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Antimicrobial Capacity of Plasma from Anurans of the

Atlantic Forest

Authors: Assis, Vania Regina de, Titon, Stefanny Christie Monteiro,
Barsotti, Adriana Maria Giorgi, Spira, Beny, and Gomes, Fernando
Ribeiro

Source: South American Journal of Herpetology, 8(3) : 155-160

Published By: Brazilian Society of Herpetology

URL: https://doi.org/10.2994/SAJH-D-13-00007.1

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Antimicrobial Capacity of Plasma from Anurans of the

Atlantic Forest

Vania Regina de Assis1,*, Stefanny Christie Monteiro Titon1, Adriana Maria Giorgi Barsotti1,

Beny Spira2, Fernando Ribeiro Gomes1

1 Departamento de Fisiologia, Instituto de Biociências, Universidade de São Paulo. Rua do Matão, travessa 14, nº 321,

CEP 05508900, São Paulo, SP, Brazil

2 Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo. Avenida Professor Lineu Prestes, nº 1.374, Edifício Biomédicas II,

CEP 05508900, São Paulo, SP, Brazil

* Corresponding author. Email: v.regina.a@gmail.com

Abstract. The viability and interpretation of techniques for the evaluation of immunocompetence of animals in their natural environment

has been largely debated. One of these methods is based on testing the antimicrobial capacity of the blood and/or plasma in vitro, which

could rapidly and effectively assess the immunological conditions of natural populations. We tested the applicability of the antimicrobial

capacity of plasma (ACP) assay in anuran amphibians from the Atlantic Forest. The assay was performed by measuring both the turbidity

(in a spectrophotometer) and the colony forming units (CFU) of the remaining bacteria (Escherichia coli) following exposure to amphibian

plasma. Although both assays were correlated, the ACP assay by spectrophotometry showed 10 times lower intra-assay variation. We also

found interspecific variation in ACP, as well as the maintenance of ACP values in males from the same population, collected in different

breeding seasons. Thus, the estimation of ACP by spectrophotometry provides a convenient and accurate method for evaluating innate im-

munocompetence in comparative and ecophysiological studies of anuran amphibians.

Keywords. Amphibians; Anura; Ecological immunology; Frogs, Immunity; Immunocompetence; Toads.

Resumo. A viabilidade e interpretação de técnicas para a avaliação da imunocompetência dos animais em seu ambiente natural têm sido

amplamente debatidas. Um destes métodos baseia-se em testes da capacidade antimicrobiana do sangue e/ou plasma, in vitro, o qual pode

rápida e efetivamente promover a avaliação das condições imunológicas de populações naturais. Testamos a aplicabilidade do ensaio de

capacidade antimicrobiana do plasma (CAP) em anfíbios anuros. O ensaio foi realizado tanto através da medição da turbidez (em espectrofo-

tômetro) quanto através do número de unidades formadoras de colônias (UFC) de bactérias remanescentes após a exposição ao plasma dos

anfíbios. Embora ambos os ensaios tenham sido correlacionados, o ensaio de CAP por espectrofotometria mostrou variação intra-ensaio 10

vezes menor. Encontramos também variação interespecífica nos valores da CAP, bem como a manutenção dos valores para machos de uma

mesma população, coletados em diferentes estações reprodutivas. Assim, a estimativa de CAP por espectrofotometria constitui um método

prático e preciso de avaliação de imunocompetência inata para estudos comparativos e ecofisiológicos de anfíbios anuros.

INTRODUCTION

Immunocompetence is defined as an individual’s

ability to respond appropriately following exposure to a

pathogen (Demas et al., 2011). Activation of the immune

system results in fitness costs, including the reduction of

current and future reproductive potential, and reduced

rates of growth and survival (Norris and Evans, 2000; Lee

and Klasing, 2004; Verhulst et al., 2005, Hasselquist and

Nilsson, 2012). Additionally, the vertebrate immune sys-

tem consists of many components that interact, each one

with a specific value of protection and metabolic costs in-

herent of production and utilization (Millet et al., 2007).

The emerging field of ecological immunology seeks to

understand the diversity in the composition and action

of animal immune systems and correlate this diversity

with aspects of ecology and life history through an evo-

lutionary approach (Norris and Evans, 2000; Ricklefs and

Wikelski, 2002; Verhulst et al., 2005).

Among the challenges of this emerging field is

the determination of biologically relevant and viable

techniques for assessing immunocompetence of animals

in their natural environment. One proposal that has

been considered suitable for rapid and effective assess-

ment of the immune conditions of natural populations

is the evaluation of the antimicrobial activity of blood/

plasma in vitro. Usually, this test involves mixing blood/

plasma with a standard number of microorganisms, in-

cubation of the mixture to allow the blood/plasma to

kill microorganisms, and quantification of the remain-

ing microorganisms. Antimicrobial activity represents

a combination of several mechanisms of the innate im-

mune system, such as the antimicrobial activity of hu-

moral proteins (when the test is conducted only with

plasma) and phagocytic activity of leukocytes (when the

test is performed with blood), representing a general

and integrative estimate of innate immunocompetence

(Matson et  al., 2006; Millet et  al., 2007). An additional

advantage of this assay is the utilization of non-species-

specific reagents, which makes it a useful tool to assess

the innate immunity of a wide range of species (Millet

et al., 2007).

South American Journal of Herpetology, 8(3), 2013, 155–160

© 2013 Brazilian Society of Herpetology

Submitted: 26 February 2013

Accepted: 27 September 2013

Handling Editor: Taran Grant

doi: 10.2994/SAJH-D-13-00007.1

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Studies conducted with different vertebrates (main-

ly in birds) have demonstrated that the antimicrobial ca-

pacity of blood/plasma varies among individuals within

a population (Tieleman et al., 2005) and between species

(Merchant et  al., 2006; Millet et  al., 2007), being repro-

ducible (Tieleman et  al., 2010). Additionally, variability

in antimicrobial capacity is associated with different as-

pects of physiology and life history, such that it might be

a useful tool to understand the evolutionary divergence

of the immune system and its trade-offs with other bio-

logical traits (Millet et al., 2007; Lee et al., 2008). Studies

with birds have also shown that the antimicrobial capac-

ity of blood is reduced in response to a stress protocol by

restriction (birds kept in small fabric bags for 60  min;

Matson et al., 2006) and is negatively correlated with cor-

ticosterone plasma levels (Millet et al., 2007).

Among the few recent studies with anuran amphibi-

ans, Graham et al. (2012) reported an increase in corticos-

terone plasma levels and a decrease in antimicrobial capac-

ity of plasma (ACP) in Rhinella marina after application of

a stress protocol by restriction whereby toads were kept in

labeled cloth bags, moistened, and placed in buckets (ap-

proximately 8 bags per bucket) for the night. Gomes et al.

(2012) found a negative relationship between ACP and

corticosterone plasma levels in three species of Rhinella

with different degrees of dependence on forested habi-

tats. According to this study, R.  ornata, the species with

the greatest dependence on forested habitats, showed the

highest baseline and post-stress corticosterone plasma

levels (after 18 h in captivity, toads were individually kept

in small wet fabric bags for 60  min). Rhinella schneideri,

the species most associated with naturally open or de-

forested areas, presented the highest ACP. These results

point to a possible compromise between the function of

the hypothalamic-pituitary-interrenal axis and innate im-

munocompetence, which could be an important determi-

nant of the ability to occupy areas modified by humans

(Gomes et al., 2012).

Most ecoimmunological studies that measure the

antimicrobial activity of blood or plasma have analyzed

the interaction of blood/plasma with microorganisms

(usually bacteria), followed by plating on agar-filled pe-

tri dishes and quantification of colony forming units

(CFU). An alternative method consists of reading the

optical density of blood/plasma samples inoculated with

microorganisms (Merchant et  al., 2003; Liebl and Mar-

tin, 2009). This methodology presents a number of ad-

vantages, including reduction of volume and processing

time, elimination of manual quantification, and most

importantly, a drastic reduction of intra-assay variation

(Liebl and Martin, 2009). Considering the advantages of

using spectrophotometry to determine ACP, we tested the

applicability of this method for assessing the ACP of an-

uran amphibians. Tests were performed to (1) evaluate if

the methodology used for birds (Liebl and Martin, 2009)

could be employed to amphibians, adjusting if necessary

the quantities of plasma and bacteria used and (2) make

a compare ACP estimates by spectrophotometry and by

CFU quantification, as in Gomes et  al. (2012). Addition-

ally, we assessed interspecific variation in ACP of male

anurans during calling activity using spectrophotometric

analysis.

MATERIALS AND METHODS

Animals and study site

The plasma of males from six different species were

used: Hypsiboas albopunctatus (n  =  13), H.  faber (n  =  24),

Rhinella icterica (n = 46), R. jimi (n = 4), R. ornata (n = 18),

and Scinax fuscovarius (n  =  5). Three R.  icterica and all

R. jimi individuals were kept in an animal facility and were

used in other studies as well. The plasma from these ani-

mals was used only for standardization tests and was not

included in the analysis of interspecific variation. Scinax

fuscovarius and R. ornata specimens were collected in de-

pendencies of the Biosciences Institute at the University

of São Paulo (23°33’45’’S, 46°43’40’’W); R.  icterica and

H.  albopunctatus were collected around the city of São

Luiz do Paraitinga (23°13’2’’S, 45°18’38’’W), and H.  fa-

ber specimens were collected in the Botanical Garden of

São Paulo (23°38’08’’S, 46°38’00’’W). All these localities

are in the state of São Paulo, Brazil. Eleven individuals of

R. ornata were collected in 2011 and 7 individuals in 2012.

The specimens were collected with authorization of IB-

AMA (process 178951) and the Ethics Committee on the

use of Vertebrate Animals in Experimentation (protocol

053/2008).

Blood collection

Animals were located by visual seaching, captured,

and bled (about 100–150  μL) via cardiac puncture with

1 mL syringes and previously heparinized 26Gx1/2’’ nee-

dles. Only blood samples collected within 3 min of capture

were analyzed because after 3 min of capture corticoste-

rone plasma levels can be affected by the stress of capture

and handling (Romero and Reed, 2005), and increased

corticosterone may reduce ACP (Millet et al., 2007).

Processing of blood samples

All blood samples were labeled and kept on ice until

transfer to microcentrifuge tubes for plasma separation

(4  min at 3000 rpm) on the same night. The separated

plasma was pipetted off into cryotubes and kept in liquid

nitrogen and/or stored at 80°C for later analysis.

Antimicrobial Capacity of Plasma from Anurans of the Atlantic Forest

Vania Regina de Assis, Stefanny Christie Monteiro Titon, Adriana Maria Giorgi Barsotti, Beny Spira, Fernando Ribeiro Gomes
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Growth of bacteria

Pellets of non-pathogenic Escherichia coli (Microbio-

Logics, #24311–ATCC 8739) were resuspended in 1  mL

sterile PBS. Then, 100 μL of this solution were transferred

to 5 mL TSB (Tryptic Soy Broth), and incubated overnight

at 37°C. On the next day, bacteria concentration was mea-

sured in a spectrophotometer (Spectra Max 250). Serial

dilutions in sterile amphibian Ringer solution were per-

formed to obtain the working concentration of 106 bac-

teria mL1.

ACP assay by spectrophotometry

Plasma samples diluted (1:  20) in Ringer’s solution

(10 μL plasma: 190 μL Ringer) were mixed with 10 μL of

Escherichia coli working solution (~104 microorganisms),

followed by incubation for 60  min at 37°C. The positive

control consisted of 10  μL of E.  coli working solution in

200 μL of Ringer’s solution (no plasma), and the negative

control contained only 210  μL of Ringer’s solution. All

samples were incubated under the same conditions.

After the incubation period, 500 μL of TSB were add-

ed to each sample. The bacterial suspensions were thor-

oughly mixed and 300  μL of each one were transferred

(in duplicate) to a 96 well microplate. The microplate was

incubated at 37°C for 2 hours, at which time the optical

density of the samples was measured hourly using a plate

spectrophotometer (600 nm wavelength), totaling four

readings. ACP was calculated according to the formula 1 –

(optical density of sample/optical density of positive control),

which represents the proportion of killed microorganisms

in the samples compared to the positive control. Antimi-

crobial capacity was evaluated at the beginning of the bac-

terial exponential growth phase.

In addition, ACP measured by spectrophotometry

was tested in 16 individuals of Rhinella icterica by using

5  μL, 8  μL, and 10  μL of plasma diluted in Ringer’s so-

lution (1:20) to 10 μL of bacteria (~104 microorganisms)

in order to evaluate differences in results according to

the amount of plasma. And 10 individuals of R.  icterica

were also used to compare ACP after 30  min, 60  min,

and 90 min with the objective of detecting differences in

results according to the interaction time between 10  μL

diluted plasma and 10 μL (~104 microorganisms) suspen-

sion of Escherichia coli.

ACP assay by CFU quantification

The procedure for ACP assay by CFU quantification

was essentially the same as described for spectropho-

tometry, except that after 60  min of Escherichia coli and

plasma incubation, 100 μL of each sample were plated in

duplicate on Petri dishes containing TSB medium. Plates

were incubated at 37°C for 14 h and the CFU were count-

ed. ACP was calculated according to the formula: 1 – (num-

ber of CFUs of the sample plate/number of CFUs in the posi-

tive control plate).

Statistical analyses

Data were initially submitted to descriptive analyses

and the Shapiro-Wilk test of normality. Given that data

were not distributed normally, the equivalence of ACP de-

termined by spectrophotometry and CFU quantification

was tested by non-parametric correlation (Spearman).

The coefficient of variation between duplicates was used

to assess the intra-assay variability of ACP determined

by the two methods. The effect of different plasma dilu-

tions and different incubation times was analyzed by the

Friedman test. The existence of interspecific variation in

ACP was performed by the Kruskal-Wallis test, excluding

Hypsiboas albopunctatus, since individuals of this species

showed no ACP. Comparison of ACP from individuals of

Rhinella ornata collected in different years was performed

using the Mann-Whitney test. All analyses were per-

formed using SPSS version 17.

RESULTS

The ACP of 10 μL plasma from Rhinella icterica di-

luted in Ringer’s solution (1:20) in the presence of ~104

bacteria was significantly higher than when 8 μL or 5 μL

plasma were used (S = 10.20, P = 0.006; Fig. 1A). Incuba-

tion time also affected ACP (S = 15.20, P ≤ 0.001), being

lower at 30 min than at 60 or 90 min (Fig. 1B).

ACP values determined by spectrophotometry and

by CFU quantification were equivalent (rho  =  0.406,

P  =  0.012; Fig.  2), and the coefficient of variation of the

former was 10 times lower (CV
turbidity

 = 0.03; CV
CFU

 = 0.30).

Male frogs during peak calling activity showed inter-

specific variation of ACP determined via spectrophotom-

etry [H (3) = 19.20, P ≤ 0.001; Fig. 3]. Moreover, male Rhi-

nella ornata collected during calling activity in the years

2011 and 2012 did not differ by ACP level (U  =  36.50,

P = 0.853; Fig. 4).

DISCUSSION

The tests performed in this study validate the meth-

od of ACP determination for anuran amphibians by spec-

trophotometry, modified from Liebl and Martin (2009).

Additionally, we confirmed the equivalence of ACP deter-

minations via spectrophotometry and CFU quantification

as in Gomes et al. (2012). However, as pointed out by Liebl

Antimicrobial Capacity of Plasma from Anurans of the Atlantic Forest

Vania Regina de Assis, Stefanny Christie Monteiro Titon, Adriana Maria Giorgi Barsotti, Beny Spira, Fernando Ribeiro Gomes
157

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and Martin (2009) in a study conducted with birds, the

determination of ACP by spectrophotometry has a num-

ber of advantages, such as reduced sample volumes and

reduced time and expense of sample processing, and a

substantially lower variation between sample duplicates.

Our results also demonstrate the existence of a large in-

terspecific diversity in ACP from male anurans during

calling activity, as well as similar values for calling male

ACP from the same species collected in different years

(

Rhinella ornata).

The use of plate spectrophotometry for determin-

ing ACP enabled the sample volume reduction used from

7.5 μL, as in Gomes et al. (2012), to 5 μL of plasma by du-

plicate. Although this reduction in sample volume seems

small, it allows measurements to be taken on a much wid-

er range of anuran species characterized by reduced body

size. Even greater reductions in sample plasma volumes,

such as the 1.5  μL per replicate employed by Liebl and

Martin (2009), might be possible by using a Nanodrop

Figure 1. Differences in antimicrobial capacity of plasma (ACP) for Rhi-

nella icterica. (A) Plasma volume used in the assay (P = 0.006). The num-

ber of individuals tested was the same in all cases (N = 16). (B) Kinetics

of ACP with a mixture of 10  μL diluted plasma and 10  μL of an Esche-

richia coli suspension (~104 microorganisms) (P ≤ 0.001). The number of

individuals tested was the same in all cases (N = 10). The bars represent

mean ± standard error.

Figure  2. Antimicrobial capacity of plasma (ACP) determined by

spectrophotometry (yaxis) and by colony forming units (CFU) quanti-

fication (xaxis). The results of both methods are positively correlated

(rho = 0.406, P = 0.012). However, variability in the spectrophotometry

method is 10 times lower than by CFU quantification.

Figure  4. Antimicrobial capacity of plasma (ACP) of males of Rhinella

ornata collected during calling activity in different years, showing no dif-

ference between years (P = 0.853). The bars represent the mean ± stan-

dard error with N in parentheses.

Figure 3. Interspecific variation of the antimicrobial capacity of plasma

(ACP) in five species of anurans (P ≤ 0.001). The bars represent the mean

± standard error with N in parentheses. Ha  = Hypsiboas albopunctatus;

Hf  = Hypsiboas faber; Sf  = Scinax fuscovarius; Ri  = Rhinella icterica; Ro  =

Rhinella ornata).
Antimicrobial Capacity of Plasma from Anurans of the Atlantic Forest

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158

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spectrophotometer, further expanding the diversity of

frog species for which these tests can reliably be done.

Male anurans exhibit interspecific variation in ACP

during their vocal activity. Among the species studied,

Hypsiboas albopunctatus was particularly interesting be-

cause it showed null ACP against Escherichia coli. The

absence of antimicrobial activity was maintained even

when the proportion between plasma volume and diluted

E.  coli (~104 microorganisms) was 10 times higher than

that used in the present study (results not shown). It is

possible that individuals from this species have a greater

investment in the production and maintenance of other

components of the immune system, such as blood cell

components and antimicrobial peptides synthesized by

granular glands in the dermis (Rollins-Smith, 2009). The

second species tested with lower ACP also belongs to the

genus Hypsiboas, suggesting the possibility of phylogenet-

ic signal for this trait in anurans (Blomberg et al., 2003).

However, inclusion of a greater number of species is re-

quired to test this hypothesis.

In contrast, ACP remained constant when calling

males from the same population of Rhinella ornata were

evaluated in two consecutive breeding seasons. It is also

interesting to note that individuals of R.  ornata collected

for this study showed lower ACP values than those of

R. icterica, a result similar to that obtained by Gomes et al.

(2012) with other populations of these species, and mea-

sured by CFU quantification. These results suggest the ex-

istence of interspecific differences in ACP of male anurans.

Accordingly, studies have shown the existence of substan-

tial variation in antimicrobial capacity and other aspects of

the immune response associated with different stages of

life history, energy demand, and nutritional status (Nor-

ris and Evans, 2000; Crommenacker et  al., 2010, Moore

et al., 2011, Beechler et al., 2012). Thus, it is important to

emphasize that interpopulation and interspecific compari-

sons should be restricted to samples in the same life-his-

tory conditions during the breeding season, for example.

We conclude that the measurement of ACP by spec-

trophotometry is a reliable and accurate method of esti-

mating the immunocompetence of anuran amphibians

that can be used in comparative and ecophysiological

studies. Additional tests including other strains of micro-

organisms, the use of blood samples, as well as antimicro-

bial peptides synthesized by granular glands in the dermis

can provide a more complete evaluation of the antimicro-

bial activity of these animals. The effects of ecology and

life history on interspecific ACP variation also remain to

be investigated in anurans.

ACKNOWLEDGMENTS

We would like to thank the people who assisted

us during fieldwork, especially Braz Titon Junior. This

research was funded by Fundação de Amparo à Pesquisa

do Estado de São Paulo through grant JP2006/546991

to F.R.G. and a Ph.D. fellowship 2011/105608 to V.R.A.;

Conselho Nacional de Desenvolvimento Científico e Tec-

nológico through a Ph.D. fellowship 140272/20119 to

V.R.A. and a master’s scholarship to A.M.G.B.; and Coor-

denação de Aperfeiçoamento de Pessoal de Nível Superior

through a master’s fellowship to S.C.M.T.

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Comparative physiology

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