Recruitment and Establishment of the Gut Microbiome in Arctic Shorebirds Kirsten Grond1,∗, Richard B Lanctot2, Ari Jumpponen1 and Brett K Sandercock1
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FEMS Microbiology Ecology, 93, 2017, fix142 doi: 10.1093/femsec/fix142 Advance Access Publication Date: 24 October 2017 Research Article RESEARCH ARTICLE Recruitment and establishment of the gut microbiome in arctic shorebirds Kirsten Grond1,∗, Richard B Lanctot2, Ari Jumpponen1 and Brett K Sandercock1 1Kansas State University, Division of Biology, Manhattan, KS 66506, USA and 2US Fish and Wildlife Service, Migratory Bird Management, Anchorage, AK 99503, USA ∗Corresponding author: Department of Molecular and Cell Biology 91 North Eagleville Road University of Connecticut Storrs, CT 06269-3125, USA. Tel: +7854776545; E-mail: [email protected] One sentence summary: Shorebird embryos hatch with negligible gut microbiota, and chicks acquire a stable bacterial community within three days of hatching Editor: Cindy Nakatsu ABSTRACT Gut microbiota play a key role in host health. Mammals acquire gut microbiota during birth, but timing of gut microbial recruitment in birds is unknown. We evaluated whether precocial chicks from three species of arctic-breeding shorebirds acquire gut microbiota before or after hatching, and then documented the rate and compositional dynamics of accumulation of gut microbiota. Contrary to earlier reports of microbial recruitment before hatching in chickens, quantitative PCR and Illumina sequence data indicated negligible microbiota in the guts of shorebird embryos before hatching. Analyses of chick feces indicated an exponential increase in bacterial abundance of guts 0–2 days post-hatch, followed by stabilization. Gut communities were characterized by stochastic recruitment and convergence towards a community dominated by Clostridia and Gammaproteobacteria. We conclude that guts of shorebird chicks are likely void of microbiota prior to hatch, but that stable gut microbiome establishes as early as 3 days of age, probably from environmental inocula. Keywords: 16S rRNA gene; bacteria; Calidris; gut microbiota; qPCR; precocial young INTRODUCTION initial composition of gut microbiota in mammalian offspring (Stevens and Hume 1998). Gut microbiota contribute to maintaining organismal health It remains unclear whether birds can acquire gut microbiota through nutrient uptake (Leser and Mølbak 2009), detoxification while inside the egg, and how microbial communities accumu- of digestive byproducts (Kohl 2012), energy and fat metabolism late after initial recruitment. Bacterial colonization can theo- (Velagapudi et al. 2010), and interactions with the host immune retically occur before or after hatching, and we propose two system (Rescigno 2014). Environmental exposure early in life alternative hypotheses to describe the process of microbial col- can shape the gut microbiota and aid in defending against onization: the ‘head start’ and the ‘sterile egg’ hypothesis. The pathogens while the immune system is immature (Bar-Shira, ‘head start’ hypothesis posits that microbes enter the gastroin- Sklan and Friedman 2003). In mammals, maternal vaginal and testinal tract of avian embryos through transovarian transmis- fecal microbe transmission are crucial in the recruitment and sion during oogenesis as a maternal effect, or possibly from establishment of microbiota in the digestive tract of a neonate the environment by penetration through eggshell pores and (Palmer et al. 2007). Thus, maternal effects strongly control the Received: 17 July 2017; Accepted: 20 October 2017 C FEMS 2017. All rights reserved. For permissions, please e-mail: [email protected] 1 2 FEMS Microbiology Ecology, 2017, Vol. 93, No. 12 embryonic membranes after egg laying (Gantois et al. 2009;Cox ples collected from shorebird chicks from hatch to up to 10 days et al. 2012; Martelli and Davies 2012). The ‘sterile egg hypoth- of age. esis’ predicts that microbiota recruitment in the avian gut oc- curs after hatch because the chorion membrane maintains a sterile environment within the egg (van der Wielen et al. 2002; MATERIALS AND METHODS Kohl 2012). The development of the avian embryo within the Sample collection and preparation membrane-enclosed yolk supports the latter hypothesis, but en- teric bacteria have been cultured from the guts of embryos of do- We collected 27 eggs from seven clutches of dunlin (Calidris mestic chicken (Gallus gallus domesticus; Kizerwetter-Swida´ and alpina) and 18 eggs from five clutches of semipalmated sand- Binek 2008), and vertical transfer of pathogenic Salmonella enter- pipers (Calidris pusilla), at Utqiagvik˙ (formerly Barrow), Alaska ◦ ◦ ica has been documented between mother and embryo in do- (71 17 26 N, 156 47 19 W) in June–July 2013. We estimated incu- mestic chickens (Guard-Petter 2001; De Buck et al. 2004). The two bation stage via changes in egg buoyancy that occur with em- hypotheses differ in the maternal contribution to gut microbiota bryonic development (Liebezeit, Smith and Lanctot 2007)and of offspring and in the source of inoculum. The ‘head start’ hy- collected eggs 1–3 days before the predicted hatch date to en- pothesis predicts that maternal or environmental inoculation sure near complete embryo development. After collection, em- occurs before hatching, possibly resulting in metabolic and im- bryos were removed from eggs, euthanized and kept frozen at ◦ munological advantages at hatching. In contrast, if gut micro- –20 C until dissection. Before dissection, embryos were washed biota are absent in embryos, as posited by the ‘sterile egg’ hy- in a weak solution of 0.6% sodium hypochlorite (10% bleach) to pothesis, chicks must recruit microbiota from the environment minimize contamination by external microorganisms. We asep- after hatch. tically removed the lower intestinal tract between the gizzard ◦ Developmental strategies after hatching vary within birds and cloaca and stored samples at –80 C. We also collected the and range from chicks hatching with complete dependence invaginated yolk sacs from the embryos of five dunlin and four on their parent(s), like most altricial species, to superprecocial semipalmated sandpipers. species that are independent of parental care after hatching. Al- To investigate gut microbiota of mobile broods after hatch- tricial and precocial birds likely differ in the degree of mater- ing, we selected dunlin and red phalaropes (Phalaropus fuli- nal influence on microbial inoculation of their chicks. Anal- carius) as study species because dunlin use mesic terres- tricial bird species, the Barn Swallow (Hirundo rustica)showed trial habitat, whereas red phalaropes use freshwater habitat slight maternal, but not paternal, effects on the fecal micro- (Cunningham, Kesler and Lanctot 2016). Different habitats and biota of their offspring (Kreisinger et al. 2017). Altricial birds have associated microbiomes could affect the recruitment of gut mi- undeveloped young that require parental feeding and brooding crobiota in chicks. We fit the attending adults with 1.55 g very until fledging, whereas precocial chicks hatch fully developed high frequency (VHF) radios, and, for dunlin, one chick per brood with invaginated yolk sacs, leave the nest immediately, forage with 0.26 g VHF radios (Holohil Systems Ltd., Carp, Ontario, independently, but rely on parental brooding and defense un- Canada; Fig. S3.1, Supporting Information). Red phalarope chicks til able to thermoregulate (Sibly et al. 2012). Thus, altricial birds were too small at hatch to apply transmitters so we located have higher potential for parental influence on gut microbiota young by tracking the attending male. We attempted to locate through salivary transfer and a longer period of nest occupa- mobile broods every 3 days, using radio telemetry and by search- tion, whereas precocial parents have little direct influence on ing areas where broods were last seen. Once broods were lo- their offspring’s microbiota aside from exposure to bacteria on cated, chicks were captured and feces were collected by placing feathers during brooding, and leading young to brood-rearing the chicks in individual compartments in an insulated, ther- areas. mally heated bag, lined with sterile wax paper. Chicks were held In addition to uncertainty about the timing of initial recruit- for <5 min to minimize stress and then released together. At ment of microbiota, the early successional dynamics of the avian several capture sites, we also collected environmental samples gut microbiota are unclear. For example, it remains unknown consisting of a mix of water and soil in a 1.5-ml Eppendorf tube how rapidly chick guts are colonized, in what order bacterial using a flame-sterilized spatula. All samples were stored in 100% ◦ taxa become established and when convergence towards an ethanol at –20 C until further analyses. adult gut microbiome occurs. Precocial young of most Arctic- To remove ethanol from fecal samples, we centrifuged sam- breeding shorebirds are nidifugous, self-feeding and dependent ples for 10 min at 10 000 rpm and discarded the supernatant. We on parents for thermoregulation, and could benefit from recruit- repeated this step twice with 1 ml of RNase/DNA-free molec- ing gut microbiota before hatching, because symbionts can pro- ular grade water to minimize ethanol contamination (Grond mote nutritional uptake and growth immediately after hatch et al. 2014;Ryuet al. 2014). We shredded embryonic gastroin- (Angelakis and Raoult 2010). Efficient use of nutrients may be testinal tissues, and extracted DNA from embryo and chick fecal particularly important in the Arctic because a short and syn- samples using the MoBio