Bull. Fr. Pêche Piscic. (1996) 343:175-182
— 175 —
ISOLATION AND CHARACTERIZATION OF MITOCHONDRIAL DNA
FROM THE ENDANGERED WHITE-CLAWED CRAYFISH
AUSTROPOTAMOBIUS PALLIPES PALLIPES, LEREBOULLET, 1858.
F. G R A N D J E A N , C. S O U T Y - G R O S S E T .
Laboratoire d e Biologie Animale, URA C N R S n° 1975, Université de Poitiers,
40 avenue du Recteur Pineau, 8 6 0 2 2 Poitiers C e d e x (France).
Reçu le 29 mars 1996
Accepté
Received 29 March,
le 20 mai 1996
Accepted
20 May,
1996
1996
ABSTRACT
Mitochondrial DNA (mtDNA) variation in the w h i t e - c l a w e d crayfish,
Austropotamobius
pallipes pallipes, w a s e x a m i n e d by restriction e n d o n u c l e a s e analysis of s a m p l e s obtained
from three geographical locations representing t w o very différent habitats : the Crochatière
and the M a g o t for b r o o k s and the Pinail for p o n d s . This s t u d y used a new type of molecular
m a r k e r in c r a y f i s h . T h e m e t h o d of m t D N A e x t r a c t i o n w a s not b a s e d on the c l e a r lysate
m e t h o d or o n ultracentrifugation a n d used no end-labelling d é t e c t i o n . It is discussed according
t o t h e literature a b o u t m a r i n e c r u s t a c e a n s . M t D N A w a s d i g e s t e d w i t h 6 e n d o n u c l e a s e s .
The molécule size of the m t D N A of the w h i t e - c l a w e d crayfish w a s approximately 17750 ± 5 8 0
b a s e pairs. T w o r e s t r i c t i o n e n z y m e s p r o d u c e d p o l y m o r p h i c d i g e s t i o n p a t t e r n s , d e f i n i n g
a t o t a l of t h r e e h a p l o t y p e s (1-3). T h e r e w a s a s h a r e d h a p l o t y p e 2 b e t w e e n i n d i v i d u a l s
a m o n g the t w o brooks. The haplotype 3 w a s only f o u n d in individuals obtained from ponds of
Pinail.
K e y - w o r d s : Austropotamobius
pallipes,
m t D N A , genetic variation, RFLR
ISOLATION ET CARACTÉRISATION DE L'ADN M I T O C H O N D R I A L C H E Z
L ' É C R E V I S S E À P A T T E S B L A N C H E S AUSTROPOTAMOBIUS
PALLIPES
PALLIPES,
LEREBOULLET, 1858, ESPÈCE M E N A C É E .
RÉSUMÉ
La variabilité d e l'ADN m i t o c h o n d r i a l ( A D N m t ) c h e z l'écrevisse à p a t t e s b l a n c h e s ,
Austropotamobius
pallipes pallipes, est examinée par l'utilisation d ' e n d o n u c l é a s e s d e restriction
à partir d'échantillons issus d e trois sites caractérisant d e u x t y p e s d'habitats : la Crochatière et
le M a g o t pour les ruisseaux et le Pinail pour les mares. Cette é t u d e met en oeuvre un nouveau
t y p e d e marqueur moléculaire chez les écrevisses. La m é t h o d e d'extraction d e l'ADNmt n'est
pas basée sur la m é t h o d e d e lyse alcaline ou sur d e s ultracentrifugations et n'utilise pas d e
m a r q u a g e terminal. La t e c h n i q u e d'extraction est discutée par rapport aux m é t h o d e s employées
chez les crustacés marins. L'ADNmt est digéré à l'aide d e six endonucleases. La taille de la
molécule d ' A D N m t chez l'écrevisse à pattes blanches est a p p r o x i m a t i v e m e n t de 17750 ± 580
paires de base. Deux e n z y m e s d e restriction d o n n e n t d e s profils de restriction polymorphes,
définissant trois haplotypes (1-3). L'haplotype 2 est présent dans les populations provenant d e s
deux ruisseaux. L'haplotype 3 est exclusivement trouvé chez les écrevisses provenant d e s mares
d u Pinail.
M o t s - c l é s : Austropotamobius
pallipes,
A D N m t , variabilité génétique, PLFR.
Article available at http://www.kmae-journal.org or http://dx.doi.org/10.1051/kmae:1996014
Bull. Fr. Pêche Piscic. (1996) 343 :175-182
— 176
INTRODUCTION
T h e natural distribution of Austropotamobius
pallipes pallipes (Lereboullet, 1858) covers
W e s t e r n E u r o p e from 5 6 ° N in Great Britain south t o 38°S in Spain and from 8°W in Ireland t o 16°E
in f o r m e r Yugoslavia. During the last century, native populations of A. pallipes t h r o u g h o u t Europe
a n d in particular in France (VIGNEUX et al., 1993) have been greatly r e d u c e d in number. This
d é c l i n e is d u e t o acidification of rivers, pollution, exotic crayfish acclimatization, and other
h u m a n interférence w i t h natural habitats (WESTMAN, 1985). It has recently been classified
b y t h e I.U.C.N. as being vulnérable a n d rare (GROOMBRIDGE, 1994).
If t h e c o n s e r v a t i o n of this s p e c i e s required t h e préservation of existing populations, it must
a l s o i n c l u d e restocking o p é r a t i o n s in suitable habitats. Before restocking a n d s t o c k i n g , it is
i m p e r a t i v e t o k n o w genetic différences between p o p u l a t i o n s , because local p o p u l a t i o n s m a y be
a d a p t e d t o their local e n v i r o n m e n t a n d introduction of genetically différent individuals c o u l d
a d v e r s e l y alter the gene p o o l .
F e w s t u d i e s have been p e r f o r m e d to appreciate the level of m o r p h o m e t r i c variation
in A. pallipes. A L B R E C H T (1982) f o u n d morphological différences in a n u m b e r of characters
b e t w e e n p o p u l a t i o n s s a m p l e d f r o m d i f f é r e n t r é g i o n s of E u r o p e . A T T A R D a n d
V I A N E T (1985) s h o w e d m o r p h o l o g i c a l différences between French, Irish a n d Italian p o p u l a t i o n s .
H o w e v e r , e l e c t r o p h o r e t i c analysis of gênerai proteins d i d not reveal any différences within
t h e s p e c i e s (NEMETH a n d T R A C E Y, 1979 ; ALBRECHT a n d V O N H A G E N , 1981 ; A G E R B E R G ,
1 9 9 0 ) . A T T A R D a n d V I A N E T (1985) revealed a l o w level of h e t e r o z y g o s i t y r a n g i n g f r o m
0 . 0 1 3 t o 0 . 0 2 6 b e t w e e n p o p u l a t i o n s . In gênerai, very little is k n o w n a b o u t p o p u l a t i o n s
g e n e t i c s in c r a y f i s h . A t t h e p r é s e n t t i m e , o n l y e l e c t r o p h o r e t i c a n a l y s i s h a s b e e n
p e r f o r m e d a n d this m e t h o d s e e m s n o t t o be a p p r o p r i a t e f o r t h e s t u d y of i n t r a s p e c i f i c
g e n e t i c variation whatever t h e crayfish species ( B R O W N , 1980 ; B U S A C K , 1988 ; FEVOLDEN
a n d H E S S E N , 1989). This lack of a p p l i e d genetic research is harmful f r o m a conservation (as well
a s s t o c k m a n a g e m e n t ) point of view.
In t h e l a s t d é c a d e , a n o t h e r m e t h o d o f i n v e s t i g a t i n g g e n e t i c r e l a t i o n s h i p s h a s
b e e n d e v e l o p e d from the analysis of mitochondrial DNA (mtDNA) using analysis of Restriction
F r a g m e n t L e n g t h P o l y m o r p h i s m (R. F. L. P.). T h e m t D N A has several characteristics w h i c h
m a k e it useful as a s p e c i e s - s p e c i f i c marker. T h e usually maternai a n d n o n - r e c o m b i n a t i o n a l
t r a n s m i s s i o n a n d t h e f a s t e r rate of évolution o f m t D N A , c o m p a r e d w i t h n u c l e a r g é n o m e
(5 t o 10 t i m e s more rapid) ( B R O W N et al., 1979), make it useful for t h e study of phylogenetic
r e l a t i o n s h i p s o r of microevolutionary processes within species (WILSON ef al., 1985 ; AVISE
ef al., 1 9 8 7 ; MORITZ et al., 1987). Generally, genetic variation a m o n g p o p u l a t i o n s is often
m o r e d r a m a t i c for m t D N A t h a n for nuclear DNA or nuclear gene p r o d u c t s , s u c h as isozymes
(AVISE, 1 9 8 5 ; Z W A N E N B U R G et al., 1992). The scarcity of papers on c r u s t a c e a n s , indeed even
n o n e o n c r a y f i s h , is p r o b a b l y related t o t h e difficulty of extracting total m t D N A for RFLP analysis.
A f e w s t u d i e s have been p e r f o r m e d o n marine Decapods. K O M M et al. (1982) s h o w e d preliminary
r e s u l t s in Panulirus argus a n d revealed some heterogeneity b e t w e e n individuals. In Jasus
verreauxi, BRASHER ef al. (1992a) s t u d i e d 25 individuals with six restriction e n z y m e s : t w o
r e s t r i c t i o n sites (on 4 5 obtained) w e r e sufficient t o distinguish New Zealand p o p u l a t i o n s from
A u s t r a l i a n o n e s . In Jasus edwardsii, OVENDEN et al. (1992) have c o m p a r e d 13 p o p u l a t i o n s a n d
o b t a i n e d a nucleotides s u b s t i t u t i o n m e a n of about 0.78 whereas the heterozygosity w a s low in
t h i s s a m e s p e c i e s (0.015 a c c o r d i n g t o S M I T H ef al., 1980). BRASHER et al. (1992b) c o n f i r m e d a
high level of m i t o c h o n d r i a l p o l y m o r p h i s m in the g e n u s Jasus by studying 5 species. Seeing that
O V E N D E N (1990) specified that s o m e marine species have less variable m i t o c h o n d r i a l g é n o m e s
t h a n p r e v i o u s l y studied terrestrial a n d freshwater species, w e can hope that m i t o c h o n d r i a l DNA
will b e a s u i t a b l e molecular marker in crayfish.
T h e objectives of this s t u d y w e r e t w o f o l d : firstly, to outline a new molecular t e c h n i q u e for
s t u d y i n g g e n e t i c variability of m i t o c h o n d r i a l DNA in crayfish a n d , secondly, t o appreciate the level
of g e n e t i c variability within a n d b e t w e e n natural p o p u l a t i o n s of A. pallipes.
Bull. Fr. Pêche Piscic. (19%) 343:175-182
— 177 —
MATERIALS AND METHODS
A d u l t crayfishes were c o l l e c t e d f r o m t w o s t r e a m s situated in t h e région of PoitouCharentes (France) : t h e Crochatière, the M a g o t a n d from a p o n d : the Pinail. 20 animais f r o m
each brook were used for genetic study, but only 5 animais of Pinail have been analysed because
this p o p u l a t i o n s e e m s t o be relie. Heart, green glands a n d testes obtained f r o m fresh animais
were u s e d for the m t D N A extraction.
Each sample w a s h o m o g e n i z e d in a Potter-Elvehjem homogenizer with a glass/teflon
pestle after a d d i n g 15 ml of sucrose-TE-buffer (0.25 M sucrose, 60 m M EDTA, 30 m M Tris-HCI
and 1.5% NaCI, p H 8). The h o m o g e n a t e w a s c e n t r i f u g e d in a 15 ml centrifuge t u b e at 700 g for
20 min at 4°C t o pellet nuclei and cellular débris. The supernatant, containing t h e mitochondria,
w a s transferred t o a s e c o n d t u b e and centrifuged again under the s a m e c o n d i t i o n s . The remaining
supernatant w a s centrifuged at 20000 g for 20 min at 4°C a n d a first c r u d e mitochondrial pellet
was o b t a i n e d . To purify the first resulting m i t o c h o n d r i a l pellet, it w a s resuspended in 15 ml of
TE-buffer (60 m M EDTA, 30 m M Tris-HCI a n d 1.5% NaCI, p H 8) a n d s p u n again under the previous
c o n d i t i o n s . The pellet containing the m i t o c h o n d r i a w a s r e s u s p e n d e d in 980 ul of TE-buffer a n d
20 ul of 2 0 % Nonidet P-40 (Sigma) w a s a d d e d (final c o n c e n t r a t i o n of 0.4%) on ice d u r i n g 10 min
with the aim of disrupting the mitochondrial m e m b r a n e s . The t u b e s were centrifuged f o r 10 min
at 12800 g t o 4 ° C . T h e supernatant w a s p h e n o l - e x t r a c t e d t w i c e by a d d i n g an equal volume
of s a t u r a t e d phénol (pH 8) t o eliminate the proteins in the s a m p l e . After each e x t r a c t i o n ,
the s a m p l e w a s s p u n for 8 min at 12000 g t o separate t h e a q u e o u s and phénol phases.
Residual phénol was removed by a single c h l o r o f o r m - isoamyl extraction. S o d i u m acétate
(pH 6) w a s a d d e d t o a final concentration of 0.3 M a n d the m t D N A precipitated by addition
of o n e v o l u m e of 1 0 0 % isopropanol f o l l o w e d by s t a n d i n g for 1 hour at - 2 0 ° C . The mtDNA
w a s pelleted at 15000 g for 30 min t o 4°C in a n E p p e n d o r f centrifuge and w a s h e d in 7 0 % (v/v)
ethanol. The s a m p l e w a s dried under v a c u u m for 45 min and dissolved in 40 ul H 2 0 for ovaries,
20 ul for green gland a n d 10 ul for heart.
A m o n g the six enzymes u s e d for the m t D N A analysis, five restriction endonucleases
eut 6-base pairs : Pst I, Xho I, Bam Hl, Eco RI and Hind III, and one eut 4-base pairs : Hpa II.
The restriction fragments obtained were separated in 1.2 % agarose gels in TE-buffer f o r 15 h at
30 v o l t s . G e l s w e r e s t a i n e d w i t h e t h i d i u m b r o m i d e a n d v i s u a l i z e d w i t h a UV light
transilluminator. The restriction fragment p a t t e r n s f r o m each of the six endonucleases were
identified by a letter, each individual being characterized by a c o m p o s i t e haplotype of several letters.
RESULTS
The size of the mitochondrial g é n o m e w a s estimated by s u m m i n g the restriction fragment
sizes obtained after digestion with the following enzymes Pst I, Eco RI, Xho I (table I). T h e size of
the m t D N A of the w h i t e - c l a w e d crayfish w a s f o u n d t o be approximately 17750 ± 580 base pairs.
H i n d III a n d H p a II p r o d u c e d the greatest n u m b e r of b a n d s , many of small size. As the
s u m s of t h è s e lengths are comparatively low, it is likely that even smaller f r a g m e n t s w e r e
u n d e t e c t e d by the coloration m e t h o d . For t e s t e d e n z y m e s Pst I, B a m Hl, Eco RI, no différence
in the restriction pattern between individuals c o u l d be d e t e c t e d (table I). B a m Hl had no restriction
site in the molécule.
Two patterns w e r e observed for the e n z y m e s H p a II and Hind III (table I a n d figure 1). For
thèse e n z y m e s , différent m o r p h s w e r e separated by a single loss or gain of a restriction site. In
total, three différent c o m p o s i t e g é n o t y p e s (haplotypes) were d e t e c t e d a m o n g the 45 crayfishes
representing the three populations (table II).
No intraspecific variability of the mitochondrial g é n o m e w a s apparent in M a g o t and in
Pinail, ail crayfishes exhibited patterns 2 a n d 3, respectively. The haplotype 3 w a s only found in
Pinail. The population of Crochatière s h o w e d an intraspecific variability by using the restriction
e n z y m e H i n d III. The haplotype 1 w a s more fréquent in this population than h a p l o t y p e 2, w i t h
respectively 7 5 % and 2 5 % .
Bull. Fr. Pêche Piscic. (1996) 343 :175-182
— 178 —
Table I
: Estimated sizes (in base pairs) of mtDNA fragments resulting f r o m digestion w i t h
restriction endonucleases in A. pallipes. For each enzyme, letters refer to the différent
génotypes revealed.
Tableau I
: Tailles estimées (en paires de base) des fragments de restriction obtenus après digestion
de l'ADNmt d'A. pallipes par différentes enzymes de restriction. Pour chaque enzyme, les
lettres correspondent aux différents types de profils de restriction observés.
H i n d III
Total
Table II
H p a II
Pst I
Xho I
E c o RI
A
B
A
B
A
A
A
5520
5520
4330
4330
8235
9200 x 2
13545
2710
1590
1730
1460
7460
1865
1425
1425
1460
1250 x 2
935 x 2
935 x 2
1050
1130
1250
1160 x 2
935
1050
1160 x 2
950
845
935
950
630
615
845
885
560
530
615
630
530
560
13640
11280
13630
10455
17565
18400
17280
: Three enzyme haplotypes of A. pallipes. The letter désignations refer to single enzyme
génotype in the order presented in table I. (Bam Hl does not eut the mtDNA).
Tableau II : C o m p o s i t i o n des t r o i s h a p l o t y p e s o b t e n u s chez A. pallipes. L'ordre d e s l e t t r e s
correspond à celui présenté dans le tableau I. (Bam Hl ne coupe pas l'ADNmt).
Location
Type
Haplotype
Crochatière
AAAAA
Crochatière / M a g o t
BAAAA
Pinail
ABAAA
Bull. Fr. Pêche Piscic. (1996) 343:175-182
— 179 —
Figure 1
: Example of ethidium bromide coloration stained agarose gel of mtDNA digestion patterns
of A. pallipes. On the right side, are given fragment sizes in Kilobase pairs (Kb) of
Lambda-phage DNA digested by Hind III. Lanes : 1-2 = Hind III (patterns A and B,
respectively), 3 = Hpa II (pattern A), 4 = Pst I, 5 = Xho I, 6 = Eco RI, 7-8 = Bam Hl, M = size
standard.
Figure 1
: Exemple d'un gel coloré au bromure d'éthidium montrant des profils de restriction
obtenus après digestion enzymatique de l'ADNmt d ' A . pallipes. Sur la droite sont
indiquées les t a i l l e s d e s f r a g m e n t s (kb) o b t e n u s après d i g e s t i o n par Hind III
du marqueur de référence (ADN du phage Lambda). Lignes 1-2 : Hind III (profils A et B
respectivement) ; 3 : Hpa II (profil A) ; 4 : Pst I ; 5 : Xho I ; 6 : Eco RI ; 7-8 : Bam Hl ; M :
marqueur de taille.
DISCUSSION
Up t o date, no study has been carried out c o n c e r n i n g m t D N A diagnostic marker in
p o p u l a t i o n genetics of freshwater crayfish. The establishment of a reliable m t D N A extraction
m e t h o d in crayfish was indeed an essential a n d difficult step of our work. Papers, essentially
p e r f o r m e d on marine crustaceans, related very t i m e - c o n s u m i n g m e t h o d s giving low yields of
extraction ; in Panulirus argus, K O M M et al. (1982) o b t a i n e d only three restriction profiles using
radiolabelled détection from m t D N A extracted by a c é s i u m chloride gradient. In our laboratory,
Bull. Fr. Pêche Piscic. (1996) 343 :175-182
— 180 —
w e have d e v e l o p e d a rapid extraction m e t h o d for Penaeus japonicus mtDNA, more suitable to
p o p u l a t i o n g e n e t i c s s t u d i e s and w h i c h c o r r e s p o n d e d t o a modified version of alkaline lysis
c o m m o n l y e m p l o y e d to extract p l a s m i d s ( B O U C H O N et al., 1994). Unfortunately, the m e t h o d is
t o o d r a s t i c if it is applied t o freshwater crayfish. We p r o p o s e d here another rapid m e t h o d of
e x t r a c t i o n of m t D N A especially a d a p t e d in crayfish. The m e t h o d involved using an a d a p t e d
e x t r a c t i o n buffer (concentration of EDTA is relatively high c o m p a r e d to other buffers used in
c r u s t a c e a n s ) a n d a spécifie lysis of mitochondrial m e m b r a n e s by NonidetP40. The use of this
d é t e r g e n t w a s essentially m a d e for use w i t h molluscs (see BLOT ef al., 1990, in mussels and
M U R R A Y ef al., 1 9 9 1 , in Partula) a n d in t h e crustacean Jasus (BRASHER ef al., 1992b), but its
c o n c e n t r a t i o n w a s higher (from 1 t o 2 % ) . Our m e t h o d has permitted the use of six restriction
e n z y m e s w i t h o u t the radiolabelled m e t h o d as used by K O M M et al. (1982), M e LEAN et al. (1983),
S I L B E R M A N et al. (1994).
The preliminary results presented here have p e r m i t t e d , firstly, the characterization of the
size of m t D N A in A. pallipes (17750 ± 580 bp) and, secondly, to reveal a genetic variability b e t w e e n
three p o p u l a t i o n s . The size of the mitochondrial g é n o m e in A. pallipes was that generally f o u n d
in a n i m a i s a n d more particulary in Crustacea D e c a p o d a ( B O U C H O N et al., 1994). In France,
generally, A. pallipes is f o u n d in the s a m e habitat that trout prêter (running water, rich in c a l c i u m
a n d d i s s o l v e d o x y g e n , w i t h refuges) (LACHAT and LAURENT, 1987) as in the Crochatière and in
t h e M a g o t . However, in t h e Pinail, A. pallipes was f o u n d in very small p o n d s (100 m2) with physical
a n d c h e m i c a l c o n d i t i o n s very particular for this species. Physical a n d chemical m e a s u r e m e n t s ,
carried out this year in t h è s e p o n d s , have revealed very low rates of dissolved oxygen and calcium
w i t h t e m p é r a t u r e s u p t o 2 9 ° C . In several freshwater d e c a p o d s , t h e discontinuous nature of
h a b i t a t s b e t w e e n p o p u l a t i o n s w a s a s o u r c e of genetic divergence (HEDGECOCK ef al., 1976 ;
F U L L E R a n d LESTER, 1980 ; A U S T I N , 1986 ; FEVOLDEN and HESSEN, 1989) although s o m e
o t h e r s have s h o w n l o w inter-populations divergence (BUSACK, 1989 ; HORWITZ et al., 1990). In
our c a s e , t h e l o w n u m b e r of s a m p l e d animais in this population d o not allow us to j u d g e if this
g e n e t i c differentiation w a s d u e either t o a différence in t h e nature of habitat or t o the f o u n d e r
effect.
This s t u d y has revealed an intrapopulational genetic variability within the Crochatière.
However, t h e t w o h a p l o t y p e s w e r e very similar a n d only differ from o n e another by a gain or loss
of o n e restrictions site. T h e lack of genetic variation in Pinail and M a g o t c o u l d be explained by
several m e a n s . B A R T O N a n d C H A R L E S W O R T H (1984) have s h o w e d that, at the p o p u l a t i o n level,
natural levels of m t D N A diversity m a y be drastically r e d u c e d by either strong sélection pressures
or a r é d u c t i o n in p o p u l a t i o n size. AVISE et al. (1988) s h o w e d that intrapopulational m t D N A
diversifies are directly proportional t o effective sizes of females. In Pinail, it seems that the nature
of t h e habitat limited the p o p u l a t i o n size (small surface, lack of hides, présence of several
predators). T h e i m p a c t of t h e f o u n d e r effect c o u l d be as important as sélective pressures.
However, t h e l o w n u m b e r of animais t a k e n for this analysis f r o m the Pinail and the low n u m b e r
of restriction e n z y m e s t e s t e d c o u l d explain this lack of genetic variation in populations of w h i t e c l a w e d c r a y f i s h . A c c o r d i n g t o O ' C O N N E L L e f al. (1995), a s a m p l e of 2 5 - 3 0 individuals is n e e d e d
f o r i n v e s t i g a t i n g m t D N A diversity in a population. In Pinail, only five crayfishes have been
analysed.
A m t D N A analysis is in progress o n a great n u m b e r of populations from Poitou-Charentes
a n d will give or not a c o n f i r m a t i o n of the low levels of genetic variability of A. pallipes populations
o n a régional scale.
ACKNOWLEDGEMENTS
We w o u l d like to t h a n k M. B R A M A R D , m e m b e r of the délégation régionale d u Conseil
Supérieur d e la P ê c h e d u P o i t o u - C h a r e n t e s (C.S.R), f o r f i e l d assistance d u r i n g f i s h i n g opérations.
This w o r k w a s s u p p o r t e d by financial support f r o m C.S.R
Bull. Fr. Pêche Piscic. (1996) 343:175-182
— 181
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