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Growth rate and survival of terrestrial isopods is related to possibility to acquire symbionts
European Journal of Soil Biology 69 (2015) 52e56
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European Journal of Soil Biology journal homepage: http://www.elsevier.com/locate/ejsobi
Original article
Growth rate and survival of terrestrial isopods is related to possibility to acquire symbionts zia Horva thova *, Jan Kozłowski, Ulf Bauchinger Tere w, Poland Institute of Environmental Sciences, Jagiellonian University, ul. Gronostajowa 7, 30-387 Krako
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 November 2014 Received in revised form 13 May 2015 Accepted 13 May 2015 Available online 17 May 2015 Handling editor: S. Schrader
The acquisition and maintenance of symbiont-host associations is considered an important prerequisite for the successful colonisation of land by animals because symbionts allowed the hosts to dwell on lowquality food sources. Digestive tract symbionts are suggested to either enhance digestive efficiency of cellulose or supply the host with nutrients otherwise limited in the food. Terrestrial isopods are the most successful land colonisers among the Crustacea, and although symbiotic bacteria have been identified in digestive tract, the mechanism of transmission and the nutritional role of the symbionts are at present poorly understood. We used a novel in vitro approach to isolate and culture juveniles of Porcellio scaber at a time when only the vertical mode of symbiont acquisition could have occurred, which allowed us to follow juvenile growth and to simulate alternative modes of symbiont acquisition through feeding manipulations. Thus, we experimentally obtained groups of juveniles that could have acquired symbionts only through vertical transfer (mother-offspring), or additionally, through horizontal (through faeces or contact with conspecifics) and environmental (through leaves) transfer. We quantified survival and growth rates over two months for the different experimental acquisition modes, and significantly different growth rates were observed (p < 0.001). Growth of juveniles suggests that first, symbiont inoculation is mediated through horizontal and environmental transfer and second, the symbionts may in fact serve as a source for fatty acids and vitamins. The growth rates further question that vertical transfer occurs in woodlice. Although survival did not differ significantly between different acquisition modes (p ¼ 0.051), juveniles supplemented with potential sources of symbionts showed a tendency towards increased survival. The successful invasion of land may thus have been facilitated through the uptake of symbionts from the surroundings. © 2015 Elsevier Masson SAS. All rights reserved.
Keywords: Symbiont transmission Nutrition Cellulose Terrestrial isopods Woodlice
1. Introduction According to the original definition by Anton de Bary [1], symbiosis can be described as the association (temporal or spatial) between individuals who do not belong to the same species, independent of the effects on organisms involved, be they negative (parasitism), neutral (commensalism) or positive (mutualism). In animals, host-symbiont associations include transient gut passengers that pass through the digestive tract with ingested food, permanent gut residents, gardening of fungi, and surface-associated and/or intracellular symbionts [2]. The acquisition of novel
* Corresponding author. ), jan.kozlowski@ E-mail addresses: [email protected] (T. Horv athova uj.edu.pl (J. Kozłowski), [email protected] (U. Bauchinger). http://dx.doi.org/10.1016/j.ejsobi.2015.05.003 1164-5563/© 2015 Elsevier Masson SAS. All rights reserved.
biological properties via symbiosis played an important role in adaptation, evolution and diversification of animals, e.g., allowing the host to explore new ecological niches [3]. Symbiotic associations are widespread in diverse taxa in both aquatic and terrestrial environments [2,4,5]. In the largest animal group on Earth, insects, up to half of the species are estimated to harbour obligatory symbionts [2]. Protection against pathogens, improved growth and survival, and provisioning of limiting nutrients are examples of the beneficial effects of symbionts [5e8]. Nutritional symbiosis appears to be common in terrestrial arthropods that feed on diets scarce in vitamins, sterols, and essential amino acids or on diets rich in cellulose and lignin [7,9e13]. However, the modes of symbiont acquisition and their potential nutritional contributions are at present not understood. In general, the establishment of the symbiont-host relationship can be facilitated through three transmission mechanisms:
thova et al. / European Journal of Soil Biology 69 (2015) 52e56 T. Horva
vertically transmitted symbionts are passed from mother to progeny by infecting milk glands, eggs or embryos [14], horizontally transmitted symbionts may be acquired through feeding on infected corpses, faeces, exuviae or through physical contact between conspecifics [14,15], and environmentally transmitted symbionts are acquired through contact with or uptake of any matter [14,16,17]. The present understanding of the mechanisms that mediate symbiont transmission is based on empirical evidence available from only a few model systems. Vertical transmission appears as the predominant mechanisms for symbiont acquisition in insects [2,18], and environmental transmission is described for species living in aqueous environment [4,17]. The horizontal transmission of microbial species through faecal-oral contact in ruminant herbivores has received the most attention [19]. Thus, the transmission mode can be considered to be habitat and taxa specific, but overall, the understanding has to be qualified as poor. The suborder Oniscidea (Crustacea) is composed of more than 3500 terrestrial species, and their association with symbiotic bacteria has been confirmed only in seven species [20]. The microbial contribution to cellulose and lignin digestion has hereby received the most attention [3,6,11,21]. Symbionts are assumed to improve the ability of the host to digest a terrestrial diet by providing necessary enzymes or essential nutrients such as fatty acids and vitamins [22]. The mode of microbe transmission in terrestrial isopods is generally assumed to be environmental [23], however due to the methodological approach this contribution cannot fully exclude the horizontal (through contact with mother) mode of transfer. We experimentally excluded and/or facilitated symbiont transmission by dissecting embryo stages from the marsupium and subsequent in vitro culturing of the juveniles with different food supplements with the goal to determine the mode of symbiont acquisition and the beneficial effects of symbionts in the terrestrial isopod species Porcellio scaber. Different sources for symbiont inoculation were provided to juveniles through nutritional supplements to an artificial standard diet to test for the vertical (mother-offspring), horizontal (through faeces or contact with conspecifics) or environmental (through leaves) mode of transfer. Based on the assumption that symbionts positively affect growth and survival, we predicted: i) no difference in growth and survival if vertical transmission occurs; ii) difference in growth and survival if vertical transfer does not occur, and iii) dietary supplements of fatty acids and vitamins offset the lack of symbionts by improving isopod growth. 2. Materials and methods 2.1. Collection of animals Specimens of woodlice (P. scaber) were collected in spring 2013 w, Poland. The isopods were maintained in plastic boxes in Krako (52 mm 48 mm Ø, 100 ml) under a constant temperature of 20 C and a photoperiod of 16L:8 D. The progeny of 30 gravid females were used for the experiment, summing up to a total of 830 dissected mancas.
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fluid is absorbed, individuals start to move and they switch from aquatic respiration to air breathing by using pleopodal lungs. To mimic such conditions in vitro, the approach used by Surbida and Wright [25], who transferred embryos of the isopod species Armadillidium vulgare to fresh 12 5 ml well-plates and cultivated them in culture saline, was modified. Because this method did not allow individuals to freely move out from the water and thus follow the ontogenic development after hatching, a funnel made out of filter paper was attached to each well (volume of 1 ml, water level reached to approximately half of the funnel height). The novel funnel-approach allowed mancas to crawl out of the liquid bottom of the funnel and onto the funnel part above the liquid surface to survive and to breath with lungs (marsupial manca stage, [26]). We used a normal filter paper and did not use any additional measures to sterilise it, assuming that all individuals experience the same contamination conditions during this period. 2.3. Diet manipulation The day when individuals crawled freely inside the wells was defined as the time of hatching (one to two weeks after dissection). A total of 267 juveniles moved from the liquid phase into the air phase and subsequently ‘hatched‘ in vitro (32% survival of all cultured individuals). After hatching, the body mass of juveniles was determined to the nearest 0.001 mg (Mettler Toledo XP26, Greifensee, Switzerland), and one or two juveniles were subsequently kept in individual boxes (52 mm 48 mm Ø, 100 ml) Las, glass sand, class III) and an containing wet sand (Grudzen autoclaved piece of clay pot, which served as a shelter and also a feeding place. The juveniles of each clutch were split equally and randomly assigned to five experimental diet groups. The individuals in all diet groups were offered ad libitum ‘minimum diet’ [27] modified after [22]; the details on diet composition and diet preparation are presented in Appendix A1. The food provided to group A contained only this minimum diet (10e15 mg), while group B was supplemented with fatty acids and vitamins (0.4e0.6 mg), group C was supplemented with a tiny piece of alder leaf (0.2e0.4 mg), group D1 was supplemented with a single faecal pellet of an adult conspecific (0.1e0.3 mg), and group D2 was supplemented with the gut (including hepatopancreas) of a freshly killed adult (0.4e0.6 mg). The tiny amounts of supplements were used to facilitate possible symbiont inoculation without serving as significant source of extra nutrients. The supplements and the diets were provided only every 7th day in the form of a fresh artificial diet pellet placed on the clay pot; the supplements for groups C and D were placed at least 1 cm from the food pellet to avoid contamination of the artificial diet. The artificial diet was always produced fresh (agar was sprinkled to the boiling water and additional ingredients were added while keeping the fluid warm; for details see Appendix A1). Because all experimental groups faced the same risk of contamination, we expected that any growth or survival differences between groups must stem from the supplement added to the diet. 2.4. Statistical analyses
2.2. Mimicking conditions inside marsupium The embryos were removed from the marsupium (¼ motherly brood pouch) at the later stage of gravidity (stage 3, [24]) when they could not have encountered symbionts through horizontal or environmental transfer, and thus only vertical transfer could have occurred. The marsupial development is characterised by Haeckel's 'ontogeny recapitulates phylogeny' because individuals inside the brood pouch experience the change from the water to land conditions one week before hatching. During this period, the marsupial
Growth rates were determined by weighing each individual at hatching (initial body mass) and after four and eight weeks. All data were tested for normality of distribution and homogeneity of variance prior to analyses. To examine the effect of diet on growth rates, repeated-measures ANOVA and Bonferroni post-hoc tests were used with initial body mass as a covariate, diet, time points (day of hatching, four weeks and eight weeks), the interaction term between the two factors, and number of juveniles per box as explanatory variables. Because juveniles were assigned to
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thova et al. / European Journal of Soil Biology 69 (2015) 52e56 T. Horva
experimental groups randomly, mean initial body mass was compared between diet groups by ANOVA (group A, n ¼ 27; group B, n ¼ 23; group C, n ¼ 23; group D1, n ¼ 21; and group D2, n ¼ 19). The GLIMMIX procedure was used to analyse differences in survival rates. The model included survival as a binary response variable (survived or died within eight weeks) with diet, number of juveniles per box and initial body mass as explanatory variables. All statistical analyses were performed with the SAS 9.4 statistical software package (SAS Institute Inc., Cary, NC, USA). 3. Results The mean initial juvenile mass did not differ among diet groups (F4,108 ¼ 1.4, p ¼ 0.24; Table 1). The initial body mass had a significant effect on the growth rate (F1,95 ¼ 12.98, p < 0.001). The juvenile mass after two months was significantly different between diet groups (F4,95 ¼ 11.32, p < 0.0001; Fig. 1a). The juveniles fed a diet supplemented with fatty acids and vitamins (group B), a tiny piece of leaf (group C), a single faecal pellet (group D1) or the gut (group D2) grew significantly faster than juveniles of group A fed only the minimum diet (Fig. 1a). The mass in groups B, C and D did not differ significantly from each other (all p > 0.184). A significant interaction between diet groups and time points was also found (F8,95 ¼ 6.39, p < 0.0001). The difference between initial mass and mass after four weeks was significant only for group C, but not for groups A, B, D1 and D2 (group C, p ¼ 0.03; groups A, B, D1, and D2, p > 0.05; Table 1). Groups B, C and D showed significant differences between mass after four and mass after eight weeks, but not group A (group A, p ¼ 0.115; groups B, C, D p < 0.0001; Table 1). The number of individuals per box (initially either one or two) did not have a significant effect on the increase in mass (F1,95 ¼ 1.88, p ¼ 0.173). Survival did not differ significantly between the diet groups (F4,136 ¼ 2.42, p ¼ 0.051), but revealed a tendency for higher survival for the groups supplemented with potential sources for symbionts, despite the low sample sizes (group A, n ¼ 7; group B, n ¼ 6; group C, n ¼ 11; group D1, n ¼ 8; and group D2, n ¼ 10; Fig. 1b). When the data were pooled for groups without a potential source for symbionts (groups A and B) and for groups with a potential source for symbionts (groups C, D1 and D2), the groups with a source of symbionts survived significantly better than the groups without a source of symbionts (F1,136 ¼ 8.37, p ¼ 0.004). The mean initial mass and also the number of individuals per box did not have significant effect on survivorship (F1,136 ¼ 1.74, p ¼ 0.189 and F1,136 ¼ 1.74, p ¼ 0.231, respectively). 4. Discussion The juveniles of P. scaber that were fed a diet supplemented with potential sources of symbionts survived better than juveniles without a symbiont source in their diet. The possible inoculation with symbionts through leaves, faeces or isopod gut improved the growth, and growth rate did not differ compared with juveniles fed a diet supplemented with fatty acids and vitamins (Fig. 1a). The
Fig. 1. (a) Body mass increase of woodlice (mean ± 95% CI) as the difference between initial body mass and body mass after two months for the five diet groups (different letters indicate post hoc comparison with p < 0.05, in fact being < 0.001). Minimum diet (group A) was supplemented either with fatty acids and vitamins (group B), piece of leaf (group C), single faecal pellet (group D1) or fresh gut (group D2). Note that juveniles hatched at the average mass of 0.5 mg. (b) Survival after two months for the five diet groups. Survival of combined groups C, D1 and D2 (with symbiont source) versus groups A and B (without symbiont source) provides statistical evidence for the increased survival (p ¼ 0.004).
juveniles fed only the minimum diet had approximately three-fold slower growth than all other diet groups. Despite we provided isopods only with tiny amounts of supplements (0.2e0.6 mg compared to 10e15 mg of minimum diet), we cannot entirely exclude that some additional nutrients that were present in the leaves, guts or faeces had an additive nutritional value, though we consider it unlikely. These results suggest that symbiont inoculation is mediated through horizontal and environmental transfer and question the existence of vertical transfer in woodlice, at least during the phase when the marsupium is filled with liquid (see next paragraph). Because the addition of fatty acids and vitamins offset the lack of symbionts in the diet (Fig. 1, group B compared with groups C, D1 and D2), it seems that the symbionts were a source for these limited resources, as hypothesised previously [22].
Table 1 Mean juvenile mass (±95% CI) for five diet groups measured at simulated hatching and after four and eight weeks. Minimum diet (group A) was supplemented either with fatty acids and vitamins (group B), piece of leaf (group C), single faecal pellet (group D1) or fresh gut (group D2). Mass (mg) at diet group
Hatching
4 weeks
8 weeks
A B C D1 D2
0.501 0.489 0.499 0.522 0.504
0.623 0.754 0.820 0.806 0.793
0.993 1.720 1.840 1.908 1.755
(0.482e0.519) (0.466e0.512) (0.485e0.513) (0.499e0.546) (0.478e0.529)
(0.556e0.690) (0.674e0.834) (0.731e0.908) (0.674e0.937) (0.667e0.920)
(0.659e1.327) (1.213e2.227) (1.408e2.272) (1.440e2.376) (1.408e2.102)
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Environmental transfer of symbionts was suggested as a result of experiments with P. scaber maintained in sterile and nonsterile conditions [23]. Hepatopancreatic bacteria were detected only in juveniles fed with nonsterilised leaves. Potential contact with the mother and thus horizontal transfer of bacterial symbionts could not be excluded because progeny were analysed after hatching from the marsupium and thus naturally encountered the external surface of the mother and her faeces. With the novel in vitro approach used in this study, the dissection of progeny from the marsupium one to two weeks before hatching minimised the contamination from the mother and the environment, and thus allowed us to differentiate between three modes of transmission. If indeed symbionts promote isopod growth, the slower growth rate of juveniles raised on the minimum diet suggests that vertical transfer does not occur and that during the dissection contamination did not occur or was at least minimised. The significant interaction between diet group and mass increase for the two periods (accelerated growth of juveniles provided with a symbiont source and dietary supplements during the second month; Table 1) suggests that inoculation of symbionts requires time to establish and maintain the gut microbiota. This may be a general effect of newly hatched juveniles, or might be associated to the tiny pieces of diet supplements that may be too small to contain necessary amounts of symbionts for inoculation (groups C, D) or essential nutrients (group B). Our results strongly support the environmental and horizontal transfer of symbionts to occur, a process that positively affects the early growth of juvenile P. scaber. Most terrestrial insects that feed on low-quality plant matter transfer their symbionts vertically [14]. Environmental transmission generally occurs in aquatic environments and was described in groups such as beard worms, squid, hornworts and tubeworms [4,14,28]. The environmental and horizontal uptake of symbionts, with the beneficial impact on the growth in the host P. scaber, provides new understanding of how a terrestrial life style in isopods may be enabled. In terrestrial habitats, the conditions experienced by symbionts during transmission from the environment to the animal gut (e.g., desiccation, UV radiation, and temperature fluctuations) likely constrained environmental transmission, and thus vertical transmission would be favourable on land. However, we find no support for such a phenomenon in P. scaber. In land animals, the only case of environmental uptake of symbionts from the soil was reported in the bug species Riptortus clavatus, which was attributed to the protective properties of the soil environment [16]. Extant isopods of the suborder Oniscidea are almost entirely composed of terrestrial forms, and the transition from sea to land is supposed to have occurred in the Early Cretaceous through the high intertidal and supra-littoral zone [29]. The intertidal species Ligia also harbours bacteria in the hepatopancreas [30], which suggests that symbiotic associations are evolutionarily ancestral and that the major constraint during land invasion by isopods was not the acquisition of symbionts but rather their maintenance. To maintain the gut microbiota in a nonaqueous environment, isopods may have ingested symbionts from food sources, which they naturally encountered in their habitat. One of the major challenges posed to terrestrial animals is feeding on low-quality plant material, which is generally rich in cellulose and lignin. It has been suggested that terrestrial animals benefit from symbiotic associations either by increasing the digestion efficiency of cellulose or by providing nutrients otherwise limited in food. Terrestrial isopods that feed on decayed leaves can harbour symbionts in two morphologically and functionally distinct parts of the digestive tract, the hepatopancreas and the hindgut [20]. Whereas resident bacterial symbionts in the hepatopancreas possibly contribute to cellulose hydrolysis [20,31e33], the hindgut community of transient microbes and
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fungi might serve as a source of food-limited nutrients [20,34]. The high growth rates of juveniles fed supplementary fatty acids and vitamins (group B) without any additional source of symbionts suggest that symbionts of the other experimental groups provide essential nutrients either as a by-product of their metabolic activities or are simply digested and utilized as a direct food source (see Refs. [22,35]). The microbial community of hepatopancreas is considered to be very specific, only two microbial species have been identified in lumen of hepatopancreas; Candidatus Hepatoplasma and Candidatus Hepatincola [32,33]. These two bacterial species never occur together and the infection is most likely possible only through feeding on dead conspecifics; these bacteria could not be detected in faeces, soil or leaf litter [15]. The contribution to cellulose digestion in P. scaber might be negligible because the prevalence of symbiont-carrying individuals in the population is generally low (below 30%, [23]), and the hepatopancreas has been shown to produce one of the enzymes needed for cellulose digestion [36]. Although our experimental approach does not allow to differentiate between hindgut and hepatopancreatic symbionts, the similar growth rates of juveniles supplemented with fresh gut, faeces or leaves might suggest that gut passengers rather than hepatopancreatic bacteria contributed to the improved growth. Therefore, the gut symbionts acquired through environmental and horizontal uptake may provide food-limited nutrients to woodlice metabolism, rather than enhance efficiency of cellulose digestion. Symbionts may also positively affect host survival [6,8]. Individuals of P. scaber harbouring a single bacteria species in the hepatopancreas showed improved survival, but only when fed on a low-quality (cellulose-based) diet [6]. In accordance with these results, survival of combined groups C, D1 and D2, which were assumed to show increased growth because of symbiont acquisition, was higher than in groups A and B (without symbiont source). The possible acquisition of symbionts positively affects survival, a result that might be more related to the microbial community in the digestive tract than to a single bacterial strain in the hepatopancreas. Symbionts have been suggested to beneficially impact on host fitness by providing essential nutrients [7,9]; however, our results showed that supplementation with dietary fatty acids and vitamins resulted in generally low survival of juveniles (Fig. 1b). Therefore, the mechanism responsible for symbiont-mediated survival is most likely not linked to the host nutritional state but rather to other components of the host physiology such as, for example, pathogen resistance or thermal tolerance [9]. Our data provide experimental, though indirect, support that symbionts acquired through environmental and horizontal transfer help to compensate for dietary limitations and might increase the survival rate imposed by feeding on a low-quality terrestrial diet. Future investigations on symbiont acquisition modes and potential nutritional contribution may be directed towards the differentiating the nutritional contribution of hindgut vs. hepatopancreatic symbionts (providing essential nutrients vs. degradation of cellulose) and possibly fungal community by combining experimental feeding manipulation with molecular approaches.
Acknowledgements The project was supported by DS (DS/MND/WBINOZ/INOS/14/ 2013) and MAESTRO grant (2011/02/A/NZ8/00064). We thank ska for critical readings of Edyta Sadowska and Alexandra Walczyn the manuscript and for help with performing the statistical analyses. We are grateful for the anonymously provided comments that helped to improve the ms.
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Appendix A1 The composition of diet: Minimum diet (dry mass %), Minimum diet (dry mass %): casein 15%, cellulose 30%, starch 25%, sucrose 10%, maltose 5%, glucose 5%, lactose 5%, di-Potassium hydrogen phosphate 1.15%, magnesium sulphate anhydrous 0.65%, copper chloride dihydrate 0.2%, sodium dihydrogen phosphatemonhydrate 0.45%, sodium chloride 0.2%, calcium hydrogenphosphate 0.65%, calcium lactate pentahydrate 1.55%, iron citrate 0.15%. Fatty acids and vitamins (dry mass %), Fatty acids and vitamins (dry mass %): oleic acid 1.5%, palmitic acid 0.75%, linoleic acid 0.375%, myristic acid 0.375%, absorbic acid 0.5%, choline chloride 0.4%, D-tocopherol 0.05%, niacinamide 0.01%, riboflavin 0.002%, thiamine hydrochloride 0.002%, folic acid 0.001%, D-biotin 0.001%, pyridoxine monohydrochloride 0.01%, menadione 0.001%. The preparation of diet: The ingredients for minimum diet were mixed with hot destilled water and then with agar. After cooling, the diet was mixed with faccy acids and vitamins (only group B). The diet was poured into sterile Petri dish and kept at 4 C. References [1] A. De Bary, De la Symbiose, Rev. Int. Des. Sci. 3 (1879) 301e309. [2] P. Buchner, Endosymbiosis of Animals with Plant Microorganisms, Interscience, New York, 1965. [3] M. Zimmer, The role of animal-microbe interactions in isopod ecology and evolution, Acta Biol. Benrodis 13 (2006) 127e168. [4] J.M. Harris, The presence, nature, and role of gut microflora in aquatic invertebrates: a synthesis, Microb. Ecol. 25 (1993) 195e231. [5] M. Taylor, O. Mediannikov, D. Raoult, G. Greub, Endosymbiotic bacteria associated with nematodes, ticks and amoebae, FEMS Immunol. Med. Microbiol. 64 (2012) 21e31. [6] S. Fraune, M. Zimmer, Host-specificity of environmentally transmitted Mycoplasma-like isopod symbionts, Environ. Microbiol. 10 (2008) 2497e2504. [7] A.E. Douglas, The microbial dimension in insect nutritional ecology, Funct. Ecol. 23 (2009) 38e47. [8] J.R. Garcia, A.M. Laughton, Z. Malik, B.J. Parker, C. Trincot, S.S.L. Chiang, E. Chung, N.M. Gerardo, Partner associations across sympatric broad-headed bug species and their environmentally acquired bacterial symbionts, Mol. Ecol. 23 (2014) 1333e1347. [9] H. Feldhaar, Bacterial symbionts as mediators of ecologically important traits of insect hosts, Ecol. Entomol. 36 (2011) 533e543. [10] P. Maurer, D. Debieu, C. Malosse, P. Leroux, G. Riba, Sterols and symbiosis in the leaf-cutting ant Acromyrmex-Octospinosus (Reich) (Hymenoptera, Formicidae, Attini), Arch. Insect Biochem. 20 (1992) 13e21. [11] M. Zimmer, W. Topp, Microorganisms and cellulose digestion in the gut of the woodlouse Porcellio scaber, J. Chem. Ecol. 24 (1998) 1397e1408. [12] E.A. Gunduz, A.E. Douglas, Symbiotic bacteria enable insect to use a nutritionally inadequate diet, Proc. R. Soc. B-Biol. Sci. 276 (2009) 987e991. [13] J.G. Lundgren, R.M. Lehman, Bacterial gut symbionts contribute to seed digestion in an omnivorous beetle, PLoS One 5 (2010) e10831. [14] M. Bright, S. Bulgheresi, A complex journey: transmission of microbial symbionts,, Nat. Rev. Microbiol. 8 (2010) 218e230.
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Original article
Growth rate and survival of terrestrial isopods is related to possibility to acquire symbionts zia Horva thova *, Jan Kozłowski, Ulf Bauchinger Tere w, Poland Institute of Environmental Sciences, Jagiellonian University, ul. Gronostajowa 7, 30-387 Krako
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 November 2014 Received in revised form 13 May 2015 Accepted 13 May 2015 Available online 17 May 2015 Handling editor: S. Schrader
The acquisition and maintenance of symbiont-host associations is considered an important prerequisite for the successful colonisation of land by animals because symbionts allowed the hosts to dwell on lowquality food sources. Digestive tract symbionts are suggested to either enhance digestive efficiency of cellulose or supply the host with nutrients otherwise limited in the food. Terrestrial isopods are the most successful land colonisers among the Crustacea, and although symbiotic bacteria have been identified in digestive tract, the mechanism of transmission and the nutritional role of the symbionts are at present poorly understood. We used a novel in vitro approach to isolate and culture juveniles of Porcellio scaber at a time when only the vertical mode of symbiont acquisition could have occurred, which allowed us to follow juvenile growth and to simulate alternative modes of symbiont acquisition through feeding manipulations. Thus, we experimentally obtained groups of juveniles that could have acquired symbionts only through vertical transfer (mother-offspring), or additionally, through horizontal (through faeces or contact with conspecifics) and environmental (through leaves) transfer. We quantified survival and growth rates over two months for the different experimental acquisition modes, and significantly different growth rates were observed (p < 0.001). Growth of juveniles suggests that first, symbiont inoculation is mediated through horizontal and environmental transfer and second, the symbionts may in fact serve as a source for fatty acids and vitamins. The growth rates further question that vertical transfer occurs in woodlice. Although survival did not differ significantly between different acquisition modes (p ¼ 0.051), juveniles supplemented with potential sources of symbionts showed a tendency towards increased survival. The successful invasion of land may thus have been facilitated through the uptake of symbionts from the surroundings. © 2015 Elsevier Masson SAS. All rights reserved.
Keywords: Symbiont transmission Nutrition Cellulose Terrestrial isopods Woodlice
1. Introduction According to the original definition by Anton de Bary [1], symbiosis can be described as the association (temporal or spatial) between individuals who do not belong to the same species, independent of the effects on organisms involved, be they negative (parasitism), neutral (commensalism) or positive (mutualism). In animals, host-symbiont associations include transient gut passengers that pass through the digestive tract with ingested food, permanent gut residents, gardening of fungi, and surface-associated and/or intracellular symbionts [2]. The acquisition of novel
* Corresponding author. ), jan.kozlowski@ E-mail addresses: [email protected] (T. Horv athova uj.edu.pl (J. Kozłowski), [email protected] (U. Bauchinger). http://dx.doi.org/10.1016/j.ejsobi.2015.05.003 1164-5563/© 2015 Elsevier Masson SAS. All rights reserved.
biological properties via symbiosis played an important role in adaptation, evolution and diversification of animals, e.g., allowing the host to explore new ecological niches [3]. Symbiotic associations are widespread in diverse taxa in both aquatic and terrestrial environments [2,4,5]. In the largest animal group on Earth, insects, up to half of the species are estimated to harbour obligatory symbionts [2]. Protection against pathogens, improved growth and survival, and provisioning of limiting nutrients are examples of the beneficial effects of symbionts [5e8]. Nutritional symbiosis appears to be common in terrestrial arthropods that feed on diets scarce in vitamins, sterols, and essential amino acids or on diets rich in cellulose and lignin [7,9e13]. However, the modes of symbiont acquisition and their potential nutritional contributions are at present not understood. In general, the establishment of the symbiont-host relationship can be facilitated through three transmission mechanisms:
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vertically transmitted symbionts are passed from mother to progeny by infecting milk glands, eggs or embryos [14], horizontally transmitted symbionts may be acquired through feeding on infected corpses, faeces, exuviae or through physical contact between conspecifics [14,15], and environmentally transmitted symbionts are acquired through contact with or uptake of any matter [14,16,17]. The present understanding of the mechanisms that mediate symbiont transmission is based on empirical evidence available from only a few model systems. Vertical transmission appears as the predominant mechanisms for symbiont acquisition in insects [2,18], and environmental transmission is described for species living in aqueous environment [4,17]. The horizontal transmission of microbial species through faecal-oral contact in ruminant herbivores has received the most attention [19]. Thus, the transmission mode can be considered to be habitat and taxa specific, but overall, the understanding has to be qualified as poor. The suborder Oniscidea (Crustacea) is composed of more than 3500 terrestrial species, and their association with symbiotic bacteria has been confirmed only in seven species [20]. The microbial contribution to cellulose and lignin digestion has hereby received the most attention [3,6,11,21]. Symbionts are assumed to improve the ability of the host to digest a terrestrial diet by providing necessary enzymes or essential nutrients such as fatty acids and vitamins [22]. The mode of microbe transmission in terrestrial isopods is generally assumed to be environmental [23], however due to the methodological approach this contribution cannot fully exclude the horizontal (through contact with mother) mode of transfer. We experimentally excluded and/or facilitated symbiont transmission by dissecting embryo stages from the marsupium and subsequent in vitro culturing of the juveniles with different food supplements with the goal to determine the mode of symbiont acquisition and the beneficial effects of symbionts in the terrestrial isopod species Porcellio scaber. Different sources for symbiont inoculation were provided to juveniles through nutritional supplements to an artificial standard diet to test for the vertical (mother-offspring), horizontal (through faeces or contact with conspecifics) or environmental (through leaves) mode of transfer. Based on the assumption that symbionts positively affect growth and survival, we predicted: i) no difference in growth and survival if vertical transmission occurs; ii) difference in growth and survival if vertical transfer does not occur, and iii) dietary supplements of fatty acids and vitamins offset the lack of symbionts by improving isopod growth. 2. Materials and methods 2.1. Collection of animals Specimens of woodlice (P. scaber) were collected in spring 2013 w, Poland. The isopods were maintained in plastic boxes in Krako (52 mm 48 mm Ø, 100 ml) under a constant temperature of 20 C and a photoperiod of 16L:8 D. The progeny of 30 gravid females were used for the experiment, summing up to a total of 830 dissected mancas.
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fluid is absorbed, individuals start to move and they switch from aquatic respiration to air breathing by using pleopodal lungs. To mimic such conditions in vitro, the approach used by Surbida and Wright [25], who transferred embryos of the isopod species Armadillidium vulgare to fresh 12 5 ml well-plates and cultivated them in culture saline, was modified. Because this method did not allow individuals to freely move out from the water and thus follow the ontogenic development after hatching, a funnel made out of filter paper was attached to each well (volume of 1 ml, water level reached to approximately half of the funnel height). The novel funnel-approach allowed mancas to crawl out of the liquid bottom of the funnel and onto the funnel part above the liquid surface to survive and to breath with lungs (marsupial manca stage, [26]). We used a normal filter paper and did not use any additional measures to sterilise it, assuming that all individuals experience the same contamination conditions during this period. 2.3. Diet manipulation The day when individuals crawled freely inside the wells was defined as the time of hatching (one to two weeks after dissection). A total of 267 juveniles moved from the liquid phase into the air phase and subsequently ‘hatched‘ in vitro (32% survival of all cultured individuals). After hatching, the body mass of juveniles was determined to the nearest 0.001 mg (Mettler Toledo XP26, Greifensee, Switzerland), and one or two juveniles were subsequently kept in individual boxes (52 mm 48 mm Ø, 100 ml) Las, glass sand, class III) and an containing wet sand (Grudzen autoclaved piece of clay pot, which served as a shelter and also a feeding place. The juveniles of each clutch were split equally and randomly assigned to five experimental diet groups. The individuals in all diet groups were offered ad libitum ‘minimum diet’ [27] modified after [22]; the details on diet composition and diet preparation are presented in Appendix A1. The food provided to group A contained only this minimum diet (10e15 mg), while group B was supplemented with fatty acids and vitamins (0.4e0.6 mg), group C was supplemented with a tiny piece of alder leaf (0.2e0.4 mg), group D1 was supplemented with a single faecal pellet of an adult conspecific (0.1e0.3 mg), and group D2 was supplemented with the gut (including hepatopancreas) of a freshly killed adult (0.4e0.6 mg). The tiny amounts of supplements were used to facilitate possible symbiont inoculation without serving as significant source of extra nutrients. The supplements and the diets were provided only every 7th day in the form of a fresh artificial diet pellet placed on the clay pot; the supplements for groups C and D were placed at least 1 cm from the food pellet to avoid contamination of the artificial diet. The artificial diet was always produced fresh (agar was sprinkled to the boiling water and additional ingredients were added while keeping the fluid warm; for details see Appendix A1). Because all experimental groups faced the same risk of contamination, we expected that any growth or survival differences between groups must stem from the supplement added to the diet. 2.4. Statistical analyses
2.2. Mimicking conditions inside marsupium The embryos were removed from the marsupium (¼ motherly brood pouch) at the later stage of gravidity (stage 3, [24]) when they could not have encountered symbionts through horizontal or environmental transfer, and thus only vertical transfer could have occurred. The marsupial development is characterised by Haeckel's 'ontogeny recapitulates phylogeny' because individuals inside the brood pouch experience the change from the water to land conditions one week before hatching. During this period, the marsupial
Growth rates were determined by weighing each individual at hatching (initial body mass) and after four and eight weeks. All data were tested for normality of distribution and homogeneity of variance prior to analyses. To examine the effect of diet on growth rates, repeated-measures ANOVA and Bonferroni post-hoc tests were used with initial body mass as a covariate, diet, time points (day of hatching, four weeks and eight weeks), the interaction term between the two factors, and number of juveniles per box as explanatory variables. Because juveniles were assigned to
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experimental groups randomly, mean initial body mass was compared between diet groups by ANOVA (group A, n ¼ 27; group B, n ¼ 23; group C, n ¼ 23; group D1, n ¼ 21; and group D2, n ¼ 19). The GLIMMIX procedure was used to analyse differences in survival rates. The model included survival as a binary response variable (survived or died within eight weeks) with diet, number of juveniles per box and initial body mass as explanatory variables. All statistical analyses were performed with the SAS 9.4 statistical software package (SAS Institute Inc., Cary, NC, USA). 3. Results The mean initial juvenile mass did not differ among diet groups (F4,108 ¼ 1.4, p ¼ 0.24; Table 1). The initial body mass had a significant effect on the growth rate (F1,95 ¼ 12.98, p < 0.001). The juvenile mass after two months was significantly different between diet groups (F4,95 ¼ 11.32, p < 0.0001; Fig. 1a). The juveniles fed a diet supplemented with fatty acids and vitamins (group B), a tiny piece of leaf (group C), a single faecal pellet (group D1) or the gut (group D2) grew significantly faster than juveniles of group A fed only the minimum diet (Fig. 1a). The mass in groups B, C and D did not differ significantly from each other (all p > 0.184). A significant interaction between diet groups and time points was also found (F8,95 ¼ 6.39, p < 0.0001). The difference between initial mass and mass after four weeks was significant only for group C, but not for groups A, B, D1 and D2 (group C, p ¼ 0.03; groups A, B, D1, and D2, p > 0.05; Table 1). Groups B, C and D showed significant differences between mass after four and mass after eight weeks, but not group A (group A, p ¼ 0.115; groups B, C, D p < 0.0001; Table 1). The number of individuals per box (initially either one or two) did not have a significant effect on the increase in mass (F1,95 ¼ 1.88, p ¼ 0.173). Survival did not differ significantly between the diet groups (F4,136 ¼ 2.42, p ¼ 0.051), but revealed a tendency for higher survival for the groups supplemented with potential sources for symbionts, despite the low sample sizes (group A, n ¼ 7; group B, n ¼ 6; group C, n ¼ 11; group D1, n ¼ 8; and group D2, n ¼ 10; Fig. 1b). When the data were pooled for groups without a potential source for symbionts (groups A and B) and for groups with a potential source for symbionts (groups C, D1 and D2), the groups with a source of symbionts survived significantly better than the groups without a source of symbionts (F1,136 ¼ 8.37, p ¼ 0.004). The mean initial mass and also the number of individuals per box did not have significant effect on survivorship (F1,136 ¼ 1.74, p ¼ 0.189 and F1,136 ¼ 1.74, p ¼ 0.231, respectively). 4. Discussion The juveniles of P. scaber that were fed a diet supplemented with potential sources of symbionts survived better than juveniles without a symbiont source in their diet. The possible inoculation with symbionts through leaves, faeces or isopod gut improved the growth, and growth rate did not differ compared with juveniles fed a diet supplemented with fatty acids and vitamins (Fig. 1a). The
Fig. 1. (a) Body mass increase of woodlice (mean ± 95% CI) as the difference between initial body mass and body mass after two months for the five diet groups (different letters indicate post hoc comparison with p < 0.05, in fact being < 0.001). Minimum diet (group A) was supplemented either with fatty acids and vitamins (group B), piece of leaf (group C), single faecal pellet (group D1) or fresh gut (group D2). Note that juveniles hatched at the average mass of 0.5 mg. (b) Survival after two months for the five diet groups. Survival of combined groups C, D1 and D2 (with symbiont source) versus groups A and B (without symbiont source) provides statistical evidence for the increased survival (p ¼ 0.004).
juveniles fed only the minimum diet had approximately three-fold slower growth than all other diet groups. Despite we provided isopods only with tiny amounts of supplements (0.2e0.6 mg compared to 10e15 mg of minimum diet), we cannot entirely exclude that some additional nutrients that were present in the leaves, guts or faeces had an additive nutritional value, though we consider it unlikely. These results suggest that symbiont inoculation is mediated through horizontal and environmental transfer and question the existence of vertical transfer in woodlice, at least during the phase when the marsupium is filled with liquid (see next paragraph). Because the addition of fatty acids and vitamins offset the lack of symbionts in the diet (Fig. 1, group B compared with groups C, D1 and D2), it seems that the symbionts were a source for these limited resources, as hypothesised previously [22].
Table 1 Mean juvenile mass (±95% CI) for five diet groups measured at simulated hatching and after four and eight weeks. Minimum diet (group A) was supplemented either with fatty acids and vitamins (group B), piece of leaf (group C), single faecal pellet (group D1) or fresh gut (group D2). Mass (mg) at diet group
Hatching
4 weeks
8 weeks
A B C D1 D2
0.501 0.489 0.499 0.522 0.504
0.623 0.754 0.820 0.806 0.793
0.993 1.720 1.840 1.908 1.755
(0.482e0.519) (0.466e0.512) (0.485e0.513) (0.499e0.546) (0.478e0.529)
(0.556e0.690) (0.674e0.834) (0.731e0.908) (0.674e0.937) (0.667e0.920)
(0.659e1.327) (1.213e2.227) (1.408e2.272) (1.440e2.376) (1.408e2.102)
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Environmental transfer of symbionts was suggested as a result of experiments with P. scaber maintained in sterile and nonsterile conditions [23]. Hepatopancreatic bacteria were detected only in juveniles fed with nonsterilised leaves. Potential contact with the mother and thus horizontal transfer of bacterial symbionts could not be excluded because progeny were analysed after hatching from the marsupium and thus naturally encountered the external surface of the mother and her faeces. With the novel in vitro approach used in this study, the dissection of progeny from the marsupium one to two weeks before hatching minimised the contamination from the mother and the environment, and thus allowed us to differentiate between three modes of transmission. If indeed symbionts promote isopod growth, the slower growth rate of juveniles raised on the minimum diet suggests that vertical transfer does not occur and that during the dissection contamination did not occur or was at least minimised. The significant interaction between diet group and mass increase for the two periods (accelerated growth of juveniles provided with a symbiont source and dietary supplements during the second month; Table 1) suggests that inoculation of symbionts requires time to establish and maintain the gut microbiota. This may be a general effect of newly hatched juveniles, or might be associated to the tiny pieces of diet supplements that may be too small to contain necessary amounts of symbionts for inoculation (groups C, D) or essential nutrients (group B). Our results strongly support the environmental and horizontal transfer of symbionts to occur, a process that positively affects the early growth of juvenile P. scaber. Most terrestrial insects that feed on low-quality plant matter transfer their symbionts vertically [14]. Environmental transmission generally occurs in aquatic environments and was described in groups such as beard worms, squid, hornworts and tubeworms [4,14,28]. The environmental and horizontal uptake of symbionts, with the beneficial impact on the growth in the host P. scaber, provides new understanding of how a terrestrial life style in isopods may be enabled. In terrestrial habitats, the conditions experienced by symbionts during transmission from the environment to the animal gut (e.g., desiccation, UV radiation, and temperature fluctuations) likely constrained environmental transmission, and thus vertical transmission would be favourable on land. However, we find no support for such a phenomenon in P. scaber. In land animals, the only case of environmental uptake of symbionts from the soil was reported in the bug species Riptortus clavatus, which was attributed to the protective properties of the soil environment [16]. Extant isopods of the suborder Oniscidea are almost entirely composed of terrestrial forms, and the transition from sea to land is supposed to have occurred in the Early Cretaceous through the high intertidal and supra-littoral zone [29]. The intertidal species Ligia also harbours bacteria in the hepatopancreas [30], which suggests that symbiotic associations are evolutionarily ancestral and that the major constraint during land invasion by isopods was not the acquisition of symbionts but rather their maintenance. To maintain the gut microbiota in a nonaqueous environment, isopods may have ingested symbionts from food sources, which they naturally encountered in their habitat. One of the major challenges posed to terrestrial animals is feeding on low-quality plant material, which is generally rich in cellulose and lignin. It has been suggested that terrestrial animals benefit from symbiotic associations either by increasing the digestion efficiency of cellulose or by providing nutrients otherwise limited in food. Terrestrial isopods that feed on decayed leaves can harbour symbionts in two morphologically and functionally distinct parts of the digestive tract, the hepatopancreas and the hindgut [20]. Whereas resident bacterial symbionts in the hepatopancreas possibly contribute to cellulose hydrolysis [20,31e33], the hindgut community of transient microbes and
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fungi might serve as a source of food-limited nutrients [20,34]. The high growth rates of juveniles fed supplementary fatty acids and vitamins (group B) without any additional source of symbionts suggest that symbionts of the other experimental groups provide essential nutrients either as a by-product of their metabolic activities or are simply digested and utilized as a direct food source (see Refs. [22,35]). The microbial community of hepatopancreas is considered to be very specific, only two microbial species have been identified in lumen of hepatopancreas; Candidatus Hepatoplasma and Candidatus Hepatincola [32,33]. These two bacterial species never occur together and the infection is most likely possible only through feeding on dead conspecifics; these bacteria could not be detected in faeces, soil or leaf litter [15]. The contribution to cellulose digestion in P. scaber might be negligible because the prevalence of symbiont-carrying individuals in the population is generally low (below 30%, [23]), and the hepatopancreas has been shown to produce one of the enzymes needed for cellulose digestion [36]. Although our experimental approach does not allow to differentiate between hindgut and hepatopancreatic symbionts, the similar growth rates of juveniles supplemented with fresh gut, faeces or leaves might suggest that gut passengers rather than hepatopancreatic bacteria contributed to the improved growth. Therefore, the gut symbionts acquired through environmental and horizontal uptake may provide food-limited nutrients to woodlice metabolism, rather than enhance efficiency of cellulose digestion. Symbionts may also positively affect host survival [6,8]. Individuals of P. scaber harbouring a single bacteria species in the hepatopancreas showed improved survival, but only when fed on a low-quality (cellulose-based) diet [6]. In accordance with these results, survival of combined groups C, D1 and D2, which were assumed to show increased growth because of symbiont acquisition, was higher than in groups A and B (without symbiont source). The possible acquisition of symbionts positively affects survival, a result that might be more related to the microbial community in the digestive tract than to a single bacterial strain in the hepatopancreas. Symbionts have been suggested to beneficially impact on host fitness by providing essential nutrients [7,9]; however, our results showed that supplementation with dietary fatty acids and vitamins resulted in generally low survival of juveniles (Fig. 1b). Therefore, the mechanism responsible for symbiont-mediated survival is most likely not linked to the host nutritional state but rather to other components of the host physiology such as, for example, pathogen resistance or thermal tolerance [9]. Our data provide experimental, though indirect, support that symbionts acquired through environmental and horizontal transfer help to compensate for dietary limitations and might increase the survival rate imposed by feeding on a low-quality terrestrial diet. Future investigations on symbiont acquisition modes and potential nutritional contribution may be directed towards the differentiating the nutritional contribution of hindgut vs. hepatopancreatic symbionts (providing essential nutrients vs. degradation of cellulose) and possibly fungal community by combining experimental feeding manipulation with molecular approaches.
Acknowledgements The project was supported by DS (DS/MND/WBINOZ/INOS/14/ 2013) and MAESTRO grant (2011/02/A/NZ8/00064). We thank ska for critical readings of Edyta Sadowska and Alexandra Walczyn the manuscript and for help with performing the statistical analyses. We are grateful for the anonymously provided comments that helped to improve the ms.
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Appendix A1 The composition of diet: Minimum diet (dry mass %), Minimum diet (dry mass %): casein 15%, cellulose 30%, starch 25%, sucrose 10%, maltose 5%, glucose 5%, lactose 5%, di-Potassium hydrogen phosphate 1.15%, magnesium sulphate anhydrous 0.65%, copper chloride dihydrate 0.2%, sodium dihydrogen phosphatemonhydrate 0.45%, sodium chloride 0.2%, calcium hydrogenphosphate 0.65%, calcium lactate pentahydrate 1.55%, iron citrate 0.15%. Fatty acids and vitamins (dry mass %), Fatty acids and vitamins (dry mass %): oleic acid 1.5%, palmitic acid 0.75%, linoleic acid 0.375%, myristic acid 0.375%, absorbic acid 0.5%, choline chloride 0.4%, D-tocopherol 0.05%, niacinamide 0.01%, riboflavin 0.002%, thiamine hydrochloride 0.002%, folic acid 0.001%, D-biotin 0.001%, pyridoxine monohydrochloride 0.01%, menadione 0.001%. The preparation of diet: The ingredients for minimum diet were mixed with hot destilled water and then with agar. After cooling, the diet was mixed with faccy acids and vitamins (only group B). The diet was poured into sterile Petri dish and kept at 4 C. References [1] A. De Bary, De la Symbiose, Rev. Int. Des. Sci. 3 (1879) 301e309. [2] P. Buchner, Endosymbiosis of Animals with Plant Microorganisms, Interscience, New York, 1965. [3] M. Zimmer, The role of animal-microbe interactions in isopod ecology and evolution, Acta Biol. Benrodis 13 (2006) 127e168. [4] J.M. Harris, The presence, nature, and role of gut microflora in aquatic invertebrates: a synthesis, Microb. Ecol. 25 (1993) 195e231. [5] M. Taylor, O. Mediannikov, D. Raoult, G. Greub, Endosymbiotic bacteria associated with nematodes, ticks and amoebae, FEMS Immunol. Med. Microbiol. 64 (2012) 21e31. [6] S. Fraune, M. Zimmer, Host-specificity of environmentally transmitted Mycoplasma-like isopod symbionts, Environ. Microbiol. 10 (2008) 2497e2504. [7] A.E. Douglas, The microbial dimension in insect nutritional ecology, Funct. Ecol. 23 (2009) 38e47. [8] J.R. Garcia, A.M. Laughton, Z. Malik, B.J. Parker, C. Trincot, S.S.L. Chiang, E. Chung, N.M. Gerardo, Partner associations across sympatric broad-headed bug species and their environmentally acquired bacterial symbionts, Mol. Ecol. 23 (2014) 1333e1347. [9] H. Feldhaar, Bacterial symbionts as mediators of ecologically important traits of insect hosts, Ecol. Entomol. 36 (2011) 533e543. [10] P. Maurer, D. Debieu, C. Malosse, P. Leroux, G. Riba, Sterols and symbiosis in the leaf-cutting ant Acromyrmex-Octospinosus (Reich) (Hymenoptera, Formicidae, Attini), Arch. Insect Biochem. 20 (1992) 13e21. [11] M. Zimmer, W. Topp, Microorganisms and cellulose digestion in the gut of the woodlouse Porcellio scaber, J. Chem. Ecol. 24 (1998) 1397e1408. [12] E.A. Gunduz, A.E. Douglas, Symbiotic bacteria enable insect to use a nutritionally inadequate diet, Proc. R. Soc. B-Biol. Sci. 276 (2009) 987e991. [13] J.G. Lundgren, R.M. Lehman, Bacterial gut symbionts contribute to seed digestion in an omnivorous beetle, PLoS One 5 (2010) e10831. [14] M. Bright, S. Bulgheresi, A complex journey: transmission of microbial symbionts,, Nat. Rev. Microbiol. 8 (2010) 218e230.
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