Evaluation of the effect of zwitterionic buffers on termite gut protists

Proceedings IRG Annual Meeting (ISSN 2000-8953)

© 2018 The International Research Group on Wood Protection

IR/WP 18-10933

THE INTERNATIONAL RESEARCH GROUP ON WOOD PROTECTION Section 1 Biology

Evaluation of the effect of zwitterionic buffers on termite gut protists

Sónia Duarte

, Dennis Jones

, Lina Nunes

1

LNEC, National Laboratory for Civil Engineering, Structures Department, Av. do Brasil, 101, 1700-066 Lisbon, Portugal [linanunes@lnec.pt; sduarte@lnec.pt]

2

cE3c, Centre for Ecology, Evolution and Environmental Changes / Azorean Biodiversity Group and University of the Azores,

Faculty of Agrarian and Environmental Sciences, 9700–042 Angra do Heroísmo, Portugal

3

DJ Timber Consultancy Ltd, 15 Heol Pen Y Coed, Neath, SA11 3SP, United Kingdom [dr_dennisjones@hotmail.co.uk]

4

Wood Science and Engineering, Luleå University of Technology, Forskargatan 1, S-93197 Skellefteå, Sweden [dennis.jones@ltu.se]

Paper prepared for the IRG49 Scientific Conference on Wood Protection Johannesburg, South Africa

29 April – 3 May 2018

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The opinions expressed in this document are those of the author(s) and

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Evaluation of the effect of zwitterionic buffers on termite gut protists

Sónia Duarte

, Dennis Jones

, Lina Nunes

1

LNEC, National Laboratory for Civil Engineering, Structures Department, Av. do Brasil, 101, 1700-066 Lisbon, Portugal [linanunes@lnec.pt; sduarte@lnec.pt]

2

cE3c, Centre for Ecology, Evolution and Environmental Changes / Azorean Biodiversity Group and University of the Azores,

Faculty of Agrarian and Environmental Sciences, 9700–042 Angra do Heroísmo, Portugal

3

DJ Timber Consultancy Ltd, 15 Heol Pen Y Coed, Neath, SA11 3SP, United Kingdom [email:

dr_dennisjones@hotmail.co.uk]

4

Wood Science and Engineering, Luleå University of Technology, Forskargatan 1, S-93197 Skellefteå, Sweden [email: dennis.jones@ltu.se]

ABSTRACT

The thermal modification of wood is recognized as the most commercialized wood modification process. The treatment of wood at temperatures usually greater than 180 ºC results in a product with some properties enhanced (dimensional stability, durability, aesthetical colour) and some diminished (mechanical properties). The loss of mechanical strength is recognized as a limitation to the use of thermally modified wood in certain products. The mechanical strength reduction is linked in varying degrees to the release of acidic volatile species, the acid-catalysed depolymerisation of the hemicelluloses present and the plasticization and redistribution of the lignin components present. Buffers, although been designed not to influence the reaction systems, may have some interactions when exposed to some conditions, potentially acting as promoters of biological changes on different systems. In this study, two zwitterionic buffers, bicine and tricine, were chosen to be tested regarding their volatilization reduction properties, as well as tricine due to offering the potential of forming Maillard-type products with fragmented hemicelluloses/volatiles.

In order to determine if there were any resultant effects of the bicine and tricine treatments submitted or not to thermal modification, the efficacy against subterranean termites was conducted as well as an evaluation of the effect of the different combinations of treatments on the termite gut symbiotic protists. The durability of thermal modified wood is recognized as low and the results of the present work are in accordance with that perception. However, bicine and tricine treatments alone had a clear influence on the survival of the termites and the study of the protist symbionts (9 morphotypes) gave a better insight of the ability of termites to react to unfavorable diet changes.

Keywords: Bicine, tricine, subterranean termites, gut symbiotic protists 1. INTRODUCTION

The thermal modification of wood is recognised as the most commercialised of the wood

modification processes. The treatment of wood at temperatures of usually greater than 180 ºC

results in a product with some properties enhanced (dimensional stability, durability, aesthetical

colour) and some diminished (mechanical properties). The loss of mechanical strength is

recognised as a limitation to the use of thermally modified wood in certain products. The

mechanical strength reduction is linked in varying degrees to the release of acidic volatile

3

species, the acid-catalysed depolymerisation of the hemicelluloses present and the plasticization and redistribution of the lignin components present.

Zwitterionic buffers are neutral molecules with a positive and a negative electrical charge, with some advantages regarding other types of buffers, due to their stability and reduced membrane permeability (Good et al. 1966, Ferreira et al. 2015). In this study, bicine and tricine were chosen to be tested. These substances are commonly used as non-fermentable buffering agents for some reactions as, for example, manufacture of primary cells (bicine) and animal tissue culture (tricine) (Soni and Kapoor 1981, Armenante et al. 1993). The pH range of bicine is 7.6-9.0 and of tricine is 7.4-8.8 (Armenante et al. 1993), besides this range, usually the pH at the midpoint of the buffering range is used to represent and describe the substance, however, this value may decrease with temperature increase, although it doesn’t interfere significantly with the substance ionic strength (Soni and Kapoor 1981, for a temperature range between 25 ºC and 50 ºC). Tricine has been described as an efficient scavenger of -OH radicals (Hicks and Gebicki 1986).

Buffers are designed to have little influence the reactions in which they are used, so it is important to investigate the compatibility of the buffer with the reaction system not only regarding the buffering range but also to other possible buffer interactions, for example, the possible impacts of the buffer on cell growth and survival, the interactions of buffers with macromolecules (tricine may form complexes with DNA, and zwitterionic buffers influence mRNA expression of some reaction systems), and the impact of buffers on redox studies (Ferreira et al. 2015). Buffers, although been designed not to influence the reaction systems, may have some interactions when exposed to some conditions, and potentially acting as promoters of biological changes on those systems.

It has been suggested that the darkening of the wood colour during thermal modification is a result of a Maillard reaction. This reaction is the result of an amine reacting with a reducing sugar. Such a mechanism was proposed by Hauptmann et al. (2015) for the reaction of xylose with tricine. By better understanding the depolymerisation/degradation process within the hemicellulose, it may be possible to apply Maillard-type reactions and react the sugar moieties within the cell wall matrix, instead of being volatilised.

In this study, two zwitterionic buffers, bicine and tricine, were chosen to be tested regarding their volatilization reduction properties, as well as tricine due to offering the potential of forming Maillard-type products with fragmented hemicelluloses/volatiles. Furthermore, to determine if there were any resultant effects of the bicine and tricine treatments submitted or not to thermal modification, the efficacy against termites was conducted as well as an evaluation of the effect of the different combinations of treatments on the gut symbiotic protists.

2. MATERIALS AND METHODS 2.1 Wood treatment

In this study, wood samples (spruce and beech) were treated with solutions of tricine and bicine

(1 M solutions in water), respectively using a vacuum impregnation method, after which the

samples were allowed to dry under low heating conditions (50 ºC). Once dry conditions had been

achieved, samples were then thermally treated using a modified process to the one described by

Rep et al. (2012), whereby a reduced maximum temperature of 160 ºC was used instead of the

conventional 180 – 230 ºC under vacuum. This was to minimise the thermal degradation of the

bicine and tricine respectively.

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The following combinations of treatments were evaluated against subterranean termites for both wood species: Untreated; Heat treated (HT); Bicine + oven dry; Bicine + oven dry + HT; Tricine + oven dry; Tricine + oven dry + HT.

2.2 Efficacy against subterranean termites

Subterranean termites belonging to the species Reticulitermes grassei Clément were captured in a pine forest, in Sesimbra, Setúbal district of Portugal, and were brought to the laboratory and kept in a conditioned room at 24 ± 1°C and 80 ± 5 % relative humidity. Groups of 150 workers of termites were established in 200 ml glass jars with moistened sand (Fontainebleau sand and water; 4:1 v/v) as substrate. Three replicates (30 x 10 x 10 mm) per treatment were then placed in contact with the termites and the test run for four weeks at the described conditions. Maritime pine test specimens with the same dimensions were also included as internal virulence controls (EN117 2012, Esteves et al. 2017) .

The initial moisture content of the blocks was measured in sets of three additional replicates per treatment and these values were used to determine the theoretical initial dry mass (IDM) of the exposed specimens (in all tests conducted). At the end of the trial the final moisture content was recorded and the mass loss was obtained using the expression: % mass loss = (FDM-IDM)/IDM x 100, where FDM is the oven dry mass of the block at the end of the test. The survival (%) of the termites was also recorded and all wood blocks were graded in terms of termite attack using the scale: 0 = no damage; 1 = attempted attack; 2 = slight damage; 3 = superficial and inner damage; 4 = heavy inner damage.

2.3 Effects on symbiotic protists

The termites used were captured as described above and belonged to the same colony. Termite workers in the 4th instar of development were selected. The trials were established according to a method adapted from a FCBA internal protocol (undated). Groups of fifty termites were exposed to the different wood samples (one for each treatment), and five termites per trial were evaluated for their symbiotic flagellate protist community diversity and abundance after: 24 hours, one week, two weeks, three weeks and four weeks (end of the trials). For flagellate protist counting, the protocol described in Duarte et al. (2017) was followed.

Flagellate protist diversity and abundance was evaluated by direct observation under a microscope; for quantification of flagellate protists, a haemocytometer was used, in accordance with a previously described method, with an adaptation for the calculation of the number of flagellate protists per microliter of hindgut fluid (Duarte et al. in press).

3. RESULTS AND DISCUSSION

3.1 Efficacy against subterranean termites

The average results for survival, mass loss and grade of attack are presented in table 1. The survival of the termites and the mass loss showed some differences between the treatments although a high variability of the results was observed for some treatments.

The durability of thermal modified wood is recognized as low and the results of the present work

are in accordance with that perception. However, bicine and tricine treatments alone had a clear

influence on the survival of the termites. For spruce, it was observed a lower survival rate for

tricine + oven dry and bicine + oven dry + HT, together with a lower mass loss and attack grade,

which may indicate a detrimental effect of those treatments (and also bicine + oven dry) on

termite fitness. For beech, bicine treated wood (both bicine + oven dry and bicine + oven dry +

HT) showed a clear negative effect on termite survival and wood mass loss and attack grade.

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Table 1: Average results of efficacy of treatments against termites

Treatment Wood Survival (%) Mass loss (%) Grade of attack Untreated control Spruce 88.4 (6.3) 11.3 (0.8) 4.0 (0.0)

Beech 82.0 (9.7) 7.7 (0.9) 4.0 (0.0)

M. pine 84.4 (7.4) 8.0 (3.3) 4.0 (0.0)

Heat treated Spruce 90.8 (4.5) 11.8 (2.1) 4.0 (0.0)

Beech 87.2 (2.7) 8.2 (0.8) 4.0 (0.0)

Bicine + oven dry Spruce 64.2 (38.9) 2.6 (1.1) 3.0 (0.0) Beech 38.0 (22.0) 3.7 (0.3) 3.0 (0.0) Bicine + oven dry + HT Spruce 21.1 (18.6) 2.9 (1.6) 2.7 (1.5)

Beech 57.6 (47.0) 4.4 (1.4) 3.0 (0.0)

Tricine + oven dry Spruce 11.3 (17.4) 2.4 (1.2) 3.0 (0.0) Beech 71.4 (17.2) 5.8 (0.3) 3.7 (0.6) Tricine + oven dry + HT Spruce 62.3 (31.2) 10.1 (1.9) 4.0 (0.0)

Beech 79.9 (16.8) 6.2 (0.0) 4.0 (0.0)

3.2 Effects on symbiotic protists

Tricine and bicine had an effect on termite survival and the study of the protist symbionts gave a better insight of the ability of termites to react to unfavorable diet changes. Nine morphotypes were found on untreated termites and variations of these were evaluated for all treatments tested.

Some morphotypes showed to be susceptible to some treatments and for example, n1 was susceptible to all treatments, although tricine seemed to influence positively their abundance.

Morphotype n5 seemed to be responding negatively not only to the treatments, but probably also to the laboratory environment and the trial setting. Morphotype 4 showed to be quite sensitive to any kind of treatment, either with or without bicine and tricine. On the other side, morphotype 6 only showed to be negatively affected by bicine on spruce. Morphotype n7 showed a negative response to beech wood, and to spruce treated with both bicine and tricine (with and without heat treatment). Morphotype n8 abundance decreased for all trials, although the effects were more visible for spruce treated with bicine (and heat-treated wood after bicine treatment) and tricine;

for beech it was more visible for both bicine and tricine (as well as wood heat treated after both bicine and tricine treatment). Similar effects were observed for n9, although for spruce, also heat-treated wood treated previously with tricine also had a visible negative impact on their abundance.

These changes showed termites’ ability of adapting to new conditions, although in some cases,

termite mortality is not avoided despite this adaptation effort.

6 4. CONCLUSION

Bicine and tricine treatments alone had a clear influence on the survival of the termites. The trials showed the plasticity of termite hindgut flagellate protist community to the exposure to different wood treatments, involving two zwitterionic buffers and the heat treatment of two different wood species. The changes in abundances and diversity of the flagellate protist community of the subterranean termites tested are a possible result of termite workers adaptation to the laboratory conditions and new diet, already observed in previous studies (Duarte et al.

2017). These changes prove their ability of adapting to new conditions, although in some cases, termite mortality is not avoided despite this adaptation effort. Severe changes may lead to termite mortality, despite the possible redundancy of some protist species, although ideally each morphotype performs its own role on lignocellulolytic process. These trials were performed in a force-feeding environment; further trials with choice tests should also be pursued.

5. ACKNOWLEDGEMENTS

The work described on the present paper was conducted under LNEC P2I project ConstBio.

SD’s work was supported by the Portuguese Foundation for Science and Technology (FCT) (SFRH/BD/84920/2012). Samples were prepared under a Short Term Scientific Mission funded to Dennis Jones in COST Action FP1407, which is hereby acknowledged. Assistance in preparation and treatment of samples within the STSM was provided by members of the University of Ljubljana, Biotechnical Faculty, Ljubljana, Slovenia (Davor Kržišnik, Miha Hočevar, Miha Humar, Nejc Thelar), without whom this study would not have been possible.

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