Download 114_2017_1437_MOESM1_ESM

1
Tail regeneration affects the digestive performance of a Mediterranean lizard
2
3
Kostas Sagonas*1, Niki Karambotsi2, Aristoula Bletsa2, Reppa Katerina2, Panayiotis Pafilis3,
4
Efstratios D. Valakos2
5
6
7
Materials and Methods
8
9
Lizard housing conditions
10
Lizards were housed individually in plastic terraria (20 cm x 25 cm x 15 cm) that contained
11
stones as hiding places and a sandy substrate. Room temperature was kept at 25oC. A
12
controlled photoperiod (12 light: 12 dark) was provided by fluorescent bulbs, while heat was
13
provided through a 60 W heating lamp upon each terrarium allowing animals to
14
thermoregulate behaviorally for 8 h/d. Lizards were fed mealworms (Tenebrio molitor)
15
every other day and had access to water ad libitum.
16
17
Apparent digestive efficiency
18
To estimate ADE, for proteins, lipids and sugars we employed the methodology proposed by
19
Pafilis et al. (2007). We fed each lizard with two weighted mealworms using a digital scale
20
(i500 Backlit Display, My Weight, accurate to 0.01 g) every second day for two months,
21
while two other identical mealworms of the exactly same mass and size were stored at -80oC
22
for subsequent analyses. Fecal material was collected, frozen in liquid nitrogen and stored at
23
-80oC. Before freezing urate material was removed from the fecal samples.
24
To estimate total lipids we weighted 30–40 mg and homogenized them with 1.5 ml of a
25
2:1 mixture of chloroform and absolute methanol. The homogenate was centrifuged at 3,000
26
rpm for 10 min in 4oC. We discarded the pellet formed. The supernatant was used for the
27
determination of lipid levels using a dilution of phosphovaniline against an olive oil and
28
corn oil standard (2:1 v/v) (Alexis et al. 1985). Absorbance was read at 530 nm using a
29
spectrophotometer (Novaspec II, Pharmacia Biotech).
30
Total proteins were estimated using the pellet obtained from the lipid analysis following
31
the Biuret method (Layne 1957). Samples were compared to a standard of bovine serum
32
albumin (0.5–10 mg/ml). Tissue was dissolved with 0.5 ml of 0.1 N NaOH and incubated at
33
37oC for 30 min. We then took 50 μl and diluted them with 950 ml H2O and afterwards
34
added 4 ml of Biuret Reagent. The mixture was incubated for 30 min at room temperature
35
and then the absorbance was read at 550 nm using a spectrophotometer (Novaspec II,
36
Pharmacia Biotech).
37
Concentrations of sugars were determined according to Dubois et al. (1956). Tissue
38
samples (150 mg) were homogenized with H2O at a 1:10 w/v ratio and then boiled for 30
39
min. 20μl of this sample were then diluted in H2O (1: 500 v/v) and incubated with 1 ml of
40
phenol (5% w/v) and 5 ml of 95% H2SO4 for 10 min at room temperature. A final incubation
41
was carried out for 40 min at 30oC. The absorbance was read at 490 nm and glucose content
42
was estimated against a known glucose standard.
43
44
Concentrations of lipids, proteins and sugars in mealworms and feces were used to
calculate ADEs according to the following equation:
ADEx =
45
100 (Ix − Ex )
,
Ix
46
where Ix is the amount of the nutrient (x = proteins, lipids or sugars) ingested and Ex is the
47
amount of the nutrient (x = proteins, lipids or sugars) remained in the fecal material.
48
49
Tail autotomy
50
Prior to the beginning of the experiment lizards were allowed to thermoregulate for two
51
hours, (caudal autotomy is affected by body temperature; Bustard 1967; Bustard 1968).
52
Lizards were placed on a cork substrate to maintain traction during the predation simulation
53
(Pérez-Mellado et al. 1997). To simulate the bite of a predator we used a pair of calipers and
54
grasped the tail of the lizard 15 mm behind the cloaca.
55
56
References
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
Alexis MN, Papaparaskeva-Papoutsoglou E, Theochari V (1985) Formulation of practical
diets for rainbow trout (Salmo gairdneri) made by partial or complete substitution of
fish meal by poultry by-products and certain plant by-products. Aquaculture 50: 6173.
Alibardi L (2010) Morphological and cellular aspects of tail and limb regeneration in
lizards. A model system with implication for tissue regeneration in mammals.
Springer Heidelberg, New York.
Bustard RH (1967) Activity cycle and thermoregulation in the Australian gecko Gehyra
variegata. Copeia 1967(4): 753-758.
Bustard RH (1968) Temperature dependent tail autotomy mechanism in gekkonid lizards.
Herpetologica 24(2): 127-130.
Dubois M, Gilles KA, Hamilton JK, Rebers BA, Smith F (1956) Colorimetric method for
determination of sugars and related substances. Anal Chem 28: 350-356.
Layne E (1957) Spectrophotometric and turbidimetric methods for measuring proteins.
Methods Enzymol 10: 447-455.
Pafilis P, Foufopoulos J, Poulakakis N, Lymberakis P, Valakos E (2007) Digestive
performance in five Mediterranean lizard species: effects of temperature and
insularity. J Comp Physiol B 177(1): 49-60.
Pérez-Mellado V, Corti C, Lo Cascio P (1997) Tail autotomy and extinction in
Mediterranean lizards. A preliminary study of continental and insular populations. J
Zool 243(3): 533-541.
78