Mangroves as biofilters:

how do crabs physiologically react to

enhanced ammonium inputs?

Dimitri Theuerkauff*, Sophia Lambert, Georgina A. Rivera-

Ingraham, Yann Mercky, Elliott Sucré and Jehan-Hervé Lignot

2018 meeting
 Context

Mangroves used widely as wastewater (WW) treatment

! Missing data on mangrove

macrofauna

2
 Context

The most abundant

=> crabs matter! Key engineers

10 cm

Herteman

How do crabs react to WW?

3
 Context

Experimental site
Comoros
archipelago Mayotte
island

7 crabs species
Bimodal breathers 4
 Context

Experimental site Pre-treatment and retention tank

Since 2008
 Wastewater discharge on parcels
 every day at low tide: 10m3 / day

Chemical profile:
• Biogeochemical background: Fe, Mn
• Low pollutant load compared to mainland France
5
 Context

Salinity 2015 Burrow density

mOsm Burrow m-2

62%

10m 10m

(R² ajusted ; F1,30 = 51.59 ; p = 5.419x10-8)

Crab ecology is impacted by WW

6
 Water Respiration rate (μmol O2 min-1 g fBW-1)

0,3

0,25
*
0,2

* *
0,15

0,1
+300% Neosarmatium meinerti

0,05

0 Seawater
0 20 40 60 80 100 120

Freshwater

BURST in O2 Wastewater
consumption

7
 Water Respiration rate (μmol O2 min-1 g fBW-1) Air
0,06

0,3
0,05
0,25 +300% b
* 0,04
0,2

* * a a 0,03
0,15 N. meinerti
0,02
0,1

0,01
0,05

0 0
0 20 40 60 80 100 120

Seawater After seawater exposure

BURST in O2
Freshwater After freshwater exposure
consumption
Wastewater After wastewater exposure

Crabs are physiologically impacted by WW

WW also affects osmoregulation & redox balances (Theuerkauff et al., 2018) 8
Water Air
0,3
Respiration rate (μmol O2 min-1 g fBW-1)
0,05
0,25

0,2
* b 0,04

0,15
* *
a a 0,03

0,1
+300% 0,02

0,05 0,01

0
N. meinerti 0
20 40 60 80 100 120
0,18
* 0,05
0,15 *
0,04
0,12 *
b
0,09 +400% 0,03
a a
0,06 0,02

0,03 Tubuca urvillei 0,01

0 140 0
0 20 40 60 80 100 120

9
Water Air
0,3
Respiration rate (μmol O2 min-1 g fBW-1)
0,05
0,25

0,2
* b 0,04

0,15
* *
a a 0,03

0,1
+300% 0,02

0,05 0,01

N. meinerti 0
0 20 40 60 80 100 120
0,18
0,05
*
0,15 *
0,04
* b
+400%
0,12
0,03
0,09
a a
0,02
0,06

0,03
T. urvillei 0,01

0 20 40 60 80 100 120 140 0

0,12 0,06

0,1 a a a
0,05
0,08 0,04
0,06
Perisesarma guttatum 0,03
0,04 *
-100% 0,02
0,02
* 0,01
*
0 20 40 60 80 100 120
0
 Context

Where does the toxicity come from? …. A puzzle with infinite interactions

Salinity
… Nutrients

Pharmaceutics Wastewater
Surfactants
toxicity

Pesticides Microorganisms

Metals
11
 Context

Hemolymph osmolality
(mOsm/kg)
Salinity Mortality (%)
… Nutrients

All hyper-hypo osmoregulators

Pharmaceutics Wastewater
Surfactants
toxicity
3/15 tested

Pesticides Microorganisms
4/25 tested Metals

LOW
External medium osmolality (mOsm/kg) 12
(no industry)
 Context

Hyper-hypo osmoregulators
Salinity
… Nutrients

Pharmaceutics Wastewater
Surfactants
toxicity
LOW Work in progress

Pesticides Microorganisms
LOW Metals Work in progress
LOW
13
(no industry)
 Focus on ammonia

Field data Salinity

… Nutrients

100
max

Pharmaceutics
10 Wastewater
toxicity
3/15 tested
1

min

0,1 Pesticides
4/25 tested
LOG
Metals
scale

HIGH HETEROGENEITY LOW

14
(no industry)
 Focus on ammonia

Field data Salinity

WW measurements
… Nutrients

100
8 – 80 mg L-1
max min max
HIGH HETEROGENEITY TOO

Pharmaceutics
10 Wastewater
8-
toxicity
3/15 tested SPATIAL AND TEMPORAL
1

HETEROGENEITY
min

0,1 Pesticides But reach high levels

4/25 tested
LOG known to be toxic for
scale Metals crustaceans
LOW
HIGH HETEROGENEITY
(no industry)
How? 15
 Ammonia excretion
(Modified from Leone et al. 2017)

Hemolymph Gill epithelium Cuticle External medium

ATP required:
H+-ATPase pumps
Na+/K+ ATPase pumps (NKA)

Also involved in osmoregulation

Hypothesis 1: Ammonia affects osmoregulation

Hypothesis 2: Ammonia increases metabolic rates
Hypothesis 3: Ammonia affects NKA/H +-ATPase activities
16
 Experimental design
Osmoregulatory function
Osmotic pressure ♂ N. meinerti
NKA activities in anterior and posterior gills
H+-ATPase activities in anterior and posterior gills

NH4-N in hemolymph
Hemolymph sampling

Energy balance
Animal respiration rates 4 5 6
2 3 7 8
Mitochondrial activities in anterior & posterior gills 1
(Electron transport chain, ETC)
Gill sampling 17
 Experimental design

Acute transfer (2h)

Respirometry & hemolymph
Acclimation
72h @ 0 ppt
Sampling Control (<0.1)
Short term
40 mg L-1
72h
80 mg L-1
72h

18
 NH4–N in hemolymph (mg L-1)

0.16 b

0.12
ab

a a
0.08

2h
After
exposure Respiration rate (μmol O2 min-1 g fBW-1)

0.06
b b
b
0.05

0.04
a

0.03

Control 10 40 80 mg L-1

N.B. No difference in hemolymph osmotic pressure 19

 NH4–N in hemolymph (mg L-1)

0.16 b

ab
NH4–N
increases
0.12

a a
0.08

2h
After
exposure Respiration rate (μmol O2 min-1 g fBW-1)

0.06
b b
b
0.05

a
RR
0.04
increases
0.03

Control 10 40 80 mg L-1

N.B. No difference in hemolymph osmotic pressure 20

 Short term
72h
Hemolymph osmotic pressure constant
Ammonia accumulates in hemolymph at 80 mg L-1
Electron transport
NKA activity
activity

b ab
NKA activity (μmol Pi h-1 mg prot-1)

60
a

ETC (nmol min-1 gFW-1)

6 b
a ab
50 control
40 mg L-1
4

40
80 mg L-1

2
30

0
21
 H+-ATPase activity in gills

Proportion of crabs
Low detection limit
with a detectible activity

NH4-N (mg L-1) Anterior Posterior

Control 14% 14%

40 71% 0%

80 57% 42%
More in anterior gills
More after ammonia exposure 22
 Experimental design

72h
control

Acclimation 40 mg L-1
72h @ 0 ppt
Sampling 80 mg L-1

2.5 mg L-1
Long term
control
144h 144h

23
 Long term
144h
Hemolymph osmotic pressure constant
Ammonia in hemolymph constant

Electron transport
NKA activity
activity
40
* *
NKA activity (μmol Pi h-1 mg prot-1)

10
35

ETC (nmol min-1 gFW-1)

8
30

6
25 control
2.5 mg L-1
4
20

2 15

0 10 24
Hypothesis 1: Exposure to high ammonia level will affect osmoregulation

Crabs continue to maintain ion homeoastasis

Ammonia in this context does

not impair osmoregulation as
observed by WW direct exposure

25
 Hypothesis 1: Exposure to high ammonia level will affect osmoregulation

Crabs continue to maintain ion homeoastasis

Hypothesis 2: Exposure to high ammonia level will increase metabolic rates

Crabs increase RR 2h after exposure to 10 mg L-1 but electron transport

decreases

Metabolic trade-off at gill level?

26
 Hypothesis 1: Exposure to high ammonia level will affect osmoregulation

Crabs continue to maintain ion homeoastasis

Hypothesis 2: Exposure to high ammonia level will increase metabolic rates

Crabs increase RR 2h after exposure to 10 mg L-1 but electron transport

decrease
Hypothesis 3: Exposure to high ammonia level will affect NKA/H +-ATPase
Both enzymes activity may increase after ammonia exposure

27
To be continued:
Need to complete our dataset (gill RR, ventilation,…)

Effect of chronic exposures (more relevant due to tidal cycles)

Need to focus on other pollutants & pH (e.g. Surfactants)

And their interactions

Need to relate the ecotoxicological effects with some specific traits

(e.g. nutrition, breathing capacities, microhabitat preferences, evolutionary strategies)

28
Thank you !

Georgina Elliott Sucre Sophia Lambert

Jehan-Hervé Lignot
Rivera-Ingraham

Yann Mercky Charlotte Mangum Student Kissimati

Support Program Abdallah