://amphibiaweb.org/ (updated on April 6th 2014)

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Overview of Iberian amphibians

African species crossing the Strait ofGibraltar during the Messinian period(5.5 million years b.p.)

Eurosiberian species searching fromrefuge during Pleistocene glaciations(80,000 years b.p.)

The cosmopolite Iberian batrachofauna (African, Eurosiberian and endemic species)

Rana temporaria

Pleurodeles waltl

Iberian endemism originated byisolation, area reduction or habitatlimitation Chioglossa

lusitanica

Outline

INTRODUCTION


Diversity

LIFE HISTORY

Breeding migrations



Larval development

Metamorphosis







Gaseous exchange

ECOLOGY

Habitat

Feeding ecology


CONSERVATION

Global decline

Threats

Reproductive modes variability: A key for success

The common reproductive strategy:

Terrestrial adults

Aquatic breeding, eggs and larvae

Breeding migrations

0

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Ammonium nitrate dose (kg N / Ha)

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Adults migrate from winter refuges to breeding ponds

Orientation by magnetic fields, firmament, chemical cues and conspecific or heterospecific calls.

Risks:

•Road mortality

•Pollution in fields crossed during migrations

Χ2=23.204; N=28; p=0.001

Ortiz-Santaliestra et al. (2005) Bull Environ Contam Toxicol 75, 662-669

Impact of terrestrial ammonium nitrate application on Iberian newt

Recommended level of application

Mating and fecundation in Anura

Males arrive first. Two strategies depending on size (male quality):

•Bigger males attract females (deeper calls). They wait for females within the ponds territorialism

•Smaller males do not attract females. They wait for females outside the water to couplebefore their entering the ponds

Amplexus (coupling). External fecundation

Some exceptions:Internal fecundation

(Ascaphus)

Terrestrial amplexa (e.g., Alytes)

© Brad Moon © Daniel Phillips

Inguinal

Axilar

Cephalic

Wells (2007) The Ecology and Behavior of Amphibians. Chicago University Press

Mating and fecundation in Caudata

Males arrive first and wait for females within the water

Mating after courtships which end with spermatophore deposition (internal fecundation)

Spermatophore deposition and pick up in a terrestrial courtship

© Oregon State University

Some exceptions:

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Oviposition

Although most species are oviparous, there are ovoviviparous and viviparous species

Alytes(~40 eggs)

Bottom: low predation, high asphyxia

Surface: high predation, low asphyxiaVegetation: low predation, high eutrophication

Bufo(~3000-5000 eggs)

Clutch size

Where to lay?

Streams: attached to stones, adapted to high O2 and water currents

Terrestrial eggs

© Thomas Reich

Oviposition

Isolated masses

Individual (newts)

How to lay?

Communal

© Andy Fion

© Henk.Wallays from Flickr

© Jenny Gitlitz

Parental care of the eggs

Foam nests (Chiromantis)

Wrapped by plants (Triturus)

Under stones or in cavities (Chioglossa)

Attached to the plants (Hyla)Shallow water

Direct parental protection

Carrying out (Alytes)

Covered by skin (Gastrotheca)

Gastric brooding (Rheobatrachus)

© Jack Goldfarb

© Merike Linammägi

© Louise Mentjies

© Luis Bravo

© Kevin Johnson

Egg protection beyond parental care

Jelly coat

Formed after absorptionof water once eggs havebeen laid

UnpalatabilityCamouflage

Dark top to camouflage against the bottom

Bright bottom to camouflage against the surface

Embryonic development

Gosner (1960) Herpetologica 16, 183-190

Risk of pollution during egg andembryonic stage:

•Removal of laying substrates

•Alteration of parental behaviours(oviposition, site selection, egg protection)

•Uptake of chemicals by jelly coat

•Maternal transfer of pollutants throughjelly coat

•Egg rotation

•Direct effects on embryonic development

The frog embryo teratogenesis assay - Xenopus

Standardized protocol to test teratogenesis

•Static system

•Freshly deposited embryos of Xenopus laevis

•25 embryos per glass in 140 ml + g soil

•4 replicates (total 100 embryos)

•Environment: FETAX solution

•20-25 tadpoles per tank in 10-12.5 liters

•Duration: 96 hours

•Phase 1) Screening test: control + 100% sample

•Responses are expressed as percent effect

•Phase 2) Final test

•LC50

•EC50

•Description of abnormalities

ASTM (2004) Report # E1439-98; Bantle et al. (1991) Atlas of Abnormalities: A Guide for the Performance of FETAX. OSU Press

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Larval development


Anura

Caudata

Larval morphological adaptations

Duelllman & Trueb (1994) Biology of Amphibians. John Hopkins University Press

Mouth morphology and position

Spiracle

Pond (low O2) Stream (high O2)

CAUDATA

Pond, benthonic

Pond, pelagic

Stream

ANURA

Larval development

Longer times(bigger tadpoles)

Shorter times(smaller tadpoles)

Juvenile survival

Reproductive quality

Avoid desiccation

Avoid predation

? Avoid pollution ?

Viviparity (S. salamandra)

Duration: from hours to years

Camouflage Unpalatability

Parental care

Exceptions:

Direct development

Protection© Tom Ray

Dendrobatidae Rhinoderma darwinii

© Fogdenphotos.com

Eleutherodactylus sp.

Grow or survive?

Advantages of phenotypicplasticity

Metamorphosis in Anura

Changes:

Respiratory system

Digestive track

Cranium and jaws

Pelvic girdle

Skin keratinization

Vision organs (cones rods)

Immunological modifications


Ecotoxicological interest:

Mobilization of reserves

Transport of accumulative chemicals from aquatic to terrestrial environment

Metamorphosis. Hormonal control

Amphibian metamorphosis assay for evaluation of EDCs:

Flow-through system

Water filtered and UV-treated

20-25 tadpoles per tank in 10-12.5 liters

Food: Sera Micron

Duration: 14-21 days

Endpoints:

-Periodical monitoring of developmental stage and snout-vent length

-Histological examination of thyroid gland at the end of the exposure

Duellman & Trueb (1994) Biology of Amphibians. John Hopkins University Press; OECD (2004) Report #ENV/JM/MONO(2004)17

Amphibian metamorphosis depends on thyroid hormones (major effects) and pituitary hormones

Metamorphosis. Immunological overview

-Development of adult immune system components

+

Maternal antibodies

Larval immune system

Developing adult immune system

Developed adult immune system

Rearrangement of immune defenses

Destruction of some components of the larval immune system

Thyroid hormones

Glucocorticoids

PerchlorateMalathion*Atrazine*

*Known immunotoxic effect during

amphibian metamorphosis

PCBsPCDDs

+ -

Robert & Ohta (2009) Devel Dynam 238, 1249-1270; Rollins-Smith et al. (1998) Immunol Rev 166, 221-230

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Highly complex mechanism

Genetic determination (XX/XY and ZZ/ZW systems, sometimes simultaneous inthe same species), that does not correspond to the phenotypic sex in manycases

Sex determination depends on the regulation of sex-determining genes (e.g.DM-W in Xenopus) which are different among species. Some species dependon multiple genes

Influence of temperature, although apparently only at temperatures out of theenvironmental range

Sex-reversal mediated by hormonal factors that keep population withinappropriate sex ratios

Nakamura (2009) Sem Cell Dev Biol 20, 271-282


Sexual maturity is usually achieved earlier by males than by females

Female’s have higher probability of dying before reproduction

Sex ratios of the breeding population are sometimes skewed towards males

Mechanisms of compensation:

•Females may live longer than males

•Young males are usually unsuccessful in defending a territory or unattractive to females

Davies & Halliday (1977) Nature 269, 56-58

Outline

INTRODUCTION


Diversity

LIFE HISTORY

Breeding migrations



Larval development

Metamorphosis







Gaseous exchange

ECOLOGY

Habitat

Feeding ecology


CONSERVATION

Global decline

Threats


Behavioural mechanisms © Ben Klocek

© Sandy Garfinkel

Basking to warm up

Use of refuges to cool down

Dermal uptake / elimination of water

10% of the body surface account for 70% of the tegumentary water diffusionHow will global warming

and increased UV radiation affect these behaviours?


Physiological mechanisms

•Evaporative cooling

•Regulation of osmolarity in body fluids

Salinity

Wright et al. (2004) J Exp Zool 301A, 559-568


Cree (1985) New Zeal J Zool 12, 341-348

Ammoniotelic or ureotelic depending on the life stage

L. ewingi

L. raniformis

Ammonia Urea

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Excretory physiology as a detoxification mechanism

Schmuck et al. (1994) Copeia 1994, 996-1007

< 1 mg/l NH4+-N < 1 mg/l NH4

+-N

5 mg/l NH4+-N 5 mg/l NH4

+-N

Physiological adaptations confer indirect protection against pollution

Ortiz-Santaliestra et al. (2010) Environ Pollut 158, 934-940

Low salinity site High salinity site

Same effects of N on growth occur at lower levels when animals are not adapted to salinity

Pelophylax perezi


Overwintering places:

Aquatic (muddy bottom): avoids freezing, risk of hypoxia (only cutaneous respiration)

Terrestrial: avoids hypoxia, risk of freezing cryoprotection: glucose accumulated within the cells while water is removed from the cells:

•Extracellular crystallization releases latent heat

•Intracellular crystallization is avoided because high osmotic concentration

Aestivation behaviours:

Burrowing

Cocoons (mud cover, shedded skin, mucous layer)

Ecotoxicological considerations:

Pre-hibernation/pre-aestivation are critical periods (accumulation of fatty acids, high membrane permeabilization to facilitate transport)

As periods of low metabolic rate, depuration of chemicals is kept to a minimum

Reserves (and potential accumulated pollutants) may be mobilized

Lithobates sylvaticusLives in areas reaching -40ºC

Body temperature drops till -5ºC

Costanzo et al. (1993) J Exo Biol 181, 245-255

© Doug Waylett

Gaseous exchange

Dermal: embryos, tadpoles, and adult aquatic stages

Gills: tadpoles and neotenic forms

Gill surface area may be related to O2 availability

Lungs: late tadpole stages (gulping) and terrestrial stages

Caudata

External gills

Anura

Gills internalized shortly after hatching

© Greg Dodge

Outline

INTRODUCTION


Diversity

LIFE HISTORY

Breeding migrations



Larval development

Metamorphosis







Gaseous exchange

ECOLOGY

Habitat

Feeding ecology


CONSERVATION

Global decline

Threats

Breeding habitat

Temporary ponds / streams

Artificial water bodies

Free from fish and other big predators

Problems:

Legally unprotected. Used as dumps

Unpredictable. High desiccation risk

Low water volume. High eutrophication risk

Accumulation of pollutants from adjacent fields

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Feeding ecology: diet

Herbivorous

(algae filtering, periphyton scrapping)

Carnivorous

(insects, worms, tadpoles and small vertebrates)

ANURA CAUDATA

Ecotoxicological considerations:

Generally opportunistic diet (sentinels of what is present in the environment)

Major effects of herbicides and insecticides

Main role in energy transfer within ecosystems (simultaneous predator andprey)

Feeding ecology: foraging

Feeding modes in larvae:

•Filter feeding

•Grazing from rocks or plants

•Syphoning

•Prey capture

•Oophagy

•Cannibalism

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** NS NS Density effects:

NS p > 0.05

** p < 0.05

Cannibalism has also an environmental component

Adaptive cannibalistic morphs

Larvae Adult

Normal

Cannibal

S. salamandra

Duellman & Trueb (1994) Biology of Amphibians. John Hopkins University Press

Ortiz-Santaliestra et al. (2012) Aquat Toxicol 110-111: 170-176

Feeding ecology: foraging

Sit and wait

(Ceratopryhs)

Jumping on prey

© Warren photographic

Lingual flips

Bolitoglossa

(7 milliseconds)

© Softpedia


Predator detection chemical and visual cues

Avoiding tactics Releasing tactics

Passive defenses

Camouflage / distractionAposematism / mimicry

Unpalatability

Active defenses

Running / swimmingSimulate mortality / bad smellsInflatingIntimidation

Distress callsDifficult posturesBladder emptying

© Perefct Vision Grpahics

© Brian Bevan

© Batraciens-Reptiles.com Unken reflex

© D. Maitland

© thehibbitts.net

Eleuterodactylus gaigeae

Phyllobates lugubris

Outline

INTRODUCTION


Diversity

LIFE HISTORY

Breeding migrations



Larval development

Metamorphosis







Gaseous exchange

ECOLOGY

Habitat

Feeding ecology


CONSERVATION

Global decline

Threats

Amphibian global decline: the suspicion

1989: First World Congress of Herpetology (Canterbury, UK)

Apparent extinctions or declines

Bufo periglenes (Costa Rica)

Rheobatrachus silus (Australia)

Additional clues…

Mass die-offs associated to bacterial blooms

Malformations

Blaustein & Wake (1990) Trends Ecol Evol 5, 203-204; Wake (1991) Science 253, 860

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Amphibian global decline: the evidence

2000: Almost 1000 populations studied worldwide

Houlahan et al. (2000) Nature 404, 752-755

Amphibian global decline: the drama

2004: Global Amphibian Assessment (IUCN) concussions:

•32% of amphibian species worldwide are threatened (12% of birdsand 23% of mammals)

•165 amphibian species have become extinct during last years, whileanother 130 have disappeared recently

•43% of amphibian species are declining, by only 1% of species areincreasing

Distribution of endangered species

Threats: Habitat loss or degradation (terrestrial)

Fires Urbanization

Habitat fragmentation and road mortality

1950

1990

Homogeneization

Threats: Habitat loss or degradation (aquatic)

Absence of buffer zones Dumping

Loss of traditional uses (pools, water troughs)Desiccation

Threats: Overexplotation Threats: Invasive species

© John White

American red crayfish (Procambarus clarkii)

Bullfrog (Lithobates catesbeianus)

Red-eared slider (Trachemys scripta)

Mosquitofish (Gambusia holbrooki)

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Threats: Diseases

Red-legged frogs (Aeromonas hydrophilla)

Secondary, non-etiological pathogen

Fungi

Oomycetes (water molds)

BacteriaChytriomycosis

Saprolegniasis

Virus

© www.sosanfibios.org

Chytridiomycosis

•Batrachochytrium dendrobatidis, theetiological agent, shows low genetic variabilityaround the world

•Suspected to be originally spread byXenopus because its use as a laboratoryanimal or in pregnancy tests. Nowadays,humans are the main vector

•First cause of decline in pristine areas

•It grows on the keratinized areas of the body,affecting mainly to metamorphs

•Two theories to explain the outbreaks:

•High nutrient levels favor fungal growth

•Environmental stressors weakenamphibians

Keratin in tadpoles is limited to oral disk. Bd may cause malformations, although most tadpoles are tolerant and act as reservoirs

Olson et al. (2013) PLoS ONE 8, e56802

Threats: Ultraviolet radiation

Ultraviolet radiation

•UV-A (λ = 315-400 nm): not filtered either by ozone or oxygen

•UV-B (λ = 280-315 nm): filtered by ozone

•UV-C (λ = 100-280 nm): filtered by ozone and oxygen

Threats: Climate change

Pelobates cultripes estimated distribution

Current50 years

(dispersal)50 years (no dispersal)

Araujo et al. (2006) J. Biogeogr. 33, 1712-1728; Rohr et al. (1998) PNAS 105, 17436-17441

Threats: Pollution Why amphibians?

Impacted by stressors acting on terrestrial or aquatic ecosystems

Low vagility and high philopatry: low dispersal and colonization ability

Distribution in metapopulations: high sensitivity to isolation

Naked skins very permeable to chemicals or pathogens

Intermediate position in trophic webs

amphibian biology 2014

Documents

eurosiberian species

toads6396 species

salamanders660 species

iberian peninsulahttp

years b

ammonium nitrate dose

magnetic fields

area reduction