Dietary-driven variation effects on the symbiotic flagellate protist communities of the subterranean termite Reticulitermes grassei Clement

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1 J. Appl. Entomol. ORIGINAL CONTRIBUTION Dietary-driven variation effects on the symbiotic flagellate protist communities of the subterranean termite Reticulitermes grassei Clement S. Duarte 1,2,3, M. Duarte 3, P. A. V. Borges 1,2 & L. Nunes 1,2,3 1 Departamento de Ci^encias Agrarias, ce3c - Centre for Ecology, Evolution and Environmental Changes/Azorean Biodiversity Group and Universidade dos Acßores, Angra do Heroısmo, Portugal 2 CITA-A (Universidade dos Acßores) and Portuguese Platform for Enhancing Ecological Research & Sustainability (PEERS), Angra do Heroısmo, Portugal 3 Structures Department, LNEC, Lisbon, Portugal Keywords digestion, lignocellulose, subterranean termites, symbiotic flagellate protists, thermally modified wood Correspondence Lina Nunes, Departamento de Ci^encias Agrarias ce3c - Centre for Ecology, Evolution and Environmental Changes/Azorean Biodiversity Group and Universidade dos Acßores, Angra do Heroısmo, Acßores, Portugal. linanunes@lnec.pt Received: January 20, 2016; accepted: April 10, doi: /jen Abstract The ability of subterranean termites to digest lignocellulose relies not only on their digestive tract physiology, but also on the symbiotic relationships established with flagellate protists and bacteria. The objective of this work was to test the possible effect of different cellulose-based diets on the community structure (species richness and other diversity metrics) of the flagellate protists of the subterranean termite Reticulitermes grassei. Termites belonging to the same colony were subjected to six different diets (natural diet, maritime pine wood, European beech, thermally modified European beech, cellulose powder and starvation), and their flagellate protist community was evaluated after the trials. All non-treated sound woods produced similar flagellate protist communities that were more diverse and of high evenness (low dominance). On the contrary, flagellate protist communities from cellulose-fed termites and starving termites were considered to be significantly different from all non-treated woods; they were less diverse and some morphotypes became dominant as a consequence of flagellate protist communities having suffered major adaptations to these diets. The flagellate protist communities of untreated beech and thermally modified beech-fed termites were considered to be significantly different in terms of abundance and morphotype diversity. This may be caused by a decrease in lignocellulose quality available for termites and from an interference of thermally treated wood with the chemical stability of the termite hindgut. Our study suggests that as a consequence of the strong division of labour among these protists to accomplish the intricate process of lignocellulose digestion, termite symbiotic flagellate protist communities are a dynamic assemblage able to adapt to different conditions and diets. This study is important for the community-level alteration approach, and it is the first study to investigate the effects of thermally modified wood on the flagellate protist communities of subterranean termites. Introduction Despite their importance on diverse ecosystems, termites may be considered severe pests of wood in service, and also as agricultural and forestry pests (Rouland-Lefevre 2011). Subterranean termites represent approximately 80% of the economically important species in this context (Nobre and Nunes 2007; Rust and Su 2012). Factors including the continuous growth of urbanization and climate change may 300 J. Appl. Entomol. 141 (2017) Blackwell Verlag GmbH

2 S. Duarte et al. Dietary effects on flagellate protists of subterranean termite favour the establishment or growth of termite populations. Subterranean termites have an established symbiosis with flagellate protists and bacteria, harboured in the dilated portion of the hindgut, the paunch. In this tripartite lignocellulolytic system, in a simplistic approach, the termites contribute endogenous cellulases and mechanical processing, the flagellate protists phagocytose the wood particles and digest them, and prokaryotes have, among others, an important role in maintaining the physical chemical equilibrium inside the termite hindgut (Brune and Ohkuma 2011; Xie et al. 2012). The bulk of the cellulose digestion process is performed by flagellated protists, which belong to two separate lineages of the unicellular eukaryotes: the order Oxymonadida (Phylum Preaxostyla) and the Phylum Parabasalia (Brugerolle 1991; Cepicka et al. 2010; Adl et al. 2012). There is a growing interest in the lignocellulolytic degradation process performed by the termite and symbiotic fauna holobiont (Scharf 2015). Metagenomics and metatranscriptomic studies are being performed to investigate the effects of different diets on symbiotic fauna, although the effect of these diets on flagellate protist communities is highlighted in only a few studies (e.g. Cook and Gold 2000; Tanaka et al. 2006). Flagellate protists seem to be sensitive to the wood species, possibly due to wood extractives, which may cause the total loss of flagellate protists, as well to high or low weight carbon sources, which may cause changes in the flagellate protist species composition and community structure (Mannesmann 1972; Cook and Gold 2000; Tanaka et al. 2006). Moreover, cellulose alone (as filter paper) may also impose major alterations to the flagellate protist communities (Cook and Gold 2000; Hu et al. 2011). Different diets were screened for their effects in the flagellate protists as these symbionts have a major role in cellulose digestion within the termite holobiont (Raychoudhury et al. 2013). The use of second-generation feedstocks has also been investigated for flagellate protist activity in terms of cellulose digestion, due to industrial interest in the application of these results for energy production (Rajaparu et al. 2015). Starvation has been used as a selective flagellate protist defaunation method, as each species may respond differently to termite starvation periods (e.g. Cleveland 1925; Nunes and Dickinson 1996; Belitz and Waller 1998; Cook and Gold 2000; Hu et al. 2011). The main objective of this work was to test the possible effect of different laboratorial diets on the community structure (species richness and other diversity metrics) of the flagellate protist communities living inside the subterranean termite Reticulitermes grassei Clement. Materials and Methods Subterranean termites from Lagoa de Albufeira ( N; W), in the Setubal district of Portugal, were subjected to six different diets: a natural diet (pieces of the original wood from where they were collected in the field Pinus pinaster Aiton), pine wood (P. pinaster), European beech (Fagus sylvatica L.), thermally modified beech beech TMT (subjected to 180 C for four hours), cellulose (cellulose powder mixed with deionized water) (this was a better option in comparison with filter paper) and starvation (no source of cellulose offered to the termites). The trials were established according with EN117 (2012), with some adaptations. Three replicates per each diet category were set, with 250 workers each and 1 5% of soldiers and nymphs to maintain the natural colony ratio of each caste. The test replicates were maintained in a conditioned room at 24 1 C and 80 5% relative humidity. The time of exposure was for 14 days, except for starving termites, which were left without food for 6 days. At the end of the trials, the survival rate and cellulose source mass loss (if applied) were evaluated. The mass loss (%) was obtained from the loss of the dry mass of the exposed test specimens according to a previously described protocol (Duarte et al. 2016). For flagellate protist counting, 11 termite workers per diet category were used. Termites were rinsed briefly in Trager Medium U buffer (Trager 1934) to remove surface contaminants. An incision was made in the last abdominal segment of the worker, and the digestive tract was pulled out using fine-tipped forceps. The anterior part of the digestive tract was cut out, and the posterior part was suspended in 25 ll of Trager Medium U buffer. The hindgut was cut open and slightly homogenized to release its contents. The termite tissue was removed, and the remaining solution was loaded in a Neubauer haemocytometer (8 ll) and observed in a light microscope (9400). Flagellate protists were classified into different morphotypes and counted. Flagellate protists were distinguished and identified according with species, or major taxa, descriptions (Leidy 1877; Brugerolle and Lee 2000; Brugerolle 2006; Brugerolle and Bordereau 2006; Lewis and Forschler 2006). For each termite, five small squares of mm 3 of volume were randomly chosen for counting the protists. The calculations for the number of flagellate protists per millilitre were estimated according with the formula: J. Appl. Entomol. 141 (2017) Blackwell Verlag GmbH 301

3 Dietary effects on flagellate protists of subterranean termite S. Duarte et al. C ¼ N S q D f N is the number of cells visualized, S q is the number of squares, and D f is the dilution factor. Termites subjected to different diets were collected from the same colony, and the results shown should clarify whether the null hypothesis (different diets do not significantly affect the diversity and abundance of the flagellate protists inside the termites) is to be rejected or failed to be rejected. To investigate the veracity of the null hypothesis (P < 0.05), the results obtained were subjected to a pairwise comparison using a Wilcoxon signed-rank test, with the Bonferroni P value adjustment method (for P < 0.05), as the data did not respect normality requirements even after transformations. For mass loss and survival data, a t-test was performed between the different diets (except for mass loss of starving termites, as this is not applicable). Community structure, according to the data obtained from flagellate protist communities associated with each diet, was evaluated using the following metrics: total abundance (N), species richness (S), Shannon Wiener (H 0 ), Simpson (D), Berger Parker (d) and evenness (E) (Magurran 2003). Abundance gives an idea of community biomass; species richness can be a surrogate of diversity of functions; Shannon Wiener combines the relative abundance of species with the number of species and can be viewed as a measure of community complexity; Simpson and Berger Parker are measures of dominance and give some indication of changes in the community as a consequence of single-species abundance dominance; evenness measures the equitability, and values near 1 imply similar species abundances. Berger Parker is considered the most reliable in terms of independence to sampling intensity (Magurran 2003). Results Termites were identified as specific morphotypes (table 1) and quantified in terms of their abundance (fig. 1). The results of the average survival rate of termites subjected to the trials and the average mass loss of the different diets offered to the termites show that, as expected, termites thriving on the natural diet reached a higher survival rate when compared with the other diets (fig. 1). The non-treated sound wood-based diets (pine, beech and natural diet) showed no significant differences relative to the flagellate protist communities of termites (table 2). However, the thermal modification of beech was considered to cause significant differences in the flagellate protist communities relative to the unmodified beech-fed termites. Thermally modified wood-fed termites showed a general decrease in the abundance of the majority of flagellate protist populations, except for n1 (increased), and n5 and n17 which disappeared compared to the beech-fed termites. Termites fed with cellulose had significantly different flagellate protist communities than beechand pine-fed termites. The morphotype n5 disappeared in the cellulose-fed termites, and also the abundances of some morphotypes (n2, n3, n9, for example) were reduced in these termites. However, it was verified that n7 and n8 morphotypes had a higher average abundance relative to the other diets. The Table 1 Flagellate protist identification by morphotypes based on observed morphological characters Phylum Class Order Family Genus Species N18 Parabasalia N1 Trychonymphea Trichonymphida Trichonymphidae Trichonympha T. agilis N17 Trichonymphidae N9 Spirotrychonymphea Spirotrichonymphida Holomastogotoididae Spirotrichonympha S. flagellata N4 Holomastigotes H. elongatum N8 Microjoenia M. hexamitoides N13 Trichomonadea Honigbergiellida Tricercomitidae Hexamastix N6 Hypotrichomonadea Hypotrichomonadida Hypotrichomonadidae N2 Preaxostyla Oxymonadida Pyrsonympha sp.1 N7 Pyrsonympha sp.2 N3 Dinenympha D. gracilis N5 D. fimbriata 302 J. Appl. Entomol. 141 (2017) Blackwell Verlag GmbH

4 S. Duarte et al. Dietary effects on flagellate protists of subterranean termite Fig. 1 Web chart representing the abundance and diversity of the flagellate protist morphotypes (n1-n9, n13, n17, n18) identified from termites subjected to six different diets (natural diet, pine, beech, cellulose, starving, beech TMT). The termite represents the survival rate (%) and the wood piece represents the mass loss (%) of the different cellulosic sources offered to the termites. Table 2 Results of Wilcoxon pairwise test, P values for paired comparisons (P < 0.05, grey shades indicate significant differences) of the different diets Natural diet Pine Beech Beech TMT Cellulose Pine Beech Beech TMT Cellulose < Starving <0.001 <0.001 < flagellate protist community of the starving termites was significantly different from that of all the other diets, except for cellulose. The natural diet termites showed a significantly higher survival rate than the beech TMT (t = 4.98; P = 0.004), starving (t = ; P < 0.001) and cellulose (t = 6.69; P = 0.021)-fed termites. For mass loss, cellulose reached significantly higher mass loss values when compared to natural (t = 4.57; P = 0.045), pine (t = 10.10; P = 0.009) and beech (t = 16.41; P = 0.004). In addition, the natural diet had a higher mass loss than pine (t = 5.63; P = 0.030), beech (t = 5.10; P = 0.036) and beech TMT (t = 5.74; P = 0.029). The results of biodiversity indices showed more diverse and numerous flagellate protist communities in the sound wood diets than in thermally treated wood, cellulose or starving termites (fig. 2). In contrast, the dominance indices (Simpson and Berger Parker) reached higher values in the thermally treated wood, cellulose and starving termites. J. Appl. Entomol. 141 (2017) Blackwell Verlag GmbH 303

5 Dietary effects on flagellate protists of subterranean termite S. Duarte et al. Discussion The majority of gut symbionts are considered to be autochthonous and probably coevolving with termite species (Hongoh et al. 2005; Noda et al. 2007). Therefore, a homogeneous gut symbiotic fauna should be present within a termite colony and amongst castes, as termites recover the symbiotic fauna discarded after the moult through horizontal transmission. However, the effect of different cellulose sources on flagellate protists has already been demonstrated in the laboratory (Mannesmann 1972; Cook and Gold 2000; Tanaka et al. 2006). The diversity indices showed that the natural diet, pine and beech diets maintained more complex and abundant flagellate protist communities, as a whole. The other three diets (starvation, cellulose and beech TMT) were characterized by less diverse and/or abundant flagellate protist communities, and a higher dominance of some flagellate protist morphotypes was observed, indicating a more disturbed community. For example, in starving termites, Pyrsonympha sp.1, Dinenympha gracilis Leidy and Microjoenia hexamitoides Grassi (n2, n3 and n8, respectively) were clearly dominant over the other morphotypes present (representing together over 70% of the flagellate protists identified), while in the natural diet, these three morphotypes (also the most abundant) represented less than 50%. Starving termites obviously exhibited the most depleted flagellate protist fauna in terms of abundance, because an energy source was not available for the termites or the protists. The relative resilience of the flagellate protist community and termites (33.2% survived after 6 days of starvation) to starvation may be explained by the fact that flagellate protists may feed on the bacteria living inside the termite hindgut, or eventually the practice of cannibalism on other protist species; or it may be related to the subterranean termite cannibalism/necrophagy habits (Cook and Gold 2000; Sun et al. 2013). The mechanisms that trigger termite practices of cannibalism due to a scarcity of food or to starvation remain obscure, and it has been hypothesized that the degree of relatedness of termite workers is low (decreasing the altruistic behaviour when under stressful situations). It is also suggested that cannibalism is a natural response to the survival struggle, as cannibalism may provide both an additional source of nitrogen and also provide symbiotic fauna to maintain the digestion abilities when food becomes available (Hu et al. 2011). For these reasons, necrophagy is considered to be a normal behaviour within termite colonies. However, subterranean termites practice necrophagy only with dead colony members and actively avoid the dead corpses of termites belonging to different termite species (Sun et al. 2013). This fact may be regarded, not only as a strategy of nutrient recycling and defence, but also as a mechanism to maintain the symbiotic fauna communities integrity (Sun et al. 2013). Cellulose is a less complex energy source for termites than sound wood that would presumably require a less complex digestive process to produce energy; however, it is not as rich as sound wood in terms of energy generation. The significantly higher mass loss shown by cellulose may also indicate that it is a poorer lignocellulosic source than sound wood. Although termites consumed relatively more cellulose than other food sources, this was not reflected in the Fig. 2 Biodiversity indices (total abundance (N), species richness (S), Shannon-Wiener (H ), Simpson (D), Berger-Parker (d) and evenness (E)) for the flagellate protist communities of termites subjected to the different diets. 304 J. Appl. Entomol. 141 (2017) Blackwell Verlag GmbH

6 S. Duarte et al. Dietary effects on flagellate protists of subterranean termite survival rate of the termites feeding on cellulose, as their survival was lower than that of termites fed a natural diet. The flagellate protists have different roles in the lignocellulosic digestion process; therefore, the simplification of the digestion process may cause the depletion of the flagellate protist communities, as shown by the results obtained. However, some flagellate protist morphotypes increased in abundance, namely M. hexamitoides and Pyrsonympha sp.2, suggesting that these flagellate protists have a main role in cellulose degradation. An increase in Pyrsonympha minor Powell abundance was also observed after a cellulose-based diet by Cook and Gold (2000) in the termite Reticulitermes virginicus Banks. One study stated that host termites contribute more actively to lignocellulose degradation, whereas symbiotic protists contribute more to a pure cellulose diet degradation. However, the metatranscriptomic nature of this study precludes establishing parallel conclusions with the current work (Raychoudhury et al. 2013). Interestingly, the flagellate protist community of untreated beech and thermally treated beech (beech TMT) fed termites were considered to be significantly different. Although the heat treatment was light, the effects in terms of the lignocellulosic components available to the termites seem to have been significant. The differences observed between beech and beech TMT show that a slight change in the wood properties may produce major modifications in the flagellate protist communities. The species Dinenympha fimbriata Kirby disappeared from the termite hindguts; in fact, Cook and Gold (2000) observed a decrease in the abundance of this species after subjecting R. virginicus to a filter paper diet, reinforcing the fact that this species is sensitive to cellulose only based diets. Beech thermal treatment leads to a variable degree of decomposition of wood components according to the type of heat treatment applied (Windeisen et al. 2007). The common effects of thermal treatment already observed in beech were as follows: loss of functional groups (carboxyl, hydroxyl and acetyl), an increased carbon content, solubilization of lignin, a decrease in ph and the decomposition of sugars (Windeisen et al. 2007). These factors directly affect the lignocellulose digestion process by the decrease of available energy sources. The associated ph modification may also interfere with the hindgut chemical stability (Brune and Friedrich 2000). The diversity of flagellate protist fauna associated with lower termites might be explained by a strong division of labour to accomplish the intricate process of lignocellulose digestion. A species or group of species acts on specific phases of this process (Yoshimura et al. 1996; Inoue et al. 1997; Todaka et al. 2007; Raychoudhury et al. 2013). The results obtained reinforce that termites from the same colony, when subjected to different diets, have the ability to adapt their symbiotic flagellate community to efficiently digest the lignocellulosic source available. The cooperation between termite host and its symbiotic flagellate protist microbiota increases their ability to adapt more rapidly to changing conditions (Rosenberg and Zilber-Rosenberg 2013). This fact should be further investigated using morphological and molecular data to provide additional important information to innovative termite control strategies which are being developed worldwide (e.g. Scharf et al. 2011; Sethi et al. 2014). 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