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Synthetic versus natural curcumin: bioequivalence in an in vitro oral mucositis model

  • Sonja C Lüer1Email author,
  • Jeannette Goette2,
  • Rolf Troller3 and
  • Christoph Aebi1, 3
BMC Complementary and Alternative MedicineThe official journal of the International Society for Complementary Medicine Research (ISCMR)201414:53

DOI: 10.1186/1472-6882-14-53

Received: 1 June 2013

Accepted: 30 January 2014

Published: 11 February 2014

Curcumin (CUR) is a dietary spice and food colorant (E100). Its potent anti-inflammatory activity by inhibiting the activation of Nuclear Factor-κB is well established.

The aim of this study was to compare natural purified CUR (nCUR) with synthetically manufactured CUR (sCUR) with respect to their capacity to inhibit detrimental effects in an in vitro model of oral mucositis. The hypothesis was to demonstrate bioequivalence of nCUR and sCUR.

The purity of sCUR was HPLC-confirmed. Adherence and invasion assays for bacteria to human pharyngeal epithelial cells demonstrated equivalence of nCUR and sCUR. Standard assays also demonstrated an identical inhibitory effect on pro-inflammatory cytokine/chemokine secretion (e.g., interleukin-8, interleukin-6) by Detroit pharyngeal cells exposed to bacterial stimuli. There was bioequivalence of sCUR and nCUR with respect to their antibacterial effects against various pharyngeal species.

nCUR and sCUR are equipotent in in vitro assays mimicking aspects of oral mucositis. The advantages of sCUR include that it is odorless and tasteless, more easily soluble in DMSO, and that it is a single, highly purified molecule, lacking the batch-to-batch variation of CUR content in nCUR. sCUR is a promising agent for the development of an oral anti-mucositis agent.

Curcumin Synthetic Bioequivalence Mucositis Cancer Chemotherapy

Curcumin (CUR), a natural compound extracted from the yellow root plant Curcuma longa (Linn.)[1], is a widely known spice and yellow colorant (E100) used in food and drinks worldwide[2]. Since millennia, CUR has also been highly appreciated as a medical remedy with anti-inflammatory and antimicrobial properties. Its therapeutic use in traditional medicines varies from topical application on wounds to systemic oral application[3]. Modern medicine recently became increasingly interested in the various biological effects of the active compounds extracted from the yellow root, mainly CUR and its derivatives. To test the effects of CUR in various settings in the laboratory, commercially available extracts from the root are used. The root itself contains about 3% of CUR (diferuloylmethane), several of its derivatives, and oils, resins and other co-factors from the rhizome. Thus, effects attributable to CUR as the active agent remain unclear in most experiments. Standardization of the effects of CUR and of curcuminoids remains difficult, since in the majority of studies the exact composition of the CUR powder used is not specified, and in preparations with a declared percentage of CUR content the remaining components are not outlined in detail. Thus, the effects seen could possibly be attributed in part to the combination of CUR and curcuminoids as well as to other components of the preparation. Most production processes include purification or concentration during or after the extraction from the root to achieve high concentrations of CUR. Even though standardized and regulated by authorities, there is a possibility of contamination of nCUR with substances used during plant growth, from the environment, and from the production and purification processes (e.g. fertilizers, heavy metals, spores).

To compare a synthetically manufactured high purity curcumin (sCUR) to a standard nCUR, we used our previously established in vitro mucositis system[4, 5] and tested cytotoxic, anti-inflammatory, and antimicrobial properties of both substances to determine how effective the > 99% pure sCUR acts in comparison with standard nCUR. sCUR could possibly serve as a topical agent against cancer therapy-induced and other forms of oral mucositis.

Microorganisms, cell lines and culture conditions

Microorganisms were grown in Brain-Heart Infusion broth (BHI) at 37°C in a 5% CO2 atmosphere or in air at 150 revolutions per minute (rpm). Clinical isolates included Moraxella catarrhalis ATCC 25238[4], M. catarrhalis O35E[6], Streptococcus pneumoniae serotype 6B[5] and nontypable Haemophilus influenzae[5]. The human pharyngeal cell line Detroit 562 (ATCC CCL 138) was maintained in Eagle’s minimal essential medium (MEM, Invitrogen, Basel, Switzerland) supplemented as indicated with heat-inactivated fetal calf serum (FCS), 2 mM L-glutamine, 1 mM sodium pyruvate (Sigma, St. Louis, MO), 1x nonessential amino acids (Sigma), 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in an atmosphere containing 5% CO2.

nCUR, purified from Curcuma longa (Turmeric), was purchased from Sigma (St. Louis, MO) (No. C1386). According to the manufacturer, it contains > 65-70% diferuloylmethane (CUR) and greater than 90% curcuminoids by HPLC. Commercially available sCUR was obtained from Aptuit Laurus Ltd., Visakhapatnam, India, a Good Manufacturing Practice (GMP) approved manufacturing site. The procedure of synthesis is proprietary. The batches of sCUR used were reported to be greater than 99% pure by high pressure liquid chromatography (HPLC) and to contain residual amounts of ethyl acetate, methanol, toluene, and n-butanol. sCUR from this manufacturer fulfils FDA approved GRAS (Generally Regarded As Safe) safety criteria. We confirmed the purity of sCUR using our in house HPLC. As indicated by the manufacturer (purity, 99.6%), we found an overall purity of 99.7% (98.2% and 1.5% in keto- and enol-form, respectively). For use in experimental procedures, both nCUR and sCUR were solubilized in fresh dimethylsulfoxide (DMSO) (stock solution, 73.678 mg/ml, i.e., 200 mM) and added to cell culture or growth media. Thus, the standard working concentration of 200 μM CUR contained 0.1% DMSO. Also, negative controls (phosphate-buffered saline (PBS), MEM, BHI broth) contained 0.1% DMSO, unless noted otherwise.

Detection of cytotoxicity

Cytotoxicity Detection Kit Plus (LDH)® from Roche Diagnostics GmbH, Mannheim, Germany was used to detect CUR-induced cytotoxicity to Detroit epithelial cells after 4 hours of exposure to various concentrations of CUR. Three independent experiments were performed.

Time-kill experiments of bacteria exposed to CUR

M. catarrhalis, S. pneumoniae and nontypable Haemophilus influenzae were grown in BHI to an OD600 of 0.4 (~5×107 colony forming units (cfu)/ml), aliquoted, and subsequently grown in medium supplemented with 20, 50, and 100 μM nCUR or sCUR, respectively. Growth in BHI containing 0.05% DMSO was used as control. Quantitative cultures were obtained by serial plating of 100 μl-aliquots at 0, 60, 120, 180 and 240 minutes, respectively.

Epithelial cell adherence assays

The ability of nCUR or sCUR to inhibit the attachment of M. catarrhalis to Detroit cells in vitro was measured as previously described[7]. Briefly, Detroit cells (~2.5×105 per well) grown to a confluent monolayer in 24-well tissue culture plates were exposed to various concentrations of nCUR or sCUR (0–200 μM) for 60 minutes in MEM supplemented with 10% FCS, followed by washing three times in MEM. Bacteria grown overnight were adjusted to a multiplicity of infection (MOI) of 30. Bacteria were then added to tissue culture wells in MEM without antibiotics, centrifuged for 5 min at 1500 rpm, and incubated for 30 minutes at 37°C. Wells were then washed 5 times in MEM, trypsinized, and the suspensions were cultured quantitatively to determine the number of adherent bacteria. Data were expressed as the proportion of bacteria, i.e. cfu, of the original inoculum adhering to the epithelial cells. Each assay was performed in triplicate and at least three experiments were performed.

Epithelial cell invasion assays

Bacterial invasion was estimated using a conventional gentamicin protection assay as previously described[8] with the following modifications. Cells were prepared in MEM without antibiotics and subsequently exposed to CUR as described for the adherence assays. After washing, bacteria were added at MOI 30, centrifuged for 5 min at 1500 rpm and incubated for 3 h at 37°C in 5% CO2. To determine the number of intracellular bacteria, the infected monolayer was washed three times in PBS and treated with gentamicin sulfate (200 μg/ml) for 2 h at 37°C in order to kill all extracellular bacteria. After washing, cells were detached from the plastic surface by treatment with 0.25% trypsin-EDTA, lysed by the addition of 1% saponin, and serially diluted in PBS for quantitative bacterial culture. Invasion ratios were calculated by dividing the number of cfu recovered after gentamicin exposure by the number of cfu inoculated.

Determination of cytokine/chemokine secretion by Detroit cells stimulated with live whole bacteria

Monolayers of Detroit cells in 24-well plates were prepared as described above and pre-incubated with nCUR or sCUR at 0 (i.e., MEM with 10% FCS and DMSO only), 100 or 200 μM for 0, 15, 30 or 60 min, respectively. Wells were then washed to remove CUR and subsequently infected at a MOI of 100. To exclude pro-inflammatory activation by CUR only, mock infection (MOI 0) after pre-exposure with 200 μM of CUR was performed as a negative control. Ten μg/ml of lipopolysaccharide from Salmonella enteritidis (Sigma) was used as positive control. After 4 hours, cell culture supernatants were removed, centrifuged and stored at -80°C before determination of cytokine/chemokine concentrations. Interleukin (IL)-8 was determined using a commercially available ELISA kit according to the manufacturer’s protocol (R&D Systems, Minneapolis, MN). For determination of additional cytokines and chemokines in the same cell supernatants the Luminex® xMAP® technology was used[5]. Milliplex map kits (Milliplex Corporation) were used for microsphere-based multiplex immunoassays. Using a commercially available eight-plex kit, IL-6, IL-8, IL-10, Monocyte chemoattractant protein 1 (MCP-1), Tumor Necrosis Factor α (TNF α), Vascular Endothelial Growth Factor (VEGF), Fibroblast Growth Factor 2 (FGF-2), and Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) were determined according to the manufacturer’s instructions. Supernatants from three independent experiments, each run in duplicates, were tested. Values below the detection limit were given an arbitrary value suggested by the manufacturer.

Statistical analysis

Two-way analysis of variance (ANOVA) with Bonferroni’s post-test correction was used (GraphPad Prism 5.02 statistics package, San Francisco, CA). P values < 0.05 were considered statistically significant.

Characterization of nCUR and sCUR

While nCUR is a yellow powder, which at our standard working concentration of 200 μM has its characteristic smell and musky, bitter flavor, sCUR, an orange-colored powder, is entirely odorless and tasteless. Maximum solubility in water, PBS or MEM supplemented each with 0.1% vol/vol of DMSO was somewhat better for sCUR at 500 μM. Both nCUR and sCUR demonstrated minimal and comparable cytotoxicity when LDH was determined in the supernatant after a 4-hour exposure of a monolayer of Detroit cells to up to 400 μM CUR (Figure 1). It is of note that at concentrations exceeding 200 μM nCUR was not entirely in solution. Although the fluid appeared clear, centrifugation resulted in the formation of a small pellet.
Figure 1

Cytotoxicity of CUR for Detroit 562 cells. Cytotoxicity assay. Monolayers of Detroit 562 cells were exposed to various concentrations of nCUR or sCUR, respectively, as indicated. The proportion of toxicity is indicated as mean + 1 SEM of three consecutive experiments run in triplicate, i.e., 9 data points per condition were tested. The positive control was provided by the manufacturer of the kit and led to complete cell lysis (defined as 100% lysis).

Bactericidal activity of nCUR versus sCUR

CUR is known to have antibacterial and antifungal activity, the extent of which is both species- and strain-dependent[5]. In order to compare nCUR and sCUR we chose two well characterized strains of M. catarrhalis for time-kill analyses. While strain ATCC 25238 was highly susceptible to CUR (data not shown)[5], a concentration of 100 μM CUR was required to result in a bactericidal effect in strain O35E (Figure 2). Importantly, there was no difference between nCUR and sCUR. S. pneumoniae serotype 6B and non-typable H. influenza e were also tested with no difference between nCUR and sCUR (data not shown).
Figure 2

Bactericidal effects of nCUR versus sCUR. Comparative quantitative time-kill analysis of nCUR versus sCUR for M. catarrhalis strain O35E. Quantitative cultures were obtained at 0, 60, 120, 180, and 240 minutes. Killing kinetics were similar for both nCUR and sCUR at all concentrations tested (20, 50, and 100 μM). The negative control medium consisted of BHI containing 0.05% DMSO.

Inhibition of bacterial adherence and invasion to epithelial cells

While 100 μM CUR did not significantly inhibit bacterial adherence to Detroit cells (Figure 3), both 200 μM nCUR and sCUR demonstrated a statistically significant inhibitory effect. There was, however, no difference between the two forms of CUR (nCUR, 17.1% vs. sCUR, 19.8%, not significant). Both values were significantly lower than the control (49.8%, p < 0.05). The effect of CUR on cellular invasion of bacteria using the gentamicin protection assay was more difficult to assess. In contrast to previous findings[4], CUR did not significantly inhibit invasion. Although the proportion of invading bacteria was somewhat lower in cells exposed to 200 μM sCUR, this difference was not statistically significant (Figure 4).
Figure 3

Effect of nCUR versus sCUR on bacterial adherence. M. catarrhalis adherence assay. After pre-incubation of Detroit cells for 60 minutes with nCUR or sCUR, respectively, at the indicated concentrations, bacteria (M. catarrhalis ATCC 25238) were inoculated onto the monolayers at a MOI of 30, centrifuged, and incubated for 30 min at 37°C. Non-adherent bacteria were removed and adherent bacteria were determined by quantitative culture of trypsinized cells. The overall ANOVA test statistic revealed a p < 0.0001, the between-column values calculated using Bonferroni’s multiple comparisons test were >0.05 for nCUR versus sCUR at 100 or 200 μM, respectively. Mean adherence ratios + 1 SD of three independent experiments, each run in triplicate, are shown. MEM containing 0.1% DMSO, but no CUR, was used as control.
Figure 4

Effect of nCUR versus sCUR on bacterial invasion. Determination of invasion of Detroit cells by M. catarrhalis strain 25238 using a gentamicin protection assay. Cells were pre-treated with 0, 100 or 200 μM of nCUR or sCUR, respectively, for 1 hour. Following 3 hours of infection, extracellular bacteria were killed by exposure to gentamicin for 2 hours. Following cell lysis, quantitative bacterial cultures identified viable intracellular bacteria. The ANOVA overall p value was 0.112. In-between column p values were > 0.05. Mean adherence ratios + 1 SEM of three independent experiments, each run in triplicate, are shown.

Inhibition of epithelial cytokine/chemokine release by CUR

As previously established, IL-8 was used as a representative of pro-inflammatory cytokines/chemokines[4, 5]. Figure 5 demonstrates that both nCUR and sCUR similarly inhibit IL-8 secretion by Detroit cells (no statistical difference). This effect was seen for both concentrations of CUR tested. In addition, Luminex technology was used to assess the same cell supernatants for concentrations of GM-CSF, IL-6, IL-8, IL-10, MCP-1, VEGF, FGF-2, and TNFα. As expected[5], the release of all cytokines/chemokines with the exception of IL-10 and FGF-2 (not shown) was fully suppressed by pre-exposure to nCUR or sCUR for 60 minutes followed by stimulation of the monolayer with a bacterial MOI of 100 for 4 hours (Figure 6).
Figure 5

Inhibition of IL-8 secretion by nCUR versus sCUR. IL-8 secretion by Detroit cells preincubated for 0, 15, 30 or 60 minutes with nCUR (green) or sCUR (red) and subsequently infected for 4 hours with live M. catarrhalis strain 25238 (MOI 100). □ indicates the positive control (0 μM CUR, MOI 100). (200 μM nCUR, MOI 0) and ■ (200 μM sCUR, MOI 0) are negative controls. At no time point there is was a statistically significant difference between nCUR and sCUR for a given concentration.
Figure 6

Inhibition of release of pro-inflammatory cytokines by nCUR versus sCUR. Effect of nCUR and sCUR on secretion of GM-CSF, IL-6, IL-8, MCP-1, VEGF, and TNFα by Detroit cells preincubated for 0 or 60 minutes with 200 μM nCUR (green) or 200 μM sCUR (red) and subsequently infected for 4 hours with live M. catarrhalis strain 25238 (MOI 100). □ (black) indicates the positive control (0 μM CUR, MOI 100). At no time point there is a statistically significant difference between nCUR and sCUR.

As of today, in vitro laboratory work and clinical trials with CUR (diferuloylmethane) have exclusively used nCUR purified from the root of Curcuma longa[9] or chemically synthesized derivatives or nanoparticles[10, 11]. This is a reasonable approach because highly purified nCUR can be produced in large quantities and has proved effective in many experimental assays and as a food additive and colorant (E100)[12]. However, as elaborated above, purified nCUR is always contaminated by other chemicals (e.g., demethoxycurcumin, bisdemethoxycurcumin, oils and resins)[12], which may or may not influence the biological effects attributed to nCUR. A large number of CUR analogs have thus been synthesized in recent years, and many displayed activities similar to the mother compound[12, 13]. A more recent approach, fully synthetic manufacturing of diferuloylmethane (sCUR), offers the potential to study the effects of this particular molecule without interference by contaminants.

In our line of work, i.e., the characterization of CUR as a potential therapeutic agent against cancer therapy-induced oral mucositis[4, 5], sCUR offers a number of potential advantages. In contrast to turmeric with its musky, bitter, and piquant flavor[12], it is entirely odorless and tasteless, and therefore suitable as an ingredient of an orally administered preparation, particularly for children. Since it appears somewhat more soluble in DMSO than nCUR, aqueous solutions for oral application can be produced with less DMSO (i.e., 0.04% vs. 0.1%, vol/vol). In this context, it is worth mentioning that CUR has recently also been shown to inhibit adherence to teeth and extracellular matrix of Streptococcus mutans, the major causative agent of human caries[14, 15]. In the present study we found that antimicrobial activity of sCUR in standard time-kill analyses was indistinguishable from nCUR[5, 16, 17] on a weight per volume basis for 3 major pharyngeal species (Figure 2).

With respect to prevention of mucositis, the key findings of this comparative analysis addressed the anti-inflammatory properties of the two different CUR preparations and are shown in Figures 3,4,5, and6. First, both nCUR[4] and sCUR inhibited bacterial adherence to pharyngeal epithelial cells to a similar extent. Attachment of microorganisms is the first step in initiating inflammation by avoiding their elimination by the flow of mucosal secretions. It enhances their ability to trap nutrients and their rate of multiplication[18]. Attachment also allows intimate interaction with cellular pattern recognition receptors (e.g., Toll-like receptors), which activate downstream pro-inflammatory cascades including activation of NF-κB[18, 19]. Second, there was no difference in the invasion ratio of live bacteria exposed to either nCUR or sCUR. However, in contrast to previous findings[4], we observed no CUR-induced inhibition of cellular penetration. Third, both nCUR and sCUR equally inhibited the secretion of several pro-inflammatory cytokines (e.g., IL-8, IL-6, MCP-1, GM-CSF) upon stimulation with live bacteria. This finding provides indirect evidence that nCUR and sCUR are equally potent in inhibiting the dissociation of IκB from NF-κB[20], and thus in down-regulating inflammation. To our knowledge, this is the first study, which compares sCUR and nCUR in an in vitro model designed to mimic the pro-inflammatory action of bacteria on human pharyngeal cells.

Our study has a number of limitations. We used a simple mucositis model, which may substantially deviate from true in vivo conditions. Also, CUR is reported to be more stable at pH values below 6.0[12]. We used solvents with a buffered pH around 7.0. We consider this justified because human salivary pH typically varies between 6.5 and 7.2. Our data are thus relevant for studies mimicking the topical use of CUR in the oral cavity.

The data provided here demonstrate that sCUR and nCUR are equivalent in a number of in vitro biological assays, which are designed to mimic bacteria-induced mucosal surface inflammation. The fact that the concentrations used are far below the daily allowances as a food additive for oral CUR (E100), which amounts to 3 mg/kg/d of nCUR, warrant the claim that sCUR used at a concentration of 200 μM (e.g., 10 ml of mouth rinse 4 times daily amounts to a total daily dose of 2.94 mg) is clearly in the range declared safe by the European Food Safety Authority[21]. An additional advantage in manufacturing sCUR is the fact that there is no batch-to-batch variation in the CUR content. Thus, sCUR appears to be a safe, equipotent and more palatable alternative to nCUR, and an excellent candidate for topical use in clinical oral mucositis trials.

The authors thank Suneetha K Nair, Aptuit Laurus, Inc., Visakhapatnam, India for providing sCUR free of charge (no sponsorship). A competitive grant from Vinetum Foundation, Biel, Switzerland, provided financial support.

Authors’ Affiliations

Division of Pediatric Hematology/Oncology, Department of Pediatrics, University of Bern, Inselspital
Institute of Hospital Pharmacy, University of Bern, Inselspital
Institute for Infectious Diseases, University of Bern, Inselspital

  1. Gupta SC, Patchva S, Koh W, Aggarwal BB: Discovery of curcumin, a component of golden spice, and its miraculous biological activities. Clin Exp Pharmacol Physiol. 2012, 39: 283-299. 10.1111/j.1440-1681.2011.05648.x.View ArticlePubMedPubMed CentralGoogle Scholar
  2. Tennant DR: Screening potential intakes of colour additives used in non-alcoholic beverages. Food Chem Toxicol: an International Journal Published for the British Industrial Biological Research Association. 2008, 46: 1985-1993. 10.1016/j.fct.2008.01.051.View ArticleGoogle Scholar
  3. Aggarwal BB, Sung B: Pharmacological basis for the role of curcumin in chronic diseases: an age-old spice with modern targets. Trends Pharmacol Sci. 2009, 30: 85-94. 10.1016/ ArticlePubMedGoogle Scholar
  4. Lüer S, Troller R, Jetter M, Spaniol V, Aebi C: Topical curcumin can inhibit deleterious effects of upper respiratory tract bacteria on human oropharyngeal cells in vitro: potential role for patients with cancer therapy induced mucositis?. Support Care Cancer. 2009, 19: 799-806.View ArticleGoogle Scholar
  5. Lüer S, Troller R, Aebi C: Antibacterial and antiinflammatory kinetics of curcumin as a potential antimucositis agent in cancer patients. Nutr Cancer. 2012, 64: 975-981. 10.1080/01635581.2012.713161.View ArticlePubMedGoogle Scholar
  6. Helminen ME, Maciver I, Paris M: A mutation affecting expression of a major outer membrane protein of Moraxella catarrhalis alters serum resistance and survival in vivo. J Infect Dis. 1993, 168: 1194-1201. 10.1093/infdis/168.5.1194.View ArticlePubMedGoogle Scholar
  7. Aebi C, Lafontaine ER, Cope LD: Phenotypic effect of isogenic uspA1 and uspA2 mutations on Moraxella catarrhalis 035E. Infect Immun. 1998, 66: 3113-3119.PubMedPubMed CentralGoogle Scholar
  8. Spaniol V, Heiniger N, Troller R, Aebi C: Outer membrane protein UspA1 and lipooligosaccharide are involved in invasion of human epithelial cells by Moraxella catarrhalis. Microbes Infect. 2008, 10: 3-11. 10.1016/j.micinf.2007.09.014.View ArticlePubMedGoogle Scholar
  9. Aggarwal BB, Harikumar KBM: Potential therapeutic effects of curcumin, the anti-inflammatory agent, against neurodegenerative, cardiovascular, pulmonary, metabolic, autoimmune and neoplastic diseases. Int J Biochem Cell Biol. 2009, 41: 40-59. 10.1016/j.biocel.2008.06.010.View ArticlePubMedGoogle Scholar
  10. Zheng XH, Shao YX, Li Z: Quantitative structure-retention relationship of curcumin and its analogues. J Sep Sci. 2012, 35: 505-512. 10.1002/jssc.201100903.View ArticlePubMedGoogle Scholar
  11. Alam S, Panda JJ, Chauhan VS: Novel dipeptide nanoparticles for effective curcumin delivery. Int J Nanomedicine. 2012, 7: 4207-4221.PubMedPubMed CentralGoogle Scholar
  12. Esatbeyoglu T, Huebbe P, Ernst IM: Curcumin – from molecule to biological function. Angew Chem Int Ed Engl. 2012, 51: 5308-5332. 10.1002/anie.201107724.View ArticlePubMedGoogle Scholar
  13. Zhu S, Moore TW, Lin X: Synthetic curcumin analog EF31 inhibits the growth of head and neck squamous cell carcinoma xenografts. Integr Biol. 2012, 4: 633-640. 10.1039/c2ib20007d.View ArticleGoogle Scholar
  14. Yao JW, Lin F, Tao T, Lin CJ: Affinity interactions between natural pigments and human whole saliva. Arch Oral Biol. 2011, 56: 285-293. 10.1016/j.archoralbio.2010.10.003.View ArticlePubMedGoogle Scholar
  15. Song J, Choi B, Jin EJ, Yoon Y: Curcumin suppresses Streptococcus mutans adherence to human tooth surfaces and extracellular matrix proteins. Eur J Clin Microbiol Infect Dis. 2012, 31: 1347-1352. 10.1007/s10096-011-1448-y.View ArticlePubMedGoogle Scholar
  16. Garcia-Gomes AS, Curvelo JA, Soares RM, Ferreira-Pereira A: Curcumin acts synergistically with fluconazole to sensitize a clinical isolate of Candida albicans showing a MDR phenotype. Med Mycol. 2012, 50: 26-32. 10.3109/13693786.2011.578156.View ArticlePubMedGoogle Scholar
  17. De R, Kundu P, Swarnakar S, Ramamurthy T: Antimicrobial activity of curcumin against Helicobacter pylori isolates from India and during infections in mice. Antimicrob Agents Chemother. 2009, 53: 1592-1597. 10.1128/AAC.01242-08.View ArticlePubMedPubMed CentralGoogle Scholar
  18. Svanborg C, Hedlund M, Connell H: Bacterial adherence and mucosal cytokine responses. Receptors and transmembrane signaling. Ann N Y Acad Sci. 1996, 797: 177-190. 10.1111/j.1749-6632.1996.tb52959.x.View ArticlePubMedGoogle Scholar
  19. Hauf N, Chakraborty T: Suppression of NF-kappa B activation and proinflammatory cytokine expression by Shiga toxin-producing Escherichia coli. J Immunol. 2003, 170: 2074-2082.View ArticlePubMedGoogle Scholar
  20. Van‘t Land B, Blijlevens NM, Marteijn J: Role of curcumin and the inhibition of NF-kappaB in the onset of chemotherapy-induced mucosal barrier injury. Leukemia. 2004, 18: 276-284. 10.1038/sj.leu.2403233.View ArticlePubMedGoogle Scholar
  21. Authority EFS: Scientific Opinion on the re-evaluation of curcumin (E100) as a food additive. EFSA J. 2010, 8: 1679-1725.Google Scholar
  22. Pre-publication history

    1. The pre-publication history for this paper can be accessed here:

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Is female preference for large sexual ornaments due to a bias to escape predation risk?

Contributed equally
BMC Evolutionary Biology201212:33

DOI: 10.1186/1471-2148-12-33

Received: 27 March 2011

Accepted: 13 March 2012

Published: 13 March 2012

A female preference for intense sexual visual signals is widespread in animals. Although the preferences for a signal per se and for the intensity of the signal were often regarded to have the identical origin, no study has demonstrated if this is true. It was suggested that the female fiddler crabs prefer males with courtship structures because of direct benefit to escape predation. Here we tested if female preference for both components (i.e. presence and size) of the courtship structure in Uca lactea is from the sensory bias to escape predation. If both components have the identical origin, females should show the same response to different-sized courtship structures regardless of predation risk.

First, we observed responses of mate-searching female U. lactea to courting males with full-sized, half-sized and no semidomes which were experimentally manipulated. Females had a directional preference for males with bigger semidomes within normal variation. Thereafter, we tested the effect of predation risk on the female bias in the non-courtship context. When threatened by an avian mock predator, females preferentially approached burrows with full-sized semidomes regardless of reproductive cycles (i.e. reproductive periods and non-reproductive periods). When the predator cue was absent, however, females preferred burrows with semidomes without discriminating structure size during reproductive periods but did not show any bias during non-reproductive periods.

Results indicate that selection for the size of courtship structures in U. lactea may have an origin in the function to reduce predation risk, but that the preference for males with structures may have evolved by female choice, independent of predation pressure.

Sexual selection can be used to explain the evolution of various male secondary sexual characters which are used as courtship signals [1, 2]. A number of experimental studies provide evidence that females prefer males with conspicuous sexual traits [39]. Females may benefit from choosing males with strong signals directly by reducing their own predation risk [1014] or by providing better parental care to their offspring [1517]. Females also may acquire indirect benefit by transmitting high genotypic quality of males to their offspring [3, 6, 18, 19]. The preference for the existence of the signal and the preference for signal intensity are often regarded to have the same origin [8, 2023]. However, the preferences for a signal per se and for intensity of the signal may have different origins when the two components serve different functions. No studies to date have tried to separate the origins of preferences for these two components of courtship signals.

Fiddler crabs of the genus Uca are semi-terrestrial animals that live on intertidal mud or sand flats [24]. Courting males of approximately 18 species sometimes build various kinds of mud or sand structures at their burrows such as hoods, pillars, and lips [25]. It has been demonstrated that the mud pillars in U. beebei [2628] and the sand hoods in U. musica [25, 29] function as sexual signals in attracting females. The sensory trap hypothesis has been suggested to explain the female preference for particular courtship structures [12, 30]. It was recently found that the preference for hoods in U. terpsichores [11] and for pillars in U. beebei [10] could increase with the perceived predation risk, because the structures provided a direct survival benefit to females by allowing them to escape predation. However, female U. terpsichores did not show a directional preference for exaggerated artificial hoods which were conspicuously larger than average-sized natural hoods [30]. Nevertheless, females may prefer larger structures within natural variation and this possibility has not yet been explored.

Here we tested if both the preference for a courtship structure and the preference for the size of the structure in the fiddler crab Uca lactea were shaped by the need of females to escape predation. Males of U. lactea build semidomes using mud at their burrows and wave their large claws to attract females for mating. Previous studies suggest that semidome building in U. lactea is related to courtship signaling [31] and the semidome has a function to attract females [32]. Given the evidence of previous studies, females may prefer larger semidomes over smaller ones if the courtship structure could provide a greater survival benefit to them.

Our first objective in this study is to determine if female U. lactea prefer males with larger sized courtship structures. If male attractiveness increases with the semidome size, we can predict that males with larger semidomes would mate more successfully than those with smaller semidomes. The second objective of this study is to examine if the preference for structures and preference for structure size have the same origin to function in predation avoidance [28, 29]. To test this, we conducted an arena experiment in the non-courtship context for females to choose burrows with different-sized semidomes in the presence and absence of predation cue. A previous pilot study found that female U. lactea without predator threat preferentially moved to burrows with semidomes during reproductive periods, but did not show any orientation bias during non-reproductive periods [32]. Thus, we conducted an experiment with and without a predator cue during both reproductive and non-reproductive periods to determine if the reproductive cycle influences female responses to male courtship structures.

Female preference in the context of mate choice

We observed 159 interactions between 73 females and semidome-building males with full-sized semidomes, half-sized semidomes and without semidomes (Table 1). On average each female visited 1.68 ± 1.32 males and passed 0.49 ± 0.88 males before choosing one of them as a potential mate.
Table 1

Responses of mate-searching females to courting male U. lactea with full-sized, half-sized or without semidome

Type of semidome

% visiting

without semidome

Generalized linear mixed model (GLMM) analyses revealed a significant difference in attractiveness among semidomes of different size (effect of semidome size: F2, 156 = 6.879, p = 0.001, effect of female identity as a random factor: Z = 0.654, p = 0.513). The visiting frequency of mate-searching females showed a linearly increasing trend with semidome size (X2 = 16.313, d.f. = 1, p < 0.001) in the chi-square test. Females preferred males with semidomes of any size to males without semidomes (X2 = 19.008, d.f. = 1, p < 0.001). Also, males with full-sized semidomes were significantly more attractive than males with half-sized semidomes (X2 = 5.210, d.f. = 1, p = 0.022).

The effect of predation risk on female choice for different sized-semidomes

An ordinal logistic regression test revealed that predation risk is a significant factor which influences the female choice on semidome size (Wald X2 = 6.606, d.f. = 1, p = 0.010). However, there was no marked effect of reproductive cycle (i.e., reproductive or non-reproductive period) on female choice (Wald X2 = 0.001, d.f. = 1, p = 0.975) in the ordinal regression test. The effect of interaction between predation pressure and reproductive cycle was not significant either (Wald X2 = 2.721, d.f. = 1, p = 0.099).

Under high predation risk, female choices did not differ significantly between reproductive periods and non-reproductive periods (X2 = 0.033, d.f. = 2, p = 0.983). Under low predation risk, however, there was significant difference between two periods (X2 = 9.013, d.f. = 2, p = 0.011). More females moved to burrows with full-sized semidomes than to burrows with half-sized semidomes or without semdiomes in the presence of a mock predator (X2 = 61.659, d.f. = 2, p < 0.001, Figure 1a). In the absence of a mock predator, however, females showed different orientation biases varying with the reproductive cycle (Figure 1b): females during reproductive periods preferred to move to burrows with semidomes rather than to burrows without semidomes (X2 = 8.665, d.f. = 1, p = 0.003), but did not discriminate between the sizes of semidomes (X2 = 0.385, d.f. = 1, p = 0.535); during non-reproductive periods, females did not show any orientation bias to different semidome treatments (X2 = 1.316, d.f. = 2, p = 0.518).
Figure 1

Visiting frequency by females to artificial burrows with full-sized, half-sized or without semidome under (a) predator-present condition and (b) predator-absent condition in the arena test.

Our study provides the experimental evidence that sexual attractiveness of male fiddler crabs increases with size of the courtship structures within natural variation. Compared to males with half-sized semidomes and without semidomes, those with full-sized semidomes were more attractive to mate-searching females. Males with half-sized semidomes were more attractive to females than those without semidomes. The directional preference for the size of structures suggests that there is a positive correlation between male attractiveness and semidome size within the normal range of variation.

Why do females prefer males with larger structures? Given that females in the non-courtship context showed a directional orientation bias for larger semidomes under predation threat regardless of the reproductive cycle, larger semidomes may be more salient visual cues to provide protection to females. The consistency between female choice in the mate-searching context and female orientation response in the predation context [12, 28, 29] indicates that the preference for the larger signal may have originated from the female bias to escape predation. In other words, mate choice is linked to male-built structures that can serve as a landmark to keep females safe against predators. Characteristics of the fiddler crab's visual system may also explain why females prefer larger semidomes. The horizontal resolving power of fiddler crabs' eyes is poor compared to their vertical resolving power [3335]. Accordingly, fiddler crabs are better able to resolve objects in the vertical than in the horizontal plain. Semidomes which are tall enough to be imaged within the zone of the acute vertical resolution of their eyes may be most conspicuous to fiddler crab females [36]. In the absence of predator cues, however, females responded differently to different-sized semidomes depending on the reproductive cycle. During the non-reproductive periods, females did not show any difference in the preference for burrows with different treatments. In contrast, during the reproductive periods, they showed different responses. Although females did not more preferentially move to full-sized structures than half-sized structures, they did prefer moving to burrows with structures over moving to burrows without structures. This result presents a striking contrast to the case of U. musica (later correctly identified as U. terpichores) where females have an orientation preference to structures, even without predator cue [29]. Therefore, the predator-induced sensory trap hypothesis [12] cannot fully explain the female preference on the structure building in U. lactea, as the effect of large structures decreased in the absence of a predator. Rather, structure building itself may have been selected by female choice independent of predation pressure.

This female preference for structures may have started because fiddler crab courtship structures may signal male nutritional condition. In previous studies in which males were supplemented with food, it was shown that structure building depends on male condition [3739]. The semidome building behavior may signal phenotypic condition of a male crab, which could be affected by food availability. Females may choose better males using semidomes as an indicator of genetic quality. Later, the predation pressure might enhance the selection on larger structures, which may help reduce predation risk for female crabs.

In conclusion, we have decoupled the origins of the preference on the sexual signal and signal intensity by manipulating predation risk. The preference for semidomes in U. lactea may have been selected by female choice not related with predation pressure. However, the preference for increased semidome size could have been strengthened to reduce the predation risk for females. Our results suggest that sexual signal per se and signal intensity is not necessarily selected by the same selection pressure.

Study site and species

All observations and experiments were conducted at the 'Choji' intertidal mudflat (37°35'N 126°32'E) in Ganghwa Island near the Han River in South Korea from June to August in 2007-2009. U. lactea lives on mudflats of the upper intertidal zone in the temperate, tropical and subtropical Indo-Pacific [24]. From May to October, they emerge and feed on the mudflat surface when the tide recedes. Each reproductive cycle of about 15 days has reproductive and non-reproductive periods in the study site [40]. During the breeding season, males build semidomes on the mudflat at their burrows, and then wave their large claws in order to attract females to mate. When the females are ready to mate, they leave their burrows and wander on the mudflat, searching for mates. Receptive females approach courting males, enter the burrows following them, and then leave the burrows several seconds later. A female visits and samples several burrows of courting males until she eventually selects a male as her mate. In successful courtship, the male copulates with a female underground and plugs the entrance of his burrow with mud for mate guarding. The mating pair remained in the burrow together for one to five days [41]. After the female finished ovulation, the male comes out from the burrow and departs, leaving the female in the male's burrow to incubate her eggs. In the non-reproductive periods, both males and females mostly feed on the mudflats, males do not wave claws to attract females and females do not search for their mates [40].

Semidome building behavior of the males starts at the beginning of the breeding season in early June. During 3-5 day periods when the number of courting males peaks in each semi-lunar cycle, many males construct semidomes using mud at their burrows. The mean ± SD height and width of semidomes was 17.6 ± 5.0 mm and 26.7 ± 5.6 mm [31].

The eastern Mew Gull, Larus canus, is the natural predator of U. lactea at the study site. During the whole experiment period they were rarely present at test areas.

Female preference test in the context of mate choice

We tested the effect of courtship structures on the female preference by measuring the visiting and passing frequencies of female crabs. We defined visiting frequency as the number of times mate-searching females approached and touched a male burrow with a part of their bodies. The passing frequency was defined as the number of times mate-searching females walked towards a male burrow but turned away before getting close to it and left without a contact with the burrow. As the mating rates of males might be positively correlated with the visiting rates of mate-searching females [10, 42], by recording the visiting frequency, we are able to give reliable estimates of attractiveness of males with different sized courtship structures. We compared female choice among males with full-sized semidomes, half-sized semidomes and without semidomes.

The experiment was conducted during the intense courtship period in which most of males constructed semidomes at their burrows. We selected an observation plot (approx. area: 150 m2) randomly in each experimental day. For every three adjacent actively courting males with semidomes in the plot, we removed all the semidomes already built and replaced them with full-sized, half-sized artificial semidomes, and nothing respectively. We deployed 50-70 manipulated clusters of courting males in randomly chosen plots. Thus, different-sized semidomes were randomly distributed across the observation area. We made the artificial semidomes prior to the experiment. Artificial semidomes were made out of clay to imitate natural semidomes, and colored with khaki dyestuff outside. We produced artificial semidomes in two different sizes: full-sized semidomes (17 mm/25 mm in height/width) and half-sized semidomes (8.5 mm/25 mm in height/width). In order to easily identify the three different experimental groups of males (with full-sized, half-sized, and no semidomes) from a distance, we marked each burrow with one of three colored (red, yellow and blue) wooden sticks (diameter: 2 mm; length: 10 cm) by inserting a wooden stick 10 cm away from each burrow. We daily designated and switched the color for each experimental group in a random order to control the effect of color of wooden sticks on female choice.

We sat on a chair near the plot and observed crabs directly or through binoculars. The distance between the observer and test plot was kept to at least 1.5 m so that the crabs could behave normally and would not be affected by our observation. We recorded the number of times females visited males with the different-sized semidomes and also noted whether females left or stayed in the male burrow. The observation on each mate-searching female continued until she left the experimental area or entered a burrow and did not emerge within five minutes. A total of 159 responses from 73 females were recorded. We did not identify individual females, but the probability that our tally included some repeated records by the same females should be small. While searching for a mate, females seldom visit a given male twice. Therefore each visit can be considered as a unique courtship interaction of a given male-female pair. This assumption has been confirmed in previous studies [10, 11, 25]. Since the original semidomes built by males were removed and replaced by different-sized artificial ones randomly in this experiment, we supposed that there was no difference in the male behavior and activity of each male-female courtship interaction between males with different semidome size.

Female choice for different-sized semidomes in the non-courtship context

We selected an area uninhabited by crabs to avoid the influence of interactions with other crabs, and scratched a 50 cm-diameter circle onto the mudflat. We made 12 artificial burrows on the edge of the circle at even intervals. The artificial burrows were approximately 10 mm in diameter and 15 mm in depth, which were large enough to let females in. Full-sized and half-sized semidomes were placed behind the artificial burrows relative to the arena center. The distance from the base of a semidome to the burrow edge was approximately 5 mm. For every three adjacent burrows, one was ornamented with a full-sized semidome, another had a half-sized semidome and another had no semidome. The order of semidome placement was different in every set of three adjacent burrows.

We caught females by placing a long plastic stick over the burrow entrance after the female emerged from the burrow and then catching the crab by hand. For each test procedure, a female was placed in the center of the arena. The female was randomly either exposed to a mock predator (the predator-present condition) or not (the predator-absent condition). We used a bird-shaped model (approx. volume: 1 dm3) connected to the end of a fishing rod by 1 m-long nylon thread to imitate a natural predator. When we flung the fishing rod over the experimental set, the mock predator moved in random directions. We waited until the crab either approached a burrow or left the arena. We recorded which of three kinds of burrows (with full-sized, half-sized, or no semidomes) females visited or touched. Each female was used once. The female which did not select the burrow was not counted. A total of 163 females during reproductive periods and 159 females during non-reproductive periods moved to burrows in this study.

Statistical analysis

All analyses were conducted using IBM SPSS Statistics 20. To determine whether there was a significant difference in the frequency of female visits among males with full-sized semidomes, half-sized semidomes and without semidomes, we ran a generalized linear mixed model (GLMM) with female choice (coded "visiting" as 1, and "passing" as 0) as a dependent variable, semidome size as a fixed effect, and female identity as a random factor. We additionally used a chi-square test for trend to evaluate if a linear trend exists between semidome size and visiting frequency. Given that the size of artificial semidomes is ordinal in the arena test, the categories of female responses to semidomes can be ordered in a meaningful way 'full-sized/half-sized/without semidome'. Therefore we used ordinal logistic regression [43, 44] to analyze relationships both between female choice and predation risk as well as between female choice and reproductive cycle. Then, we used a chi-square test to compare female choices between reproductive periods and non-reproductive periods under each predation risk. If female responses were significantly different between two periods, we tested the effect of semidome size on female choice separately for each period. If responses were not different, we pooled data to test the effect of semidome size on female choice.

Ethical note

The Institutional Animal Care and Use Committee, which oversees animal experimentation at Ewha Womans University, was not established when this study was designed and conducted. However, all experimental manipulations and procedures complied with recommended guidelines for the treatment of animals in behavioural research [45]. We did not sacrifice any animals for this study. In the end of each test, female crabs were released in the same areas where they captured and all of them behaved normally.

We thank Ju Hyung Lee, Na Ahn, Mi Ri Nam and Heung Jin Ryu for their assistance in the field work, as well as Susan Lappan and two anonymous referees for helpful comments on the manuscript. We are also grateful to Joong Hwan Jeon and Yunbiao Hu for giving statistical advice. Our gratitude also goes to Abby Watrous for revising the English language of the manuscript. This study was supported by the Ewha Global Top 5 Grant of Ewha Womans University, National Research Foundation Grants funded by the Korean Government (MOEHRD, Basic Research Promotion Fund, KRF-314-2008-1-C00384), and Converging Research Center Program funded by the Ministry of Education, Science and Technology (2010K001149). Zhen Zhu was supported by Ewha Global Partnership Program.

Authors’ Affiliations

Laboratory of Behavior and Ecology, Division of EcoSicence, Ewha Womans University
The Jane Goodall Institute China Office/Roots & Shoots Beijing Office, (Located inside Beijing City International School, Room 1309)
Monterey Bay Aquarium Research Institute

  1. Andersson M: The theory of sexual selection. Sexual selection. 1994, Princeton: Princeton University Press, 3-31.Google Scholar
  2. Darwin C: Sexual selection. The descent of man, and selection in relation to sex. 1871, London: John Murray, 253-385.View ArticleGoogle Scholar
  3. Rogers DW, Denniff M, Chapman T, Fowler K, Pomiankowski A: Male sexual ornament size is positively associated with reproductive morphology and enhanced fertility in the stalk-eyed fly Teleopsis dalmanni. BMC Evol Biol. 2008, 8: 236-10.1186/1471-2148-8-236.PubMedPubMed CentralView ArticleGoogle Scholar
  4. McClintock WJ, Uetz GW: Female choice and pre-existing bias: visual cues during courtship in two Schizocosa wolf spiders (Araneae: Lycosidae). Anim Behav. 1996, 52: 167-181. 10.1006/anbe.1996.0162.View ArticleGoogle Scholar
  5. Pryke SR, Andersson S, Lawes MJ: Sexual selection of multiple handicaps in the red-collared widowbird: female choice of tail length but not carotenoid display. Evolution. 2001, 55: 1452-1463.PubMedView ArticleGoogle Scholar
  6. Schantz Tv, Göransson G, Andersson G, Fröberg I, Grahn M, Helgée A, Wittzell H: Female choice selects for a viability-based male trait in pheasants. Nature. 1989, 337: 166-169. 10.1038/337166a0.View ArticleGoogle Scholar
  7. Bischoff RJ, Gould JL, Rubenstein DI: Tail size and female choice in the guppy (Poecilia reticulata). Behav Ecol Sociobiol. 1985, 17: 253-255. 10.1007/BF00300143.View ArticleGoogle Scholar
  8. Zuk M, Thornhill R, Ligon JD, Johnson K, Austad S, Ligon SH, Thornhill NW, Costin C: The role of male ornaments and courtship behavior in female mate choice of red jungle fowl. Am Nat. 1990, 136: 459-473. 10.1086/285107.View ArticleGoogle Scholar
  9. Andersson M: Female choice selects for extreme tail length in a widowbird. Nature. 1982, 299: 818-820. 10.1038/299818a0.View ArticleGoogle Scholar
  10. Kim TW, Christy JH, Dennenmose S, Choe JC: The strength of a female mate preference increases with predation risk. Proc R Soc B. 2009, 276: 775-780. 10.1098/rspb.2008.1070.PubMedPubMed CentralView ArticleGoogle Scholar
  11. Kim TW, Christy JH, Choe JC: A preference for a sexual signal keeps females safe. PLoS One. 2007, 2: e422-10.1371/journal.pone.0000422.PubMedPubMed CentralView ArticleGoogle Scholar
  12. Christy JH: Mimicry, mate choice and the sensory trap hypothesis. Am Nat. 1995, 146: 171-181. 10.1086/285793.View ArticleGoogle Scholar
  13. Møller AP: Badge size in the house sparrow Passer domesticus. Behav Ecol Sociobiol. 1988, 22: 373-378.Google Scholar
  14. Endler JA: Natural and sexual selection on color patterns in poeciliid fishes. Environ Biol Fishes. 1983, 9: 173-190. 10.1007/BF00690861.View ArticleGoogle Scholar
  15. Møller AP, Jennions MD: How important are direct fitness benefits of sexual selection?. Naturwissenschaften. 2001, 88: 401-415. 10.1007/s001140100255.PubMedView ArticleGoogle Scholar
  16. Senar JC, Figuerola J, Pascual J: Brighter yellow blue tits make better parents. Proc R Soc B. 2002, 269: 257-261. 10.1098/rspb.2001.1882.PubMedPubMed CentralView ArticleGoogle Scholar
  17. Suk HY, Choe JC: Females prefer males with larger first dorsal fins in the common freshwater goby. J Fish Biol. 2002, 61: 899-914. 10.1111/j.1095-8649.2002.tb01851.x.View ArticleGoogle Scholar
  18. Blount JD, Metcalfe NB, Birkhead TR, Surai PF: Carotenoid modulation of immune function and sexual attractiveness in zebra finches. Science. 2003, 300: 125-127. 10.1126/science.1082142.PubMedView ArticleGoogle Scholar
  19. David P, Bjorksten T, Fowler K, Pomiankowski A: Condition-dependent signalling of genetic variation in stalk-eyed flies. Nature. 2000, 406: 186-188. 10.1038/35018079.PubMedView ArticleGoogle Scholar
  20. Ryan MJ, Keddy-Hector A: Directional patterns of female mate choice and the role of sensory biases. Am Nat. 1992, 139: S4-S35. 10.1086/285303.View ArticleGoogle Scholar
  21. Garcia CM, Ramirez E: Evidence that sensory traps can evolve into honest signals. Nature. 2005, 434: 501-505. 10.1038/nature03363.PubMedView ArticleGoogle Scholar
  22. Rodd FH, Hughes KA, Grether GF, Baril CT: A possible non-sexual origin of mate preference: are male guppies mimicking fruit?. Proc R Soc B. 2002, 269: 475-481. 10.1098/rspb.2001.1891.PubMedPubMed CentralView ArticleGoogle Scholar
  23. Endler JA: Signals, signal conditions, and the direction of evolution. Am Nat. 1992, 139: S125-S153. 10.1086/285308.View ArticleGoogle Scholar
  24. Crane J: Fiddler crabs of the world (Ocypodidae: Genus Uca). 1975, Princeton, New Jersey: Princeton University PressGoogle Scholar
  25. Christy JH, Backwell PRY, Goshima S, Kreuter T: Sexual selection for structure building by courting male fiddler crabs: an experimental study of behavioral mechanisms. Behav Ecol. 2002, 13: 366-374. 10.1093/beheco/13.3.366.View ArticleGoogle Scholar
  26. Christy JH: Pillar function in the fiddler crab Uca beebei (I): Effects on male spacing and aggression. Ethology. 1988, 78: 53-71.View ArticleGoogle Scholar
  27. Christy JH: Pillar function in the fiddler crab Uca beebei (II): Competitive courtship signaling. Ethology. 1988, 78: 113-128.View ArticleGoogle Scholar
  28. Christy JH, Backwell PRY, Schober U: Interspecific attractiveness of structures built by courting male fiddler crabs: experimental evidence of a sensory trap. Behav Ecol Sociobiol. 2003, 53: 84-91.Google Scholar
  29. Christy JH, Baum JK, Backwell PRY: Attractiveness of sand hoods built by courting male fiddler crabs, Uca musica: test of a sensory trap hypothesis. Anim Behav. 2003, 66: 89-94. 10.1006/anbe.2003.2196.View ArticleGoogle Scholar
  30. Christy JH, Backwell PRY: No preference for exaggerated courtship signals in a sensory trap. Anim Behav. 2006, 71: 1239-1246. 10.1016/j.anbehav.2005.11.008.View ArticleGoogle Scholar
  31. Kim TW, Christy JH, Choe JC: Semidome building as sexual signaling in the fiddler crab Uca lactea (Brachyura: Ocypodidae). J Crustac Biol. 2004, 24: 673-679. 10.1651/C-2471.View ArticleGoogle Scholar
  32. Kim TW: Ecology and evolution of reproductive strategies and communication in fiddler crabs (genus Uca). PhD thesis. 2006, Seoul National UniversityGoogle Scholar
  33. Zeil J, Al-Mutairi MM: The variation of resolution and of ommatidial dimensions in the compound eyes of the fiddler crab Uca lactea annulipes (Ocypodidae, Brachyura, Decapoda). J Exp Biol. 1996, 199: 1569-1577.PubMedGoogle Scholar
  34. Zeil J, Nalbach G, Nalbach H-O: Eyes, eye stalks and the visual world of semiterrestrial crabs. J Comp Physiol A. 1986, 159: 801-811. 10.1007/BF00603733.View ArticleGoogle Scholar
  35. Land M, Layne J: The visual control of behaviour in fiddler crabs II. Tracking control systems in courtship and defence. J Comp Physiol A. 1995, 177: 91-103. 10.1007/BF00243401.View ArticleGoogle Scholar
  36. Christy JH, Backwell PRY, Goshima S: The design and production of a sexual signal: hoods and hood building by male fiddler crabs Uca musica. Behaviour. 2001, 138: 1065-1083. 10.1163/156853901753287145.View ArticleGoogle Scholar
  37. Backwell PRY, Jennions MD, Christy JH, Schober U: Pillar building in the fiddler crab Uca beebei: evidence for a condition-dependent ornament. Behav Ecol Sociobiol. 1995, 36: 185-192. 10.1007/BF00177795.View ArticleGoogle Scholar
  38. Kim TW, Choe JC: The effect of food availability on the semilunar courtship rhythm in the fiddler crab Uca lactea (de Haan) (Brachyura: Ocypodidae). Behav Ecol Sociobiol. 2003, 54: 210-217. 10.1007/s00265-003-0614-3.View ArticleGoogle Scholar
  39. Kim TW, Sakamoto K, Henmi Y, Choe JC: To court or not to court: reproductive decisions by male fiddler crabs in response to fluctuating food availability. Behav Ecol Sociobiol. 2008, 62: 1139-1147. 10.1007/s00265-007-0542-8.View ArticleGoogle Scholar
  40. Kim TW, Kim KW, Srygley RB, Choe JC: Semilunar courtship rhythm of the fiddler crab Uca lactea in a habitat with great tidal variation. J Ethol. 2004, 22: 63-68. 10.1007/s10164-003-0100-4.View ArticleGoogle Scholar
  41. Goshima S, Murai M: Mating investment of male fiddler crabs, Uca lactea. Anim Behav. 1988, 36: 1249-1251. 10.1016/S0003-3472(88)80091-5.View ArticleGoogle Scholar
  42. de Rivera CE, Backwell PRY, Christy JH, Vehrencamp SL: Density affects female and male mate searching in the fiddler crab, Uca beebei. Behav Ecol Sociobiol. 2003, 53: 72-83.Google Scholar
  43. Bender R, Grouven U: Ordinal logistic regression in medical research. J Roy Coll Phys Lond. 1997, 31: 546-551.Google Scholar
  44. Lall R, Campbell MJ, Walters SJ, Morgan K: A review of ordinal regression models applied on health-related quality of life assessments. Stat Methods Med Res. 2002, 11: 49-67. 10.1191/0962280202sm271ra.PubMedView ArticleGoogle Scholar
  45. Animal Behviour Society: Guidelines for the treatment of animals in behavioural research and teaching. Anim Behav. 2006, 71: 245-253.View ArticleGoogle Scholar

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