The mucous covering of fecal sacs prevents birds from infection with enteric bacteria

354 The mucous covering of fecal sacs prevents birds from infection with enteric bacteria Juan Diego Ib á ñ ezÁ lamo , Magdalena Ruiz-Rodr í guez and Juan Jos é Soler J. D. Ib á ñ ezÁ lamo ([email protected]), Depto de Zoolog í a, Facultad de Ciencias, Univ. de Granada, Avda. Fuentenueva s/n, ES-18071 Granada, Spain. – M. Ruiz-Rodr í guez and J. J. Soler, Depto de Ecolog í a Funcional y Evolutiva, Estaci ó n Experimental de Zonas Á ridas (EEZA-CSIC), Ctra. Sacramento s/n, ES-04120 La Ca ñ ada de San Urbano, Almer í a, Spain. Nestlings of many bird species produce fecal sacs, excrements encapsulated within a mucous covering. Although it facilitates parents ’ removal of feces from nests, which would improve hygienic conditions for developing nestlings, no functional (i.e. adaptive) explanation of fecal sac production has been previously investigated. We propose that the mucous covering would isolate enteric pathogenic bacteria, thereby preventing contamination of nestlings and parents. Th is antimicrobial hypothesis therefore predicts that density of bacteria would be drastically reduced from the inside to the outside of nestlings ’ droppings, and that the fecal sac covering would inhibit other bacterial grow. We tested these predictions by means of culturing bacteria obtained from diff erent parts of the sac and inhibition tests. In accordance with the hypothesis, bacterial loads of the outside of fecal sacs were signifi cantly lower than those estimated from the inside of the covering. In addition, we did not fi nd evidence of antimicrobial activity of the covering, which suggests that the hypothesized bacterial isolation function is accomplished by a physical rather than a chemical protection. Bacterial density of the liquid that permeates out after 23 min does not diff er with that estimated for the inside of the sac, suggesting short-term eff ects of fecal sacs as bacterial barrier. Th ese fi ndings highlight the major role of bacterial infections as a selective pressure for explaining the evolution of traits that, as the covering of fecal sacs, facilitate nest sanitation in this group of animals. Many bird species are characterized by a period in which off spring (eggs and nestlings) stay in a fi xed location, the nest, wherein they are completely dependent on their parents (Del Hoyo et al. 1992). Th e study of parent – off spring interactions during the nesting phase has been critical in the advances of diff erent research areas, including the evolution of traits that reduce the probability of infection, (Loye and Zuk 1991, Royle et al. 2012). Excrements are a source of potential pathogenic microorganisms and consequently may play an important role in the evolution of such traits. Up to date, however, few studies have investigated this topic. For example, although nestlings ’ droppings can act as an important source of energy and nutrients for adult birds (Morton 1979, Gl ü ck 1988, Dell’Omo et al. 1998) they may facilitate the transmission of harmful microorganisms from young to their parents. Furthermore, the accumulation of excrements in (or around) the nest may facilitate nest detection and, therefore, increase probability of predation (Herrick 1900, Weatherhead 1984, Petit et al. 1989, but see Ib á ñ ezÁ lamo et al. 2013b). Independently of adaptive functioning, nest sanitation by means of feces removal is a widespread parental behavior in birds (approximately 99% of North American passerine species according to Guigueno and Sealy 2012). One of the most intriguing adaptations regarding nestlings ’ droppings are fecal sacs (excrements encapsulated in a mucous covering, Herrick 1900, Weatherhead 1984), which are restricted exclusively to the nestling stage of many bird species (Blair and Tucker 1941, Guigueno and Sealy 2012). Some researchers have suggested that the sac surrounding the feces will help parents to carry them away from the nest (McGowan 1995). In addition, Herrick (1900) proposed that the mucous covering could avoid ‘soiling the bill’, which obviously may result in contamination by microorganisms. Microorganisms are important determinants of avian development and survival (Benskin et al. 2009, Archie and Th eis 2011, Ezenwa et al. 2012) and the perspective of animal evolution in a bacterial world has recently been claimed as imperative for the life science (McFall-Ngai et al. 2013). Bacteria can have benefi cial (Moreno et al. 2003) or detrimental eff ects in birds. Pathogenic bacteria cause several diseases in birds (Batt et al. 1996, Lombardo et al. 1996, Mills et al. 1999), some of them for instance inducing embryonic mortality (Pinowski et al. 1994), growth reduction (Potti et al. 2002) or the degradation of feathers (Gunderson 2008). Enteric bacteria of adult and nestling wild birds include pathogens (Brittingham et al. 1988, Journal of Avian Biology 45: 354–358, 2014 doi: 10.1111/jav.00353 © 2014 Th e Authors. Journal of Avian Biology © 2014 Nordic Society Oikos Subject Editor: Simon Griffi th. Accepted 7 January 2014 355 Lombardo et al. 1996, Westneat and Rambo 2000) and, consequently, the manipulation of chicks ’ excrements by adults or the contact with nestling skin would imply an increased risk of infection. We propose a new hypothesis within this theoretic framework, the antimicrobial hypothesis, which states that the mucous covering of fecal sacs will prevent infection of parents and/or nestlings by harmful microorganisms contained within nestlings ’ excrements. Th is protection could be provided by two diff erent, not mutually exclusive, mechanisms. On the one hand, the mucous covering could act as a barrier isolating enteric bacteria inside, avoiding thereby the contamination of birds during the contact, similarly to the physical defensive function of the eggshell that protects the embryo from bacterial infection (Wellman-Labadie et al. 2008a, b). On the other hand, fecal sacs may contain antimicrobial compounds, as found for example in uropygial secretions (Mart í n-Vivaldi et al. 2010), conferring chemical protections to adults and nestlings. Th e main objective of this study was to investigate whether the mucous covering of fecal sacs has an antimicrobial function. Evidence of antimicrobial properties would suggest that fecal sacs impede trans-sac bacterial contamination of adults and nestlings. To investigate several predictions of this new hypothesis, we carried out diff erent approximations using the common blackbird Turdus merula as the model species. According to the antimicrobial hypothesis, bacterial loads of the inside of fecal sacs should be higher than those of the outside (prediction 1) due to the presence of the mucous covering between these two parts of nestlings ’ droppings (i.e. physical barrier functioning). Evidence supporting this prediction would imply that adults manipulating fecal sacs would face a reduced risk of infection in comparison with those handling excrements not covered by mucous sacs. Moreover, if the antimicrobial function of this mucous layer is accomplished chemically by the presence of antimicrobial compounds (i.e. chemical antimicrobial functioning), we could predict that: a) intact fecal sacs would inhibit bacterial growing at a higher rate than excrements without covering (prediction 2). Furthermore, given that some bacteria should have been inhibited by this layer after contact, b) bacterial loads inside the sac should be higher than in the liquid that passes through the covering some time after its production (prediction 3). Th ese two possible functionings of fecal sacs are not mutually exclusive. Methods Study area and fi eld work Th is study was conducted in a population of common blackbirds located in the Valley of Lecr í n, south of Spain (36 ° 56 ′ N, 3 ° 33 ′ W; 580 m a.s.l.) from April to May 2012. Th e study area is dominated by orange groves in which blackbirds usually nest (see Ib á ñ ezÁ lamo and Soler 2010 for a more detailed description of the population). We used the common blackbird as the model species given that their nestlings produce fecal sacs and adults remove them from their nests (Ib á ñ ezÁ lamo et al. 2013a, b). We actively searched for blackbird nests since the beginning of the fi eld season (beginning of March). All nests were visited regularly and fecal sacs obtained directly from middle aged chicks (mean ⫾ SE: 6.6 ⫾ 0.4 d old; n ⫽ 46) to standardize for possible diff erences in microbiota due to age (Mills et al. 1999). Fecal sacs were taken by using new latex gloves washed with 96% ethanol for each nest to maintain sterile conditions and avoid inter-nest contamination. A sterilized plastic container (60 ml) was placed just below the cloaca of each nestling, and fecal sacs fell inside directly (blackbird nestlings easily defecate when handled, unpubl.). Containers with fecal sacs were conserved at ambient temperature until arrival at the laboratory (for up to 8 h, usually for no more than 4 – 5 h) when they were stored in a refrigerator at 5 ° C until their processing (within the next 24 h). Bacterial growing from fecal sacs In order to test the fi rst and third prediction, we used a fi rst group of containers (n ⫽ 53). We took three samples from each excrement by using sterile swabs: 1) from the outside, 2) the inside of the sac, and 3) from the liquid that percolated from the sac to the container after a mean of 23.75 ⫾ 2.82 min (n ⫽ 53). To homogenize the quantity of sample taken from each part, we made a single touch (i.e. time in contact 1 s) with the swab over the external or internal surface of the excrement, or over the liquid. We used a diff erent sterile stick to open each sac. Containers were opened under sterile conditions in the lab. Th e swab was then introduced in an eppendorf tube containing 1 ml of sterile phosphate buff er (pH 7.2, 0.2 M) and vigorously agitated in the vortex. Th en, 100 μ l of the mix was spread onto a general culture medium for mesophilic bacteria and in two specifi c media for Enterococcus and Enterobacteriaceae ; both groups are typical from intestinal microbiota of birds and include several opportunistic pathogens (Brittingham et al. 1988, Lombardo et al. 1996, Westneat and Rambo 2000). A serial dilution was performed at a factor of 100 (990 μ l of sterilized distillated water and 10 μ l of sample) to count bacterial colonies in the petri dishes. Plates were incubated aerobically at 37 ° C during 72 h, and then colonies that grew in each plate were counted. Bacterial load was estimated as the number of CFU (colony forming units) per ml of buff er. Antimicrobial properties of fecal sacs A second group of samples (n ⫽ 69) from diff erent nestlings were used for the inhibition tests to investigate our second prediction. Half of the sacs (n ⫽ 35) were directly placed on plates with brain heart infusion (BHI, see below) medium mixed with the indicator bacteria (antagonistic plates). Th e rest of the collected sacs (n ⫽ 34) were opened by breaking the covering with a sterile stick and the whole contents placed in plates as we did for the complete fecal sac. Antagonistic plates were prepared with two indicator bacteria from separated taxonomic groups, Enterococcus faecalis MRR-10 and Bacillus licheniformis D-13, both from our laboratory collection. Th e former is a typical commensal 356 of the intestine, frequently detected in bird ’ s cloaca (Moreno et al. 2003), while the latter is a keratinolytic bacteria typically found in birds plumage (Burtt and Ichida 1999). Indicator bacteria were cultured in (BHI) overnight, and 100 μ l of each culture were inoculated in 15 ml of BHI-B (1.8% BHI, 0.8% agar in 0.1 M pH 7 phosphate buff er). Th is solution was poured onto sterile petri dishes, and after solidifi cation, sacs were laid on. Plates were prepared just before the experiments. After 12 h of incubation at 28 ° C, plates were checked for inhibition halos (a transparent zone that would indicate inhibition of the indicator bacteria) around sacs. In the case of the existence of halos, they were measured from the limit of the sac until where the indicator bacteria started to grow. Statistical analyses We carried out repeated-measures ANOVAs to detect differences in bacterial loads among the three diff erent parts of each sac and test prediction 1 (inside vs outside) and 3 (inside vs liquid). CFU ml ⫺1 in each medium was the dependent variable, and as explanatory ones we used the part of the sac sampled, consistently included as the within-group factor, and age as a covariable, while considering their interaction too. Recent literature has detected a change in bacterial communities of cloacal samples in relation with nestling ages even for short-term periods (Gonz á lez-Braojos et al. 2012). Th us, we decided to include age as an additional independent factor to statistically control for diff erences in bacterial community associated with age. Th e assumptions underlying the use of these analyses were systematically checked and the log 10 transformation was applied for bacterial loads. Analyses were performed by using the Statistica 7.0 software. We used a generalized linear mixed model to determine if there were diff erences in antibacterial activity between opened and closed fecal sacs (prediction 2). Th e zone of inhibition was fi tted to a Poisson distribution. We selected the best model according to the Akaike information criteria among those built including state of the fecal sac (open or close), nestling and nest identity. Th e best model included fecal sac state as a fi xed factor and nest identity as a random factor. We used R 2.15 (lme4 package) for these analyses. Results Bacterial loads from the outside of recently collected fecal sacs were three orders of magnitude lower than that estimated for the inside or for the percolated liquid after more than 20 min, which support prediction 1. It occurs independently of the culture media (TSA: F 2,52 ⫽ 4.04, p ⫽ 0.02; KF: F 2,64 ⫽ 12.50, p ⬍ 0.001; HK: F 2,64 ⫽ 5.62, p ⫽ 0.006; Tukey HSD post hoc tests, p ⫽ 0.0001 in all cases Fig. 1). Bacterial loads of samples from the inside of the sac and the permeated liquid did not diff er for any of the three media (Tukey HSD post hoc tests, TSA: p ⫽ 0.59; KF: p ⫽ 0.10; HK: p ⫽ 0.79), which did not fi t with prediction 3. Nestling age or its interaction with the origin of samples was not signifi cant in any case (results not shown) and was therefore removed from the fi nal model. In relation to antimicrobial tests, the size of the inhibition halo produced by opened and closed fecal sacs when tested against B. licheniformis (Z 3 ⫽ 0.94, p ⫽ 0.35; n ⫽ 49; mean ⫾ SE for opened sacs ⫽ 0.58 ⫾ 0.21 mm; closed sacs ⫽ 0.84 ⫾ 0.26 mm) or E. faecalis (Z 3 ⫽ 0.30, p ⫽ 0.76; n ⫽ 20; opened sacs ⫽ 0.50 ⫾ 0.22 mm; closed sacs ⫽ 0.60 ⫾ 0.22) did not diff er signifi cantly, providing no support for prediction 2. Discussion Our fi ndings suggest that the mucous covering of fecal sacs protects birds from bacterial contamination, thus supporting this new antimicrobial hypothesis. Bacteria on the surface of fecal sacs were less abundant than in their Figure 1. Mean bacterial loads ( ⫾ SE) for each part of the fecal sac for Tryptone Soja Agar (TSA; dark-grey bars), Hektoen Agar (HK; light-grey bars) and Kenner-Faecal Agar (KF; black bars) culture media. 357 relation to this, other components of sanitation behavior have already been proposed to play a role in feather degrading bacteria-birds interactions (Lucas et al. 2005, Shawkey et al. 2007). However, this isolation function of the mucous covering will not only be restricted to detrimental bacteria but also to benefi cial microorganisms (Moreno et al. 2003). Surely, the benefi ts associated with the protection against harmful bacteria will surpass those provided by such benefi cial microbiota. Alternatively, the bacterial isolation eff ect detected here could be the byproduct of other functions of the mucous covering like for example facilitating manipulation by parents during nest sanitation tasks (McGowan 1995). However, independently of the origin of this trait, the protective eff ects against bacterial infection should have strengthened the evolution of fecal sacs enhancing such function. More experimental works are in any case necessary for further conclusions related to the evolution of fecal sacs in birds and we hope this work contribute to encourage further research. To sum up, our fi ndings indicate that the mucous covering would confer protection to adults and/or nestlings against bacteria contained in excrements, although only for a short time after chicks ’ defecation. Furthermore, this isolation function seems to be due to a physical barrier that encapsulates bacteria within a gelatinous container rather than the presence of antibiotics. Our study provides the fi rst adaptive explanation for the evolution of fecal sacs and off ers a new perspective about parent-off spring relationships in birds. It also highlights that bacteriabirds interactions could have played a major role shaping nest sanitation, a poorly understood but important parental behavior in birds. Acknowledgements – We thank F. Ruiz-Raya for his help in the fi eld, G. Roncalli off ered assistance for some statistical analyses and M. Mart í n-Vivaldi helped us with some interesting discussions about our study. Financial support to MRR and JJS was provided by the Spanish Ministerio de Educaci ó n y Ciencia/FEDER (research project CGL 2010-19233-C03-01). References Archie, E. A. and Th eis, K. R. 2011. Animal behaviour meets microbial ecology. – Anim. Behav. 82: 425 – 436. Batt, R. M., Rutgers, H. C. and Sancak, A. A. 1996. Enteric bacteria: friend or foe? – J. Small Anim. Pract. 37: 261 – 267. Benskin, C. M. H., Wilson, K., Jones, K. and Hartley, I. R. 2009. Bacterial pathogens in wild birds: a review of the frequency and eff ects of infection. – Biol. Rev. 84: 349 – 373. Blair, R. H. and Tucker, B. W. 1941. 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Th is short-term antimicrobial eff ect could partly explain the commonly observed parental behavior of stimulating nestlings ’ defecation during parental visits (Dell’Omo et al. 1998, Ib á ñ ezÁ lamo et al. 2013a), and why chicks in many species only defecate in the presence of their parents (Herrick 1900, Brooke and Birkhead 1991). Otherwise, if nestlings ’ feces production and parental disposal of excrements are not synchronized, adult birds (and probably off spring) may become contaminated with potentially harmful bacteria. We did not fi nd evidence of antimicrobial properties of fecal sacs due to chemical compounds since we did not detect any signifi cant diff erences in the inhibitory activity of feces with and without the covering (prediction 2). We found a little halo of inhibition in both (nonmanipulated and opened) fecal sacs. Several bacterial groups, including gut bacteria, produce diff erent kind of antimicrobial substances, mainly bacteriocins, to outcompete other bacteria (Riley and Wertz 2002). Th erefore, is not surprising to fi nd a low rate of inhibition, although it was similar between the two types of excrements. Additionally, the amount of bacteria was the same after the liquid passed over the fecal covering (prediction 3; Fig. 1), which means that there was no signifi cant inhibition in the mucous layer. Th us, the mucous covering seems to act as a physical barrier isolating (at least temporally) intestinal bacteria within the sac. Th is function will be similar to that of the eggshell impeding pathogenic bacteria to infect the embryo (Board et al. 1994). Feces manipulation entails the possibility of contact with potentially pathogenic microorganisms, and feces covering could prevent their transmission from nestlings to adults. Th us, the detected isolation eff ect of fecal covering sac detected here, even if temporal, seems to be adaptive, as many enteric bacteria of birds are potentially dangerous and may even provoke their death (Brittingham et al. 1988, Lombardo et al. 1996, Westneat and Rambo 2000, Potti et al. 2002). Although we have not detected evidence of antimicrobial activity against the two tested bacteria, it would be very interesting to investigate whether this isolation function of the mucous covering is also eff ective against other potentially harmful microorganisms like other bacterial groups, viruses, fungi or protists. Independently of the more or less broad eff ectiveness of fecal sacs covering preventing infections, our results suggest that it would protect nestlings and/or adults. It is interesting to note that parents dispose of fecal sacs by two diff erent mechanisms: transporting them away with their beaks or directly ingesting them (Blair and Tucker 1941, Guigueno and Sealy 2012). 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