New immunological data obtained using Libby’s photron-reflectro- metric technique confirms that Discoglossus belongs to a difíerent family (Discoglossidae Giinther, 1858) from that of Alytes and Bombina (Bombi- nidae Fitzinger, 1826).Nuovi dati immunologici ottenuti con la técnica fotronriflettometrica di Libby confermano che Discoglossus appartiene a una famiglia (Discoglossidae Giinther, 1858) diversa da quella di Alytes e di Bombina (Bombi- nidae Fitzinger, 1826).Material digitalizado en SEDICI gracias a la colaboración del Dr. Jorge Williams (FCNM-UNLP).Facultad de Ciencias Naturales y Muse

Cei, José Miguel Alfredo María

Crespo, E. G.

Lanza, B.

English

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MONITORE ZOOL. ITAL. (n.s.) 10: 311-314, 1976.
FURTHER IMMUNOLOGICAL EVIDENCE
FOR THE VALIDITY OF THE FAMILY BOMBINIDAE 
(AMPHIBIA SALIENTIA) *
* This research was supported by the Ministero delia Pubblica Istruzione.
B. Lanza, J. M. Cei and E. G. Crespo
Istituto di Anatomia Comparata, Biologia Generale e Genetica, 
Museo Zoologico dell’Università di Firenze
Instituto de Biologia Animal, Universidad Nacional de Cuyo, Mendoza 
Museu e Laboratorio Zoológico, Faculdade de Ciências, Lisboa
Received 21 May 1976
Lanza et al. (1975), using Libby’s photronreflectometric technique, 
demonstrated the existence of a relatively strong intergeneric distance 
between Alytes and Discoglossus, which is stronger than that occurring 
between Discoglossus and the Pelobatid genus Pelobates. At the same 
time they pointed out that both Alytes and Bombina difíer from Disco­
glossus in other important characters and concluded that the first two could 
be located in the family Bombinidae Fitzinger, 1826 and the latter in the 
family Discoglossidae Gúnther, 1858. In order to convalidate this hypoth- 
esis further immunological research on Discoglossus, Alytes and Bom- 
bina, was carried out and the results are reported in the present paper.
MATERIALS AND METHODS
The following samples were studied: Discoglossus pictus Otth, 1837 from Lisbon 
(Portugal); Alytes obstetricans boscai Lataste, 1879 from Minho (Portugal); Bombina 
variegata pachypus (Bonaparte, 1838) from the Rio a’ Buti stream (Calvana Mountains 
near Prato, Florence, Italy).
All the specimens were bled in the same period and utilized in the comparative 
precipitin tests.
Immunological reactions were tested according to Libby’s photronreflectometric 
technique (Libby, 1938a; 1938b; 1938c; Boyden at al., 1947). As pointed out by one 
of us in several reports since 1963 (see Cei, 1972, for the bibliography), this technique 
offers a quantitative expression (in galvanometric units) of the whole antigenic relation- 
ship between species and species-groups at a different taxonomic levei of physiological 
and genetic differentiation.
312 B. LANZA, J. M. CEI end E. CRESPO
Rabbits of a uniform strain were injected with collected sera, using microsamples 
(0.3 ml) reinforced by Freud’s adjuvant. As indicated by trial bleedings, a noticeable 
degree of immunity was obtained after 45 days. Rabbits were bled by cardiac puncture 
and the immune sera tested with progressive antigen dilutions.
Table 1.
Homo-heterologous precipitin reaction between ««íz-Discoglossus, zzwíz-Alytes and anti- 
Bombina sera and homo-heterologous specific antigens (turbidimetric measurements 
expressed as % of the homologous reaction: see text). Data obtained in 1974 (Lanza 
et al., 1975) and in 1975.
Antigens
Discoglossus pictus
Palermo (Sicily), 1974 100 92.9 — 81.1 — _ 52.7 —
Discoglossus pictus
Minho (Portugal), 1974 92.3 100 _ 82.6 61.1 _
GJJ-l
<U
Discoglossus pictus
Lisbon (Portugal), 1975 __ __ 100 _ _ 61.4 — 60.0
A
 n
 t 
i s Discoglossus sardus Tschudi, 
1837
Vizzavona (Corsica), 1974 82.7 80.6 100 63.4
Alytes obstetricans boscai
Minho (Portugal), 1975 __ __ 60.5 __ _ 100 — 74.4
'èombina variegata pachypus
Prato (Italy), 1975 59.7 71.0 — 100
D
isc
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 pic
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Pa
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ic
ily
), 19
74
 
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 pic
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M
in
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 (Po
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 197
4 
D
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 pic
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Li
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 (P
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 197
5 
D
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 sar
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V
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 (C
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 ob
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 bos
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 197
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 197
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A
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 197
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Pr
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The whole precipitin reaction with the homologous antigen is indicated as 100% 
(homologous reaction) and corresponds to the amount of the reactions of the immune 
serum with the progressive antigen dilutions. Heterologous reactions of the same 
immune serum with different specific antigens are expressed as percentages of the 
homologous reaction. Differences in the homo-heterologous precipitating systems (anti-
BOMBINIDAE IMMUNOSYSTEMATIC EVIDENCE 313
gens-antibodies) are strengthened by the difíerent scattering of the flocculated molecules 
as evidenced by the photronic photocell and its galvanometric circuit. The intensity of 
the precipitin reactions at a difíerent dilution, i.e. at a difíerent antigens/antibodies 
proportion, can thus be obtained numerically by photronreflectometric units. More 
than 90% in a homo-heterologous reaction is assumed to correspond to a conspecific 
or populational relationship (see, for instance, Cei, 1971). Lower percentage may 
correspond to interspecific or intergeneric relationships in accordance with the genetic 
or evolutionary affinities of the species tested.
RESULTS AND CONCLUSIONS
The data obtained in 1974 (Lanza et ah, 1975) and 1975 (Table 1) 
show that the following degree of immunological relationship exists in 
the genus Discoglossus and between Discoglossus, Alytes and Bombina:
a) D. pictus from Sicily versus D. pictus from Portugal: 92.3-92.9%
b) D. pictus versus D. sardus: 80.6-82.7%
c) A. obstetricans boscai versus B. variegata pachypus: 71.0-74.4%
d) Discoglossus versus Alytes and Bombina-, 52.7-63.4%.
In analogy with the data obtained studying other Anurans (see for 
instance Cei, 1971 and 1972), we may assume that the above-mentioned 
differences in percentage may correspond respectively to (a) intraspecific 
(populational or racial), (b) interspecific, (c) intergeneric and (d) family 
relationships. According to recent studies by Crespo (in preparation), Di­
scoglossus pictus also differs markedly from Alytes obstetricans boscai and 
Alytes cisternasii also in haemoglobins and lactic dehydrogenase isozymes 
pattern (analised by polyacrylamid and cellulose acetate electrophoresis).
SUMMARY
New immunological data obtained using Libby’s photron-reflectro- 
metric technique confirms that Discoglossus belongs to a difíerent family 
(Discoglossidae Giinther, 1858) from that of Alytes and Bombina (Bombi- 
nidae Fitzinger, 1826).
RIASSUNTO
Nuovi dati immunologici ottenuti con la técnica fotronriflettometrica 
di Libby confermano che Discoglossus appartiene a una famiglia (Discoglos­
sidae Giinther, 1858) diversa da quella di Alytes e di Bombina (Bombi- 
nidae Fitzinger, 1826).
7*
314 B. LANZA, J. M. CEI end E. CRESPO
REFERENCES
Boyden, A., E. T. Bolton & D. G. Gemeroy 1947. Precipitin testing with special 
reference to the photoelectric measurements of turbidity. T. Immunol. 57: 
211-227.
Cei, J. M. 1971. Analisis sero-immunológico de la variación geográfica en el 
conjuncto poblacional de Bufo arenarum. Acta zool. lilloana 28: 163-173.
Cei, J. M. 1972. Archaeobatrachia versus Neobatrachia: a first serological approach. 
Serological Mus. 48: 1-4 and 8.
Crespo, E. G. Contribução para o estudo das espécies ibéricas do gén. Alytes 
(Alytes cisternasii Boscá e Alytes obstetricans boscai Lataste) ( Amphibia, Sa- 
lientia). I. (In preparation).
Lanza, B., J. M. Cei & E. Crespo 1975. Immunological evidence for the specific 
status of Discoglossus pictus Otth, 1837 and D. sardus Tschudi, 1837, with notes 
on the families Discoglossidae Giinther, 1858 and Bombinidae Fitzinger, 1826 
(Amphibia Salientia). Monitore zool. ital. (N.S.) 9: 153-162.
Libby, R. L. 1938a. The photronreflectometer, an instrument for the measurement 
of turbid system. J. Immunol. 34: 71-73.
Libby, R. L. 1938b. A new and rapid quantitative technic for the determination 
of the potency of types I and II antipneumococcal serum. J. Immunol. 34: 
269-279.
Libby, R. L. 1938c. A simplified photronreflectometric technic for the titration of 
the antibody-potency of antipneumococcal horse and rabbit serum. J. Immunol. 
35: 289-302.
Prof. Benedetto Lanza
Museo Zoologico delFUniversità 
Via Romana, 17
50125 Firenze (Italia)
Prof. José Miguel Cei
Instituto de Biologia Animal U.N.C. 
Casilla Correo 327
Mendoza (Republica Argentina)
Dr. Eduardo G. Crespo
Museu e Laboratório Zoológico e 
Antropológico (Museu Bocage) 
Rua da Escola Politécnica, 58 
Lisboa-2 (Portugal)


Further immunological evidence for the validity of the family Bombinidae (Amphibia Salientia)

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Further immunological evidence for the validity of the family Bombinidae (Amphibia Salientia)

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