Ricerca ProgettiMECOThe ProjectMethodology and activitiesEcologyBioassay-1Isoenzyme Analysis

Isoenzyme Analysis

By Elvira De Matthaeis, Valerio Ketmaier
CoNISMa, University of Rome "La Sapienza"
Department of Animal Biology

 

The MECO project had as a central aim to evaluate the state of health of different coastal ecosystems in Malta, Morocco and Tunisia through a multidisciplinary approach and to propose a sustainable management of these areas. As to the biological characterisation of the study sites, Talitrus saltator (Crustacea, Amphipoda) were chosen as the model organism since it is a typical element of the supralittoral fauna and thus a good candidate as source of biological informations in order to yield important results on the quality of the study coastal sites. The goal of this part of the project was to genetically characterise some natural populations of T.saltator by using allozyme markers.

In the set of experiments carried out during the first year of the MECO project we compared three North-African populations of T.saltator, one from Marocco and two from Tunisia, with several other populations from the whole Mediterranean, in order to understand the pattern of genetic relationships of these newly studied populations with ones coming from different parts of the species range. T.saltator may be frequently found associated with species belonging to the genus Talorchestia such as T. deshayesii (now Deshayesorchestia deshayesii) and T.brito (now Britorchestia brito). In particular, at Tabarka beach (Tunisia) we found both T.saltator and T. brito, whereas at Zouaraa (Tunisia) T.brito was the most abundant species. In addition to the data on T.saltator populations we were able to obtain also information on the genetic structure of two populations of T.brito.

 

Electrophoretic analysis

Fig.1 - Scheme of starch gel electrophoresis

 

The basic outline of starch gel electrophoresis are shown in Fig.1. Hydrolysed starch is heated in an ionic solution and allowed to cool, forming a gel. Electrical current separating protein solutions is applied to the gel via the ionic buffers. Following electrophoresis the proteins may be visualised as bands on the gel by specific histochemical stainings. Protein electrophoresis is used to reveal the changes in the amino acid sequences in the encoding DNA locus due to mutations that may affect the net charge of the protein itself. The banding pattern of an individual contains information on the individual’s genotype at a number of structural loci. In the present work we used 12 enzymatic proteins to assess the degree of genetic polymorphism of the study populations. In Table 1 are listed the study proteins, buffer systems, stains and the number of scored loci.

 

Table 1- Enzymes tested, E.C. numbers, buffer systems, staining techniques and scored loci. For Buffer systems and Staining techniques see De Matthaeis et al. (1994; 1995; 1998).

Code	Enzyme	E.C.N°.	Buffer systems	Staining techniques	Scored loci
Acph	Acid phosphatase	3.1.3.2	A	Tracey et al., 1975	Acph1; Acph2; Acph-3
Ao	Aldehyde oxidase	1.2.3.1	B	Ayala et al., 1974	Ao
Aph	Alkaline phosphatase	3.1.3.1	A	Ayala et al., 1972	Aph1; Aph2
Ca	Carbonic anhydrase	4.2.1.1	G	Brewer & Sing, 1970	Ca1; Ca2
Est	Esterase	3.1.1.1	A	Ayala et al., 1972	Est2; Est3
Got	Glutamic-oxalacetic transaminase	2.6.1.1	B1	Ayala et al., 1975	Got1; Got2
Hk	Exokinase	2.7.1.1	C	Ayala et al., 1974	Hk
Lap	Leucine-amino peptidase	3.4.11.1	A	Ayala et al., 1972	Lap1; Lap2
Mpi	Mannose phosphate isomerase	5.3.1.8	I	Harris & Hopkinson, 1978	Mpi
Pep	Peptidase	3.4.11	A	Shaw & Prasad, 1970	Pep1; Pep2; Pep3
Pgm	Phosphoglucomutase	2.7.5.1	D	Brewer & Sing, 1970	Pgm
Phi	Phosphoexose isomerase	5.3.1.9	C	Brewer & Sing, 1970	Phi

 

 

Genetic variation within Talitrus saltator

The Ca-2 and Got-2 loci were found to be diagnostic for the North-African populations from all other geographic groups of T.saltator (Table 2). These loci completely discriminate the North-African group of populations from the Tyrrhenian, Adriatic and Aegean populations.

 

Table 2-Diagnostic loci among geographical groups of T. saltator.

Geographical groups	T	A	EM	NA
T	-
A	Acph3; Ao1; Ca2; Est1; Est2	-
EM	Acph3; Ca1; Got1	Acph3; Ao1; Ca1; Got1	-
NA	Ca2; Got2	Ca2; Got2	Ca2; Got2	-

T= Tyrrhenian group ; A= Adriatic group ; EM= Eastern-Mediterranean group ; NA= North-African group

 

Intrapopulation genetic variability

The average degree of allozyme heterozygosity is not very high in the populations sampled and this result matches those achieved on different populations of the three species (De Matthaeis et al., 1994; Scapini et al., 1995). From these previous results, on the average, western Mediterranean populations of T.saltator appeared to be more polymorphic (H_o= 0.044) than those belonging to the Adriatic group (H_o= 0.027). Mean heterozygosity appeared highly correlated with a coastal stability index, calculated as follows: presence of one/two dune belts stable or in accretion (score: 2/4), of one dune belt under erosion (score: 1), of a coastline in recession or in progression (score: -1) (Scapini et al., 1995).

Although the results achieved in the project must be considered preliminary a similar trend appears between mean level of genetic heterozygosity of T. saltator and T.brito populations and the stability of the coasts from which populations were collected.

The correlation between the coastal stability index and mean heterozygosity levels may suggest a positive effect of environmental temporal stability on genetic variability. This may be due to both stochastic and deterministic factors. For stochastic factors, the degree of coastal stability, causing fluctuations in population size, could affect the different levels of heterozygosity. On the other hand, the genetic variability of the populations could be related to the past occurrence of bottlenecks. For the deterministic factors, in temporally stable environments some forms of balancing selection could account for the differences in the level of heterozygosity of the examined populations.

 

References

• De Matthaeis E., Cobolli M., Mattoccia M., Saccoccio P., Scapini F. 1994- Genetic divergence between natural populations of Mediterranean sandhoppers. In: Beaumont A. R. (ed.). Genetics and Evolution of aquatic organisms. Chapman & Hall. London.
• De Matthaeis E., Cobolli M., Mattoccia M., Scapini F. 1995- Geographical variation in Talitrus saltator (Crustacea, Amphipoda): biochemical evidence. Boll. Zool., 62: 77- 84.
• De Matthaeis E., Davolos D. & Cobolli M. 1998- Genetic divergence between populations and species of talitrids from Aegean islands. J. Hered., 89: 37-43.
• Nei M. 1978- Estimation of average heterozygosity and genetic distance from a small number of individuals. Genetics, 89: 583- 590.
• Scapini F., Buiatti M., De Matthaeis E., Mattoccia M. 1995- Orientation behaviour and heterozygosity of sandhopper populations in relation to stability of beach environments. J. Evol. Biol., 8: 43-52