In the Name of Allah, Most Gracious, Most Merciful! STUDIES on the EFFECT of WOOD EXTRACTIVES in COMBINATION with PLANT OIL on SUBTERRANEAN TERMITES

In the name of Allah, Most Gracious, Most Merciful! STUDIES ON THE EFFECT OF WOOD EXTRACTIVES IN COMBINATION WITH PLANT OIL ON SUBTERRANEAN TERMITES

BABAR HASSAN M.Sc. (Hons.) Entomology 2006-ag-1738

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF REQUIREMENTS FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY IN ENTOMOLOGY

DEPARTMENT OF ENTOMOLOGY FACULTY OF AGRICULTURE UNIVERSITY OF AGRICULTURE, FAISALABAD PAKISTAN

2017

And when We decreed (Solomon's) death, they had no indication that he was dead until (they saw a termite), a crawler of the earth eating away his staff. And when he fell down, the jinn realized that had they known the unseen, they would not have continued in their humiliating punishment Al-Quran (XXXIV. 13) Declaration

I hereby declare that the contents of the thesis, " Studies on the effect of wood extractives in combination with plant oil on subterranean termites" are product of my own research and no part has been copied from any published source (Except the references, standard mathematical or generic models/ formulates/protocols etc.). I further declare that this work has not been submitted for award of any other diploma/degree. The university may take action if the information provided is found incorrect at any stage. In case of any default the scholar will be proceeded against as per HEC policy.

Babar Hassan

2006-ag-1738

DEDICATED

MY SWEET LATE MOTHER AND MY LOVING FATHER

Whose love is more precious than pearls and diamonds, by the virtue of whose prays, I have been able to reach at this high position ACKNOWLEDGEMENTS

Alhamdullilah, all praises to Allah for the strengths and His blessings in completing this thesis. All praises be to the Holy Prophet Muhammad (Peace be upon him), the city of knowledge, the illuminating torch and the rescuer of humanity from going astray. Without the help of the people, to whom I wish to express my gratitude this thesis could not be completed. First and foremost, I would like to thank Prof. Dr. Sohail Ahmed, Department of Entomology University of Agriculture, Faisalabad, for his sympathetic attitudes, fatherly behavior, animate directions, observant pursuit, scholarly criticism, cheering perspective and enlightened supervision. Dr. Muhammad Altaf Sabri and Prof. Dr. Shahbaz Talib Sahi, as member of supervisory committee are acknowledged for their helpful advice and suggestions. I would also like to express my special appreciation to foreign advisors, Carol Clausen, Mark, E. Mankowski and Grant Kirker, USDA-FS, Forest Products Laboratory (USA). They have been a tremendous mentors for me. I would like to thank all of them for encouraging during research and for allowing me to grow as a research scientist. Their advices on both the research as well as on learning career have been priceless. I am also thankful to Dr. Muhammad Misbah-ul-Haq (NIFA) and Dr. Khalid Zamir Rasib (FC College University) for providing me space for laboratory and field experiments against Heterotermes indicola. I would like to acknowledge and thank Higher Education Commission of Pakistan (HEC) and USDA- FS International Program, who granted me fellowship and PhD Indigenous Scholarship to conduct research at USDA-FS, Forest Products Laboratory (USA) and the Nuclear Institute for Food and Agriculture (NIFA), Peshawar (Pakistan). I am also thankful to Craig Bell, USFS FPL Starkville, MS for wood block and shaving preparation, Amy Bishell for decay tests and Dr. Hamid Borazjani, Mississippi State University, for use of his laboratory facilities. My deepest gratitude goes to my family for their unflagging love and support throughout my life; this dissertation is simply impossible without them. I want to acknowledge my brothers Anser Abbas and Qaiser Abbas and my sisters for their love, sincerity and cheerful jokes, which make possible my stay here in this University. My gratitude will remain incomplete if I do not mention the contribution of the sincere friends, Rahat Afza, Asad Ahmad, Basit Naseer, Nadeem Ahmad, Tahmmal Huassain, Ali Anwar and laboratory fellows, Ali, Nasir, Awias, Muzammal, Bilal and Uzair Saleem for their great company and advices. Thanks to all.

BABAR HASSAN CONTENTS

CHAPTER TITLE PAGES

ACKNOWLEDGMENTS vii

CONTENTS viii

LIST OF TABLES ix

LIST OF FIGURES xiii

LIST OF APPENDICES xiv

ABSTRACT xv

01 INTRODUCTION 01

02 REVIEW OF LITERATURE 07

03 MATERIALS & METHODS 27

04 RESULTS 49

05 DISCUSSIONS 145

06 SUMMARY 159

LITERATURE CITED 163

APPENDICES 194 LIST OF TABLES

Sr. No. Title of the Table Page No. 3.1. Properties of durable woods selected for current study 29 3.2. Indicators to Evaluate Antifeedant Activity of extractives (Antifeedancy %) 33 3.3. Termite rating scheme (AWPA E-1, E-7, E-26) 47 3.4. Decay rating scheme (AWPA E-10, E-7) 48 4.1. Yield of heartwood extractives from durable woods extracted with ethanol: 50 toluene (2:1) 4.2.1. Median lethal concentrations (LC50s) of filter paper treated with four type of 52 heartwood extractives against R. flavipes and H. indicola 4.2.2. Mean weight loss, area loss and mortality of R. flavipes and H. indicola after 53 toxicity test on filter paper treated with T. grandis heartwood extractives 4.2.3. Mean weight loss, area loss and mortality of R. flavipes and H. indicola after 54 toxicity tests on filter paper treated with D. sissoo heartwood extractives 4.2.4. Mean weight loss, area loss and mortality of R. flavipes and H. indicola after 55 toxicity tests on filter paper treated with C. deodara heartwood extractives 4.2.5. Mean weight loss, area loss and mortality of R. flavipes and H. indicola after 56 toxicity tests on filter paper treated with P. roxburghii heartwood extractives 4.2.6. Mean mortality (%) of R. flavipes and H. indicola after toxicity tests on 57 filter paper treated with four heartwood extractives. 4.2.7. Mean weight loss (%) of filter papers after feeding of R. flavipes and H. 57 indicola treated with four heartwood extractives 4.2.8. Mean area loss (%) of filter papers after feeding of R. flavipes and treated 58 with four heartwood extractives 4.2.9. Antifeedant activity of T. grandis heart wood extractives against R. flavipes 67 and H. indicola 4.2.10. Antifeedant activity of D. sissoo heart wood extractives against R. flavipes 67 and H. indicola 4.2.11. Antifeedant activity of C. deodara heartwood extractives against R. flavipes 68 and H. indicola 4.2.12. Antifeedant activity of P. roxburghii heartwood extractives against R. 68 flavipes and H. indicola 4.2.13. Antifeedant activity of four heartwood extractives against R. flavipes and H. 69 indicola 4.2.14. Mean GSTs activity of H. indicola exposed to IC50s concentrations of BHT, 72 quercetin and heartwood extractives 4.2.15. Mean ESTs activity of H. indicola exposed to IC50s concentrations of BHT, 72 quercetin and heartwood extractives 4.2.16. Mean CATs activity of H. indicola exposed to IC50s concentrations of BHT, 72 quercetin and heartwood extractives 4.3.1 Effect of T. grandis heartwood extractives on total population of gut 75 protozoans of R. flavipes and H. indicola 4.3.2. Effect of D. sissoo heartwood extractives on total population of gut 76 protozoans of R. flavipes and H. indicola

4.3.3. Effect of C. deodara heartwood extractives on total population of gut 77 protozoans of R. flavipes and H. indicola 4.3.4. Effect of P. roxburghii heartwood extractives on total population of gut 78 protozoans of R. flavipes and H. indicola 4.3.5. Percentage reduction of total population of gut protozoans of R. flavipes and 79 H. indicola after feeding on filter paper treated with four durable heartwood extractives 4.3.6. Number of sequences, OTUs, and percent coverage for each sample by 81 treatment 4.3.7. Sequence similarity analysis of isolated bacterial strains from H. indicola 83 with the available databases in NCBI 4.4.1. Mean weight loss (%) for extractive free and un-extracted blocks of T. 86 grandis and mortality of R. flavipes and H. indicola under Choice and No- choice tests 4.4.2. Mean weight loss (%) for extractive free and un-extracted blocks of D. 86 sissoo and mortality of R. flavipes and H. indicola under Choice and No- choice tests 4.4.3. Mean weight loss (%) for extractive free and un-extracted blocks of C. 88 deodara and mortality of R. flavipes and H. indicola under Choice and No- choice tests 4.4.4. Mean weight loss (%) for extractive free and un-extracted blocks of P. 88 roxburghii and mortality of R. flavipes and H. indicola under choice and no- choice tests 4.4.5. Average rating, retention, mortality and weight losses of SYP and CW 90 treated with T. grandis extractives after feeding of R. flavipes 4.4.6. Average rating, retention, mortality and weight losses of SYP and CW 92 treated with D. sissoo extractives after feeding of R. flavipes 4.4.7. Average rating, retention, mortality and weight losses of SYP and CW 93 treated with C. deodara extractives after feeding of R. flavipes 4.4.8. Average rating, retention, mortality and weight losses of SYP and CW 94 treated with P. roxburghii extractives after feeding of R. flavipes 4.4.9. Average rating, retention, mortality and weight losses of SYP and CW 96 treated with T. grandis extractives after feeding of H. indicola 4.4.10. Average rating, retention, mortality and weight losses of SYP and CW 97 treated with D. sissoo extractives after feeding of H. indicola 4.4.11. Average rating, retention, mortality and weight losses of SYP and CW 99 treated with C. deodara extractives after feeding of H. indicola 4.4.12. Average rating, retention, mortality and weight losses of SYP and CW 100 treated with P. roxburghii extractives after feeding of H. indicola 4.4.13. Weight losses of SYP and CW treated with four durable wood extractives 101 4.4.14. Mortality of termites after feeding of SYP and CW treated with four 102 durable wood extractives 4.4.15. Mean mortality (% ) of R. flavipes and weight losses in SYP and CW woods 104 after leaching and un-leaching tests of extractives 4.4.16. Mean rating of leached and un-leached SYP and CW after feeding of R. 105 flavipes

4.4.17. Mean mortality (%) of H. indicola and weight losses in SYP and CW woods 107 after leaching and un-leaching tests of extractives 4.4.18. Mean rating of leached and un-leached SYP and CW after feeding of H. 108 indicola 4.4.19. Weight losses, termites’ mortality and rating of SYP and CW treated with 110 T. grandis extractives and linseed oil after feeding of R. flavipes 4.4.20. Weight losses, termites’ mortality and rating of SYP and CW treated with 111 D. sissoo extractives and linseed oil after feeding of R. flavipes 4.4.21. Weight losses, termites’ mortality and rating of SYP and CW treated with 112 C. deodara extractives and linseed oil after feeding of R. flavipes 4.4.22. Weight losses, termites’ mortality and rating of SYP and CW treated with 113 P. roxburghii extractives and linseed oil after feeding of R. flavipes 4.4.23. Weight losses, termites’ mortality and rating of SYP and CW treated with 115 T. grandis extractives and linseed oil after feeding of H. indicola 4.4.24. Weight losses, termites’ mortality and rating of SYP and CW treated with 116 D. sissoo extractives and linseed oil after feeding of H. indicola 4.4.25. Weight losses, termites’ mortality and rating of SYP and CW treated with 117 C. deodara extractives and linseed oil after feeding of H. indicola 4.4.26. Weight losses, termites’ mortality and rating of SYP and CW treated with 118 P. roxburghii extractives and linseed oil after feeding of H. indicola 4.4.27. Mortality of termites after feeding on SYP and CW treated with extractives 119 of four durable woods and linseed oil 4.4.28. Comparison of weight losses of non-durable woods treated with four durable 120 wood extractives and oil at different concentration levels 4.4.29. Weight losses of SYP and CW treated with four durable wood extractives 120 and oil at different concentration levels 4.4.30. Mean weight losses of extracted and un-extracted softwood species exposed 122 to P. placenta in a 12 week soil bottle assay 4.4.31. Mean weight losses of extracted and un-extracted hardwood species exposed 122 to T. versicolor in a 12 week soil bottle assay 4.5.1. Five largest components from GC-MS analysis of solvent extracted T. 127 grandis 4.5.2. Five largest components from GC-MS analysis of solvent extracted D. 128 sissoo. 4.5.3. Five largest components from GC-MS analysis of solvent extracted C. 130 deodara 4.5.4. Five largest components from GC-MS analysis of solvent extracted P. 131 roxburghii 4.6.1. Average decay and termite resistance rating of treated SYP chunks for field 134 tests located at MS USA 4.6.2. Average decay and termite resistance rating of treated SYP chunks for 135 field tests located at Lahore Pakistan 4.6.3. Average decay and termite resistance rating of treated CW chunks for field 136 tests located at MS USA

4.6.4. Average decay and termite resistance rating of treated CW chunks for field 137 tests located at Lahore Pakistan 4.6.5. Average decay and termite resistance rating of heartwoods field chunks 138 located at (MS) USA and (Lahore) Pakistan 4.6.6. Average decay and termite resistance rating of treated SYP stakes for field 140 tests located at MS USA 4.6.7. Average decay and termite resistance rating of treated SYP satkes for field 141 tests located at Lahore Pakistan 4.6.8. Average decay and termite resistance rating of treated CW stakes for field 142 tests located at MS USA 4.6.9. Average decay and termite resistance rating of treated CW stakes for field 143 tests located at Lahore Pakistan 4.6.10. Average decay and termite resistance rating of heartwoods field stakes 144 located at (MS) USA and (Lahore) Pakistan LIST OF FIGURES

Sr. No. Title of Figures Page No. 3.1 Steps for preparation of heartwood extractives at Department of 30 Sustainable Bio products, MSU 3.2 Methods for collection of R. flavipes and H. indicola 31 3.3 Filter paper test set up (a) and repellency test set up (b) 33 3.4 Gel electrophoresis for DNA quality analysis (Photo sent by Molcare, 39 University of Agriculture, Faisalabad) 3.5 Vacuum (a), pressure chamber (b) and test set up (c) according to AWPA 41 E-1 3.6 Orbital shaker for leaching (a) and test set up (b) according to AWPA E- 42 1 3.7 Ground proximity test (AWPA- E26) set up in USA (a) and Pakistan (b) 46 3.8 Field stake test (AWPA- E7) set up in USA (a) and Pakistan (b) 47 4.2.1 Repellent activities of T. grandis heartwood extractives against R. 60 flavipes 4.2.2 Repellent activities of T. grandis heartwood extractives against H. 60 indicola 4.2.3 Repellent activities of D. sissoo heartwood extractives against R. flavipes 62 4.2.4 Repellent activities of D. sissoo heartwood extractives against H. indicola 62 4.2.5 Repellent activities of C. deodara heartwood extractives against R. 64 flavipes 4.2.6 Repellent activities of C. deodara heartwood extractives against H. 64 indicola 4.2.7 Repellent activities of P. roxburghii heartwood extractives against R. 65 flavipes 4.2.8 Repellent activities of P. roxburghii heartwood extractives against H. 65 indicola 4.2.9 Effect of heartwood extractives on DPPH radical scavenging activity (% 71 inhibition); Conc. = concentration 4.3.1 Inverse Simpson diversity index of bacterial communities of H. indicola 81 after feeding on treated filter paper 4.3.2 Major bacterial phyla identified in the guts of H. indicola separated by 82 treatments 4.4.1 Mean weight losses of softwood blocks treated with extractives of 123 durable wood species at 2.5, 5 and 10 mg ml-1exposed to Postia placenta for 12 weeks in a soil bottle test 4.4.2 Mean weight losses of hardwood blocks treated with extractives of 124 durable wood species at 2.5, 5 and 10 mg ml-1exposed to Trametes versicolor for 12 weeks in a soil bottle test 4.5.1 Chromatogram from GC-MS analysis of solvent extracted T. grandis 127 4.5.2 Chromatogram from GC-MS analysis of solvent extracted D. sissoo 128 4.5.3 Chromatogram from GC-MS analysis of solvent extracted C. deodara 130 4.5.4 Chromatogram from GC-MS analysis of solvent extracted P. roxburghii 131 LIST OF APPENDICES

Sr. No. Title Page No. 1 Extractive free (a) and un- extracted (b) heartwood blocks after feeding 194 of R. flavipes under choice and No-choice test 2 Heartwood blocks after feeding of H. indicola on extracted free (a) and 195 un-extracted (b) woods under choice and No-choice tests 3 Treatment plant used during Vacuum pressure impregnation of wood at 195 FPL, USA.

4 Galleries of termites on ground proximity test after 12 months (A), 196-197 Solvent treated chunks attacked by termites (B), Heartwoods and Positive control (C) at Pakistan site. 5 Heartwoods, SYP and CW treated with solvent attacked by termites at 197 Pakistani site in field stake test after 12 months 6 Protozoans species found in guts of R. flavipes 198 7 Protozoans species found in guts of H. indicola 199 4.1 ANOVA for % Mortality of termites after feeding on treated filter paper 201 4.2 ANOVA for % weight loss of filter paper after feeding of termites for 15 201 days 4.3 ANOVA for % area loss of filter paper after feeding of termites for 15 201 days 4.4 ANOVA for Repellent activities of four heartwood extractives against 202 termites 4.5 ANOVA for antifeedant activity of four heart wood extractives against R. 202 flavipes and H. indicola 4.6 ANOVA for % reduction of protozoans in guts of termites after feeding 203 on filter paper treated with heartwood extractives 4.7 ANOVA for % weight losses of SYP and CW treated with four heartwood 203 extractives after feeding of R. flavipes and H. indicola 4.8 ANOVA for % mortalities of termites after feeding on SYP and CW 204 treated with four heartwood extractives 4.9 ANOVA for % weight losses of SYP and CW treated with four heartwood 205 extractives + linseed oil after feeding of R. flavipes and H. indicola 4.10 ANOVA for % mortalities of termites after feeding on SYP and CW 205 treated with four heartwood extractives + linseed oil 4.11 16S rRNA sequence of Enterobacter cloacae (HE-1) isolated from the 206 gut of H. indicola (A) and its evolutionary relationships with taxa (B) 4.12 16S rRNA sequence of Bacillus cereus (HE-2) isolated from the gut of 207 H. indicola (A) and its evolutionary relationships with taxa (B) 4.13 16S rRNA sequence of Pseudomonas aeruginosa (HE-7) isolated from 208 the gut of H. indicola (A) and its evolutionary relationships with taxa (B) 4.14 16S rRNA sequence of Pantoea agglomerans (HE-8) isolated from the 210 gut of H. indicola (A) and its evolutionary relationships with taxa (B) 4.15 16S rRNA sequence of Un-cultured (HE-12) isolated from the gut of H. 211 indicola (A) and its evolutionary relationships with taxa (B) ABSTRACT

The screening of extractives from termites’ resistant woods and their utilization as preservatives was main focus of the studies; methodologies and results obtained are being presented in this piece of manuscript.

Extractives of four durable wood species, Tectona grandis, Dalbergia sissoo, Cedrus deodara and Pinus roxburghii were investigated for antitermitic activities against Reticulitermes flavipes (Kollar) and Heterotermes indicola (Wasmann). Heartwood extractives were removed from wood shavings by Soxhlet extraction with ethanol: toluene as solvent part of this procedure /system. Yields of extractives of these durable woods were 5.51, 9.11, 9.67 and 9.40 % of dry wood shavings, respectively. Results of Filter paper bioassays showed that there was concentration dependent feeding response and mortalities of termites on treated filter papers except D. sissoo which showed non-linear dependency on concentrations in terms of weight loss of filter papers and mortality of R. flavipes. Maximum repellent activities were observed on C. deodara heartwood extractive but there was non-significant difference among four types of extractives at the highest concentration. P. roxburghii extractives showed no significant repellent activities against two species of termites. In DPPH radical scavenging assay, maximum antioxidant activity was observed in D. sissoo extractives with lowest IC50 -1 (28.83 µg ml ) among the evaluated heartwoods. GC-MS results also confirmed that D. sissoo heartwood extractives contained majority of phenolic compounds, which had significantly high antioxidant activity, irrespective of the concentrations. Activities of Glutathione S- transferases and esterases were significantly reduced in the guts of workers of H. indicola fed on filter papers treated with extractives and positive controls (BHT, Quercetin). However, all extractives did not significantly reduce the activity of catalases. Maximum reduction of gut protozoans in R. flavipes and H. indicola was 45 and 82%, respectively after feeding on extractives treated filter papers. 16S rRNA bacterial gene was amplified to examine the abundance of major taxonomic groups of bacterial community of H. indicola in treated versus control groups. Treatments did not appear to show any major differences regarding phyla of gut bacteria.

Extracted and un-extracted heartwood blocks of each durable species were subjected to choice and no-choice feeding tests. Increased wood weight loss due to termite feeding on extractive free wood was observed and effect of feeding on termite mortality was significantly different on un-extracted and extractive free woods except T. grandis, which showed non-significant difference in weight losses and mortality of R. flavipes. In another test, sapwood blocks (19×19×19 mm) of SYP and CW were vacuum-pressure impregnated with different concentrations of extractives alone and in combination with linseed oil to observe feeding response and mortality of both the termite species after 28 days of exposure to the wood blocks. Overall, T. grandis and D. sissoo extractives treated SYP and CW woods had minimum weight losses but at maximum concentration (10 mg ml-1) weight losses were non-significantly different on both the SYP and CW treated woods in comparison to those treated with other heartwood extractives. Similarly at the highest concentration, all extractives performed non-significantly different from one another in terms of mortalities of both the termite specie while extractives combination with linseed oil provided significant protection to SYP and CW against termites and correspondingly in the same manner caused

mortalities in two termite’s species. There were non- significant differences in weight losses of SYP and CW treated with extractives of D. sissoo and T. grandis after leaching test (AWPA E-11) but reverse was true with C. deodara and P. roxburghii extractives treated woods. In laboratory fungus decay test, non-durable species (SYP and CW) were treated with extractives from durable species and decay resistance in susceptible woods was improved against either brown or white rot fungi, especially by D. sissoo extractives.

GC-MS analyses yielded 96, 52, 147 and 12 compounds from T. grandis, D. sissoo, C. deodara and P. roxburghii, respectively. GC-MS analysis showed high percentage of 2- methyl- 9,10 anthracenedione and Squalene, Trismethoxy resveratrol and 1,3-Diamino-8-n-butyl- 5,6- dihydrobenzoquinazoline, (E)-Atlantone and Di-epi-alpha-cedrene, 1,2,3,4-tetrahydro- 5,8- dimethyl-Acridin-9-amine and 2,3-dihydro-5,7-dihydroxy-2-phenyl-4H-1-Benzopyran- 4-one, respectively T. grandis, D. sissoo, C. deodara and P. roxburghii extractives.2-3 compounds which have been implicated in the increased termite’s mortality observed as they had been noted to be fungicidal or insecticidal

Finally, field tests were carried out according to AWPA E-7 and E-26 at Lahore (Pakistan) and Mississippi (USA). Control treatments comprising of solvent, oil treated blocks and stakes at Lahore site (Pakistan) were severely attacked by the termites (H. indicola) and partially decayed after a lapse of time period of 12 months. Linseed oil treated stakes of SYP and CW showed discoloration with average rating of 9.5 due to fungal decay at USA. P. roxburghii extractive stakes were partially deteriorated by the fungi at both sites.

CHAPTER ONE INTRODUCTION

An exclusive capability to live eusocially and cellulose digestion has permitted the termites to dominate surroundings in which they are present, at least from a biomass stand point (Potter, 2011). Termites are found in all continents, excluding Antarctica; the greatest diversity being in tropical and sub-tropical regions. Nearly 3,000 termite species, belonging to seven families and 280 genera, have been described but only 80 species pose a threat to wooden structures. Among these, subterranean termites accounted for 38 species (Eggleton, 2000; Lee and Chung, 2003; Brune, 2014) and are the major pest of woods and their products because of their cryptobiotic nature. Globally, termites invade every place where wooden structures are erected for various purposes and take a toll of billions of dollars annually (Kuswanto et al., 2015).

Wood being a biological material is readily degraded by many deteriorating agents including termites and fungi (Langrish et al., 1993; Schultz and Nicholas, 2000). The susceptible woods vulnerable to these agents must be preserved with the chemicals to enhance their service life by decreasing and/or avoiding attack of timber deteriorating agents (Evans, 2003). Wood is mostly preserved with synthetic inorganic and organic chemicals such as chromated arsenicals, propiconazole and pentachlorophenol. These synthetic compounds are costly and some are banned from several countries such as CCA and creosote. These are also frequently injurious to workforces and environments. Worse still, these wood preservatives are not easy to depollute, cannot be degraded to harmless products easily and are not readily available in developing countries. Copper based preservatives are also poor inhibitors of wood degrading fungi (Lebow et al., 2000; Green and Clausen, 2005; Arango et al., 2006).

Despite the vulnerability of woods to fungi and termites infestation, it is generally accepted that heartwoods of the several tree species are naturally resistant to biodegradation. This resistance or durability is generally due to the buildup of extractives in heartwood (Kityo and Plumptre, 1997; Kadir et al., 2014). Extractives are abundant in heartwood as compared to sapwood, latter are prone to termites’ attack due to this deficiency which predicts important role these extractives can play in natural resistance of wood species (Hinterstoisser et al., 2000). Natural resistance of woods is extremely complex phenomenon, additional factors of

1 wood density, lignin contents etc. may also aid in biocide activity of extractives (Schultz and Nicholas, 2002). Natural resistant woods were tuned into susceptible to termites’ attack after removal of extractives in many studies (Ohmura, 2000; Taylor et al., 2002; Oliveira et al., 2010) and relationships between wood properties and extractives contents have also been evaluated (Schultz et al., 1990; Reyes-Chilpa et al., 1998; Chang et al., 1999; Morimoto et al., 2006). Non-durable woods such as cotton (Populus deltoides) and Southern yellow pine (Pinus taeda) have been converted into durable by introduction of extractives from Osage orange (Maclura pomifera) and black locust (Robinia pseudoacacia) (Smith et al., 1989). Methanol extracts of redwood, Intsia bijuga (Kamden, 1994) and several tropical woods have been studied for their role in improving wood durability (Kennedy et al., 1995; Van Acker et al., 1999; Kennedy and Powell, 2000; Kennedy et al., 2000; Powell et al., 2000; Onuorah, 2002; Lukmandaru and Ogiyama, 2005; Tascioglu et al., 2012; Dungani et al., 2012; Kirker et al., 2013; Kadir et al., 2014).

Extractives are non-structural wood components and generally signify a minor part in woods. Many families of tree species share similar nature of extractives, however, differences in composition of these extractives among closely related tree species are also widespread. Additionally, various parts of a tree such as needles, stem, roots, bark and branches vary significantly with respect to quantity and composition of the extractives. (Hillis, 1978; Walker, 1993; Haygreen and Bowyer, 1996). Wood extractives may account for 3-30% by weight depending on wood type. Cellular differentiation of extractives exists within a tree; same extractives in different cell types or different extractives in similar cell types in different parts of a tree have been reported (Desch and Dinwoodie, 1996). There is, however, an overall reduction in extractives contents with an increase in tree height and from the pith to bark (Walker, 1993).

Chemically wood extractives range from low to high molecular weight substances and from simple hydrocarbons to complex polyphenolic structures (Walker, 1993). Generally, extractives are copious and chemically diverse in heartwoods of both hard and softwoods, than sapwoods with one exception of Pinus sp. in which same extractives are common to both types. Heartwood extractives are found in the highest quantity in innermost growth rings adjacent to pith (Lloyd, 1978), particularly in butt log of mature trees.

Lignocellulosic tissues of both hard and softwoods contain a vast diversity of triglycerides, steryl esters, fatty acids, sterols, neutral compounds (fatty alcohols), sterols, phenolic compounds [tannins (Fava et al., 2006), flavonoids (Ohmura et al., 2000; Chen et al., 2004; Morimoto et al., 2006; Sirmah et al., 2009)] and other compounds such as alkaloids (Kawaguchi et al., 1989), quinones (Ganapaty et al., 2004. Fatty acids and alcohols similar to softwoods have been reported from hardwoods (Fengel and Wegener, 1984).

Further, sapwoods of softwood pines possess high amount of resins and terpenoids in the inner zone due to a process of enrichment via transverse resin canals (Harris and Sand, 1965), while resin acids preponderate in heartwood of pines and comprise 70-80% of total extractives. Pine sapwoods have equal amount of resin and fatty acids, but in heartwood which in turn have flavonoids (C6-C3-C6) and stilbene (C6-C2-C6) compounds as well (Walker, 1993).

Terpenoids extractives in hardwood of Cinnamonum camphora comprise of monoterpenoids. Diterpenoid compounds occur in hardwoods occasionally. Diterpenoid, phenol ferruginol, contributes to durability of Cryptomeria japonica and Dacrydium colensoi. Triterpenoid compounds (C-30 compounds) occur frequently in hardwoods either as alcohols or acids, for example, alcohol betulin and methyl ester of acetylated betulininic acid from Betula species. Triterpene acids are often present in Quercus and Terminalia species while sterols such as ß-sitosterol are present in many other hardwoods.

A wide variety of highly hydroxylated flavonoids including flavones, flavanols, flavanones, isoflavones, and chalcones from hardwoods have been isolated. Polyphenols, i.e., stilbenes such as pinosylvin, in Pinus sp. are considered to be responsible for the better durability of pine heartwood as compared to its sapwood. Dihydroquercetin is responsible for durability of Pseudotsuga menziesi heartwood whereas tectoquinone, 2-methyl anthraquinone, is responsible for stability of Tectona grandis. Cedrol contributes resilience in Chinese fir, Cunninghamia lanceolata (Huang et al., 2006).

Extractives are usually removed from finely-ground woods by Soxhlet extraction with solvents for time periods ranging from 4-18 hours. High volatile extractives can be separated by water distillation. They are mainly composed of monoterpenes and other volatile terpenes, terpenoids as well as of many different low molecular compounds. Techniques such as gas chromatography, mass spectrometry and H1 and C13 nuclear magnetic resonance (NMR)

3 spectroscopy are often used for identification and structural elucidation of the extractives (Walker, 1993; Kirker et al., 2013).

Biological functions of wood extractives include antifeedant, antioxidant, antiviral, bacteriacide, cytotoxin, fungicides etc. (Walker, 1993; Ragon et al., 2008). Heartwood extractives can protect the woods by direct toxicity to the termites as well (Walker, 1993; Hinterstoisser et al., 2000; Schultz and Nicholas, 2002).

The use of naturally produced extractives in wood for the management of termites and borers has been premeditated in the field of wood protection and preservation. Recent studies have implicated the role of extractives as toxicant to gut symbionts and bacterial communities of the termites and to fungi as well, in addition to being directly toxic and repellent against several termites species (Thevenon et al., 2001; Lukmandaru and Ogiyama, 2005; Asaomoh et al., 2007; Ragon et al., 2008; Dungani et al., 2012; Tascioglu et al., 2012; Kirker et al., 2013; Kadir et al., 2014, 2015).

Interest in natural wood preservatives has augmented the significance for testing oils isolated from foliage and seeds of several plant species as a wood protectant (Fatima and Morell, 2015). Oils have such similar action on insects and other organisms as exhibited by extractives. Oils are antimicrobial, antioxidant, antifeedant and repellent and a number of vegetable oils have been tested against termites. One property of oils, besides being toxic, was their ability to transfer toxicant deep into woods for protection against termites and fungi (Ahmed et al., 2013, 2014; Fatima and Morrell, 2015). Several reports in scientific literature illustrate their efficacy against termites.

Linseed oil extracted from ripened seeds of flax plant has been extensively investigated for wood protection against several wood destroying agents including fungi and termites (Fatima and Morrell, 2015). Boiled linseed oil, rape oil and three modified waxes were very effective against decay fungi during laboratory testing (Treu et al., 2004). Several linseed oil treated non-durable woods with high retention performed well against decay in both ground proximity and field stake tests (Edlund and Jermer, 2007). Beech wood and Norway spruce treated with Tung and linseed oils were protected against brown and white rot fungi (Humar and Lesar, 2012, 2013). Mixtures of linseed oil with andiroba, copaiba and mineral oil gave the promising results as wood protectant against cellulolytic fungi (Silveira et al., 2012). Still

4 other studies have revealed the wood protectant potential of linseed oil. Linseed was also used to increase the retention of boron in wood and increases the resistance of wood against termites (Lyon et al., 2007). Linseed oil was found to be a good solvent for pentachlorophenol owing to excellent drying properties and provided adequate penetration to the wood (Fatima and Morell, 2015). Epoxidized linseed oil treatment significantly reduced the water absorption and improved the durability of scot pine wood treated with bio-oil against R. flavipes (Temiz et al., 2013).

Oils derived from the Kukui nut have proved to be feeding deterrent and had good antitermitic properties (Nakayama and Osbrink, 2010). Neem oil with the most toxic ingredient, azadirachtin, is used as insect sterilant, antifeedant and repellent, and has fairly good biocide potential against termites (Achio et al., 2012; Adebawo et al., 2015). Cashew nut shell liquid and neem oil in combination with copper were very effectives against decay and termites. Cinnamon, citrus peel and Tung seed oils have recently been explored as potential wood protectant against termites and fungi (Laredo et al., 2015). Previously inexpensive and locally available plant oils in Pakistan such as rape, soya, linseed, castor bean, sunﬂower etc. have been verified for their ability to protect woods against several species of subterranean termites when compared to control treatments (Ahmed et al., 2014).

Retrospectively, research on plant oils and wood extractives has been amply studied for their potential as preservatives. It is generally assumed that they may emerge as partial solutions to control wood deteriorating organisms such as fungi, bacteria and termites. However, current concern to avoid the use of toxic chemicals and develop new technologies based on low environmental impact agents on sustainable principles in each pest control program, an anticipated state-of-the-art adjustment to heartwood extractives can still offer further investigation on potential to develop stable and effective molecules for wood preservation.

The definitive applied objective of current studies was screening of heartwood extractives from T. grandis, D. sissoo, C. deodara, P. roxburghii and their usefulness as termiticide on SYP (Southern Yellow Pine) and CWs (Cotton wood). Moreover, improvement in delivery of extractives in the presence of oils was another objective to develop logical complete wood protection program.

To achieve this objective, the studies proposed herein are as follows:-

 Filter paper bioassay for the determination of concentration dependent repellency, antifeedancy and mortality of termites  DPPH radical scavenging assay to determine the antioxidant activity of extractives  Choice and no choice test on extractive-free wood  Effect of wood extractive on total population of termite’s gut symbionts  Termite bioassay on southern yellow pine and cotton wood pressure treated with extractives and linseed oil  Determination of leach resistance of wood impregnated with wood extractives  Characterization of extractives by GC/MS  Evaluation of extractives as wood preservatives using ground proximity test  Evaluation of extractives as wood preservatives using filed stake test

CHAPTER TWO REVIEW OF LITERATURE

2.1. Subterranean termites as pest of wood

Evolutionary confirmation from provisional termite species, Mastotermes darwiniensis, shows that termites ascended from highly adapted and wood-eating social cockroaches and now these are re-classified as Blattodea. Presently, economically important termite species as pests in agriculture, forestry and urban ecosystems constitute 6.1% of recorded species. These termite pests damage man-made wooden structures and wood products worth $22–40 billion worldwide, though approximations vary significantly by the regions. Economic losses due to termites are conservatively estimated at $11 billion in USA, $4.0 billion in Australia, and $35 million per year in India (Su, 2002; Forsythe, 2004; Ghaly and Edwards, 2011).

Cost of control of termites’ damage to household wooden structures, respectively, in Japan, Indonesia and Malaysia have been estimated to tune of $0.8-1.0 billion, $6-7 million and 10-12 million annually (Tsunoda, 2005; Yeoh and Lee, 2007; Nandika, 2014). China represented the worst case of damage ($289 million annually) of subterranean termites and witnessed destruction of 80% buildings in Guangdong province (Zhong and Liu, 2002). In Pakistan, termites cause significant economic damage to wooden structures in rural and urban areas but the annual cost associated with termite damage has not been estimated.

The economically important termite species can be categorized into three ecological groups: subterranean termites, damp wood termites and dry wood termites. Subterranean species differ from dry- and damp-wood termite species in a way that they need interaction with either soil or moisture. Out of three groups, subterranean termites account for at least 80% of the losses (Su and Scheffrahn, 1990). Subterranean termite pests constitute 38 species as serious threats. Several major termite pest species belong to Genera; Odontotermes Coptotermes, Heterotermes and Reticulitermes. Rhinotermitidae is one of the most economically important family as huge cost to prevent losses ($3 billion per annum) is associated with these termites (Su, 2002).

Termites have become an object of public interest under recent urbanization and promotion of wood structures in household after famous earthquake, in the year 2005, in Pakistan. Thirteen species of termites out of pool of fifty were identiﬁed as pests of constructions in rural and urban areas of Pakistan, which had caused economic losses. Microtermes mycophagus, Odontotermes guptai, Odontotermes obesus, Heterotermes indicola and Microtermes obesi, (Akhter, 1983; Manzoor and Mir, 2010), Coptotermes heimi, Microtermes unicolor were previously recorded as the most dreaded termite species damaging woods in forests and residential areas (Manzoor et al., 2011). Literature on nature and extent of wood damage and its prevention illustrates Odontotermes obesus as the most damaging termite in Pakistan (Ahmed et al., 2004, 2006, 2008, 2014; Fatima et al., 2015). Microcerotermes championi has recently invaded vast residential areas in Lahore city and has become a menace. Wood damaged by the subterranean termites is frequently invisible for the reason that the external surface generally must be detached to see impairment (Koehler and Tucker, 2003). The terrestrial environment in subtropics and tropics has been witnessing considerable degradation and impairment of woods and other cellulosic materials by the termites (Peralta et al., 2003, 2004).

2.2. Conventional control methods

A strategy called “Termites Exclusion Program” currently being advocated include physical obstacles to entry, treatment of timbers and structural voids, pressure treated wood, soil termiticides and baiting systems (Lewis, 1997; Hu, 2005; Huang et al., 2006; Mulrooney et al., 2006; Mulrooney et al., 2007; Horwood et al., 2012; Koehler et al., 2011; Eger et al., 2012; Jordan et al., 2013). Biological control of termites has been explored, in addition, but was either unsuccessful or impractical (Chouvenc et al., 2011).

Physical methods mainly focus on increasing the difficulty for termites to access the wood. For example, Behr et al. (1972) demonstrated an inverse relationship between amount consumed by R. flavipes and the specific gravity and hardness of wood; constructions made of high-density wood can influence termites’ ability to fragment the wood mechanically. Typical physical method also includes proper management of the moisture in wood which determine termite’s preference on woods.

Soil termiticides provide leading method of termite exclusion and deterrence. Soluble concentrates of termiticides are mostly applied pre- or post-construction barriers against subterranean termites (Su and Scheffrahn, 1998). The method of application of soil termiticides has not changed much what have been used for several decades. These are applied after mixing with water as vertical and horizontal treatments. All non-repellant soil termiticides have dual qualities of efficacy and residual effect, but a bit expensive (Wagner et al., 2003). Another drawback to use of liquid termiticide is concern over environmental impact (Potter et al., 2001; Potter and Hillery, 2001; Verkerk and Bravery, 2001). Though prevalent liquid termiticides have lower mammalian toxicity than organochlorine insecticides but regulatory/legislative measures against environmental deterioration now place serious constraints on the use of such liquid termiticides.

Inorganic substances like chromated copper arsenate (CCA) has been the chemical of choice since 1970. It is no longer used for residential uses since 2004 due to a voluntary industrial support to phase it out. For example, according to European Commission Directive No. 2003/02/EC, Copper Chrome Arsenate (CCA) is not allowed for residential construction and for all those uses which involve direct human and/or animal contact (Braunschweiler, 2002). Several more kinds of preservatives are also suspected to meet the same fate. In this era of biotechnological intervention, society is pressurizing the industry to replace the synthetic wood preservatives with biodegradable substances. To achieve this objective, research is focused on the use of plant oils and extractives as wood preservatives.

2.3. Wood Extractives

Low molecular weight extractives in woods can be separated with polar and non- polar solvents (Chang et al., 2001b; Sjostrom and Alen, 2013). Polyphenol extractives in heartwoods of several tree species are mostly formed during the process of growth and development (Arango et al., 2006). Natural durability of individual wood species against biotic factors depends mainly on the chemical structure and amount of extractives present (Jelokava and Sindler, 2001). The higher the proportion of extractives, the greater the durability of heartwood (Hillis, 1978). In general, hardwoods have higher extractive contents than softwoods (Telmo and Lousada, 2011). Extractives in both the hard- and softwoods are major contributor to color,

9 durability and cologne of woods. Extractives also affect the hygroscopicity, pulping, adhesion, drying and acoustic properties of woods (Monica et al., 2009). Several wood extractives have particular biological activities and are sources of crude drugs and medicines for centuries (Hon and Shiraishi, 2001). Since the work of Hawley et al. (1924) about the correlation among natural durability and extractives of wood, importance of mechanism of natural durability of wood has not been lessened (Taylor et al., 2006; Santana et al., 2010).

Wood extractives and their role as preservatives have been reviewed in detail in section 2.4.3.

2.3.1. Classification and components of wood extractives

Organic extractives of woods are categorized into three main classes:

(1) Aliphatic and alicyclic compounds - terpenes and terpenoids such as resin acids and steroids, esters of fatty acids, (fats and waxes), fatty acids.

(2) Phenolic compounds - simple phenols, stilbenes, lignans, isoflavones, condensed tannins, flavonoids, hydrolysable tannins.

(3) Miscellaneous compounds - sugars, cyclitols, tropolones and amino acids, alkaloids, coumarins and quinones (Sjostrom, 1981; Holmbom and Stenius, 2000).

2.3.1.1. Aliphatic and alicyclic compounds:

2.3.1.1.1. Terpenes and terpenoids

Terpenes are known as the large group of hydrocarbons made up of isoprene units (C5H8), whose derivatives with hydroxyl, carbonyl, and carboxyl functions are the terpenoids. Terpenoids can be divided into Mono-, sesqui-, di-, tri-, and polyterpenoids, which have been abundantly found in the parenchyma resins of both the hard- and softwoods (Sjostrom, 1981; Hon and Shiraishi, 2001; Monica et al., 2009). Monoterpenoids and monoterpenes in softwood as oleoresins are volatile complexes and impart significantly to the aroma of wood (Fengel and Wegener, 1984). More than 2500 sesquiterpenoids have been identified, representing a wide variety of compounds of different skeletal types which are mostly found as constituents of canal resin and are heartwood deposits of softwoods. Sesquiterpenes are found in several tropical hardwoods but are rare constituents of hardwoods from temperate zones (Rowe, 2012).

Diterpenoids and diterpenes are principal part of canal extractives and limited to softwood species mostly as resin acids and few diterpenoids have been found in tropical hardwoods. Triterpenoids are structurally and biogenetically related to steroids, and both also exist in several hardwoods of temperate and tropical zones. For example, triterpene acids are frequently present in Termilnalia and Quercus species whereas sterols (steroids) such as ~sitosterol are found in several soft- and hardwoods, and a possible raw material for making wood based chemicals (Sjostrom, 1981; Walker, 2006) used as additive agents, repellents to insects, fungicides and for therapeutic purposes (Obst, 1998; Agatha, 2006). Polyterpenoids are abundant in higher plants, especially in the leaves, but not in wood. However, a special type of polyprenols, called betulaprenols occur as fatty acid esters in Betula verrucosa (silver birch) (Sjostrom, 1981). 2.3.1.1.2. Fats and Waxes Aliphatic extractives comprised of fats, waxes, alkanes, fatty acids and fatty alcohols are also present in wood. Both saturated and un-saturated compounds resides in parenchyma resin of soft and hard wood extractives (Sjostrom, 1981). 2.3.1.2. Phenolic compounds

Phenolic compounds, as glycosides in numerous tree species, provide resistance to attack by insects and fungi (Obst, 1998), and are categorized into sub-classes: stilbenes, lignans, tannins, and flavonoids (Pereira et al., 2009).

2.3.1.2.1. Stilbenes

Traditionally, the term stilbene is stated to the compounds holding 1, 2-diphenylethene structure, but recently discovered benzyls and phenanthrenes comprising of C6,-C2,-C6 skeleton are also part of this group (Hon and Shiraishi, 2001). Resveratrol, pinosylvin, elydxangeic acid, lunularin and batatasin found in Moraceae, Pinaceae, Leguminosae and Betulaceae family are examples of stilbenes in their woods. Two isomeric forms of 1, 2- diphenylethylene: cis-stilbene and trans-stilbene as hydroxylated trans-stilbene provides heartwood durability especially for resistance to termites and fungal decay. Shibutani et al.

(2004) found 3, 4′, 5-trihydroxystilbene-3-β-D-glucoside and 3′-methoxy-3, 4′, 5- trihydroxystilbene-3-β-D-glucoside from the bark of Picea glehnii as termite feeding deterrent at a very low concentrations of 0.63 to 2.5 μmol/disc. Pinus sylvestris in response to external

11 stress such as fungal infections or UV light produce pinosylvin which is cause of durability and resistance to decay (Hon and Shiraishi, 2001). 2, 4, 3’, 5’tetra and 3, 4, 5, 3’, 5’-penta- hydroxystilbenes are responsible for wood resistance against brown-rot and white rot fungi (Schultz et al., 1995; Royer et al., 2012).

2.3.1.2.2. Lignans

Lignans are categorized as neolignans and lignans. Phenyl propane dimers (C6 C3- C6

C3) linked at the ß-positions are referred as lignans and those linked at a position different from ß are occasionally termed neolignans. Lignans are abundantly present in bark of trees, Ostrya japonica (neolignan rhamnoside and lignin xyloside), Betula ermanii (lignan glycosides and dicaffeoylated lignan) and Magnolia kobus var. borealis (magnolin and yangambin) (Fuchino et al., 1995). Lignans present in woody angiosperms are thomasic acid and syringaresinol. Heartwood is known to be a rich source of lignans than sapwood (Hon and Shiraishi, 2001). Physical injury or fungal and insect attack on trees also produce resins rich in lignans as a response to the injury (Obst, 1998). Lignans exhibit significant range of biological activity such as fungal enzyme and growth inhibitor and insect antifeedant.

2.3.1.2.3. Tannins

Tannins are water soluble phenolic compounds of molecular weights ranging from 500 to 3000, which have distinct properties to precipitate gelatin, alkaloids and proteins besides giving the usual phenolic reactions (Hon and Shiraishi, 2001). These form the most significant groups of defensive secondary metabolites in higher plants (Haslam, 1988), distributed usually in bark, wood, and leaves and are mostly classified into hydrolysable and condensed tannins. Condensed tannins are natural preservatives and antifungal agents acting as inhibitor of fungal secreted enzymes (lignases and cellulases) (Zucker, 1983). Extracts from numerous woods and barks rich in tannin have been used as adhesives and wood preservatives for a long time (Thevenon, 1999). For example, tannin extracted from Acacia mearnsii in combination with boron provided high resistance against decay fungi and termites (Reticulitermes santonensis) (Tondi et al., 2012).

2.3.1.2.4. Flavonoids

Flavonoids are the major phenolics, which are widely distributed secondary metabolites in all parts of each and every plant species (Harbone, 1973). These compounds offer defense against pathogens, herbivores and ultraviolet radiation (Harbone and Willians, 2000). Flavonoids are categorized into flavones, flavanones, flavonols, chalcones, dihydroflavonols, flavan-3, 4-diols aurones, flavan-3-ols, isoflavonoids, isoflavonoids, flavan- 3,4-diols, anthocyanidins and neoflavonoids. These have significant influence on the durability of wood being free radical scavengers (Schultz and Nicholas, 2000; Chang et al., 2001b; Wang et al., 2004; Pietarinen et al., 2006; Gupta and Prakash, 2009). Principal flavonoids (taxifolin and aromadedrin) present in the wood of Larix leptolepis (Pinaceae) exhibit strong feeding deterrent activity against C. formosanus Shiraki. Similarly, wood of Lonchocarpus castilloi Standley was highly resistant to dry wood termites (Cryptotermes brevis) due to presence of castillen D and E in heartwood (Reyes-Chilpa et al., 1995). The antifeedant activity and structure-activity relationships of some flavonoids was tested in choice and no-choice tests against subterranean termite C. formosanus. Compounds holding two hydroxyl groups at C-5 and C-7 in A-rings exhibited higher antifeedant activities against termites (Ohmura et al., 2000).

Pterocarpans (-)-homopterocarpin, (-)-pterocarpin, and (-)-hydroxyhomopterocarpin) isolated from heartwood of Pterocarpus macrocarpus Kruz showed antifeedent activity against subterranean termite, Reticulitermes speratus Kolbe (Isoptera: Rhinotermitidae) (Morimoto et al., 2006). Neoflavonoids, dalbergiphenol, latifolin, and 4-methoxydalbergione isolated from heartwood of Dalbergia latifolia (Leguminosae) were active against white rot fungi (Trametes versicolor) and termites. Dalbergiphenol also exhibited relatively high antifungal activity against the brown-rot basidiomycete, Fomitopusis palustris. Latifolin showed high termiticide and antifeedant activities against Reticulitermes speratus (Kolbe). Dalbergiphenol and 4-methoxydalbergione exhibited moderate termite antifeedant activity (Sekine et al., 2009). Durability of heartwood of Acacia species and Morus mesozygia has been found due to presence of flavonoids, aromadedrin, morin, pinobanksin, dihydromorin and proanthocyanidins contents (Schultz et al., 1995; Barry et al., 2005; Toirambe and Ouattara,

2008). Durability of heartwood of Prosopis juliflora against wood rot fungus and termites was due to presence of (-)-mesquitol (Sirmah et al., 2009; Pizzo et al., 2011).

2.3.1.3. Miscellaneous compounds

2.3.1.3.1. Quinones

Anthraquinones, benzoquinones and naphthoquinones are found in various plant families. p-quinone is the natural form of quinones but o-quinone also exists (Hon and Shiraishi, 2001). These pigments perform many biological activities. Tectoquinone are well known allelochemical and impart resistance in wood against termites due to toxic and repellent properties (Dungani et al., 2012).

Naphthoquinone (7-methyljuglone) are mostly studied for antitermitic activities (Carter et al., 1978). Lapachol isolated from heartwood of Tabebuia impetiginosa (Bignoniaceae) was active against Microcerotermes crassus (Isoptera: Termitidae) and Kalotermes flavicollis (Isoptera: Kalotermitidae) but did not show any repellent activity against Reticulitermes spp (Becker et al., 1972).

Naturally durable teak wood (Tectona grandis) contains a large amount of quinones such as 4’, 5’–dihydroxyepiisocatalponol, naphthoquinone, anthraquinone which exhibit strong repellent and antitermitic activities against Cryptotermes brevis and Reticulitermes flavipes (Haupt et al., 2003; Kokutse et al., 2006). Quinones such as catalponol and catalponone and 7- methyl juglon isolated from many heartwoods showed antitermitic activity against R. flavipes (Castillo and Rossini, 2010).

2.3.1.3.2. Inorganic components

Ash represents <1% of dry wood weight of various salts of metals, carbonates, oxalates, silicates and phosphates deposited in the cell walls and lumina of needles, leaves and bark. Further, ash contents play no significant role in resistance to degrading agents (Hon and Shiraishi, 2001).

2.3.2. Solvents and isolation techniques for wood extractives

Large number of polar and non-polar solvents have been used for extraction and isolation of extractives from the woods. Solvents and their mixtures yield particular extract,

14 which cannot directly be analogous with extracts acquired while using other solvents (Desh and Dinwoodie, 1996; Sjöström and Alen, 2013). No single solvent is proficient of extracting all constituents considered as extractives (David and Nobuo, 2000). It is customary to use solvents such as methanol, tailed by aqueous extraction where the total extractive content is needed (Walker, 1993). Sequential steam distillation and extraction with ethanol, ether and water remove diverse types of extractives. Thus, standardization is very important and it would be extremely required to fix the ratio of solvents. Following ratios (expressed on the volume basis), 9:1 of ethanol: acetone or acetone: water, 1:2 of ethanol: benzene, 2:1 of ethanol: toluene, are usually used for extraction purpose. Combination of dichloromethane and acetone has been extensively used solvent extraction of the wood but each has its own merits and demerits. Ethanol: toluene (2:1) mixture of solvents is standardized solvent for preparation of extractive free wood (ASTM D 1106-96).

Different types of extractors/equipment are used for extraction of wood extractives such as Soxhlet extractor, Soxtec extractor and Accelerated solvent extraction (ASE). Former two methods are traditional extraction methods while ASE is modified equipment for good extraction. ASE combines elevated pressures and temperatures with the standard solvents used in Soxhlet extraction. ASE is much quicker and requires significantly less solvent than traditional techniques. ASE system is costly, but it is the promising efficient extraction method. Its technology helps in increasing productivity, sample throughput and reduce preparation cost (Sjöström and Alen, 2013).

Supercritical extraction (SFE) has been suggested as substitute to old solvent extraction for wood extractives (Jansson et al., 1993). Due to improved solute distribution and an effective mass transfer, this method of extraction is significantly faster with supercritical fluids than with conventional solvents. Cost of SFE restricts its use for repetitive extraction (Sjöström and Alen, 2013).

2.3.3. Identification and characterization of wood extractives

Wood extractives can be analyzed at three different levels, i.e. gravimetric method or estimation of total extractives, determination of different component groups and exploration of individual components. Component groups in extracts can be determined by several chromatographic techniques; gas chromatography (GC), high performance liquid

15 chromatography (HPLC), size-exclusion chromatography (SEC), supercritical fluid chromatography (SFC) and thin-layer chromatography (TLC). Quantitative analysis of extracts is also possible by 13C NMR. In addition, Fourier transform infrared spectroscopy (FTIR) is currently preferred (Sjöström and Alen, 2013).

The extremely high resolution achieved by capillary open-tubular columns makes GC an excellent technique for complex extractives. Flame ionization detector (FID) of GC is sensitive and reliable, which has a wide linear range and gives nearly the same response for different organic compounds. Combination of GC and mass spectrometry (popularly known as GC/MS) is an excellent technique for component identification of complex mixtures. GC is particularly powerful for analysis of individual resin components (Holmbom 1977; Foster and Zinkel, 1982; Ekman and Holmbom, 1989; Han and Zinkel, 1990; Lee and Peart, 1991; Sithole et al., 1992; Dethlefs and Stan, 1996; Wallis and Wearne, 1996). In addition, GC can also provide convenient quantitative component group determination including steryl esters and triglycerides by employing short columns.

HPLC has been used for group analysis of extractives in both the size-exclusion (SE) and reversed-phase (RP) modes (Suckling et al., 1990). A fairly good separation of wood resin groups can be obtained in SE mode using cross-linked polystyrene resin columns and tetrahydrofuran (THF) solvent. HPLC-SE enables the isolation of separated fractions for further analysis into individual components (Sjöström and Alen, 2013).

Supercritical Fluid Chromatograph (SFC) combines advantages of GC and HPLC (Jansson et al., 1993). FID can be used in SFC, which enables rapid direct analysis of wood and pulp extracts without hydrolysis.

Thin-Layer Chromatography (TLC) is an inexpensive and convenient technique providing a clear visual image of the sample composition (Laamanen, 1984). Many samples can be analyzed simultaneously. TLC is also well suited for preparative isolation of component groups prior to GC over HPLC. TLC on silica plates gives excellent separation of groups of wood resins, namely, hydrocarbons, steryl esters, diterpene aldehydes, triglycerides, fatty, resin acids, various terpene alcohol and sterol components.

NMR Spectroscopy has been used for determination of group composition of resin samples C-NMR (Suckling and Ede, 1990). This non-destructive technique can give the amounts of fatty acids, resin acids, triglycerides, and fatty acid esters in wood and pulp extracts (Sjöström and Alen, 2013).

2.4. Plant extracts and oils as wood preservatives 2.4.1. Extracts from seed, fruit and herbaceous plants Extracts and essential oils from botanical sources have been tested against wood degrading fungi and termites (Vanneste et al., 2002; Maoz et al., 2007) and several had termiticide activities (Sakasegawa et al., 2003; Park and Shin, 2005; Jembere et al., 2005; Cheng et al., 2007; Ding and Hu, 2010; Supriadi and Ismanto, 2010) or repellent action from extracts of Eucalyptus globules, Cymbopogon sp., Eucalyptus citrodora, Cedrus deodara, Syzygium aromaticum and Chrysopogon zizanioides (Zhu et al., 2001a, b), Taiwania cryptomerioides Hayat (Chang et al., 2001a), Ocimum basilicum L., Cymbopogon winterianus, Cinammomum camphora, Rosmarinus officinalis (Sbeghen et al., 2002) and Dodonaea viscosa (Nisar et al., 2012).

Few examples of plant extracts having potential for wood preservative are presented in the following lines. Black pepper fruit extracts, Piper nigrum in hexane, ethanol, and petroleum ether were used against drywood termite, Cryptotermes brevis, and hexane extracts had maximum efficacy against these termites (Moein and Farrag, 2000). Aqueous extracts of some common plant products for their anti-termite properties by exposing treated bamboo baits in Karnataka, India showed significant reduction against termite damage (Mokabel and Gowda, 2000). Decoction of seven plant species like Thevetia peruviana, Cassia fistula, Calotropis procera, Datura metel, Lantana camara, Sapium sebiferum and Myrtus communis @ 5% provided significant protection against termites (Bajwa and Rajpar, 2001). Phytoextracts of Adhatoda vasica, Cynodon dactylon, Pongamia pinnata, Rauvolfia serpentina, Cleistanthus collinus, Tamarindus indica and Eichhornia crassipes controlled the termites, Microcerotermes mycophagus (Nilanjana and Chattopadhyay, 2003). Acetone, ethyl alcohol and methyl alcohol extract of neem leaves inhibited growth of Poria menticola (a brown rot) and Polyporus versicolor (a white rot) (Dhyani et al., 2005). Woods treated with cinnamaldehyde from cinnamon leaf extracts showed excellent decay and termite resistance in

17 laboratory exposure tests in Taiwan (Lin et al., 2007). Leaf, fruit, wood, bark, seed and flower extracts of Cerbera odollam showed resistance against fungi and termites. Acetone and hexane extracts of Vetiveria zizanoides were very effective against Trinervitermes geminatus and more than 90 % survival of termite was reduced after feeding on treated diet (Hashim et al., 2009).

2.4.2. Bark Extracts

Waxes, resins and tannins extracted from barks of many tree species are a rich source of termiticidal, antioxidants and antimicrobial agents. Tannins have been widely used as adhesives and were recognized as wood preservatives for some time. One major problem with tannins and tannin derived compounds is their difficulty to fix in wood after treatment. Use of additives (ferric chloride) and metallic salts have been reported for satisfactory wood protection. Patents using tannins and tannic acid, particularly woods soaked in hot water tannins extract from oak bark as an early technique have been summarized to improve wood resistance and durability (Lotz and Hollaway, 1988; Lotz, 1993).

Bio-oils from barks using pyrolytic methods have been tested as wood preservatives (Suzuki et al., 1997; Mourant et al., 2005). Extraction of barks and wood wastes with the pyrolysis process and their utilization has been studied extensively. Pyrolysis is a thermal degradation process where biomass (bark from sawmills) is broken down into small molecular compounds in absence of oxygen. The nature and yield of the compounds obtained by the pyrolysis process vary depending on the biomass source, moisture content (MC), temperature and pressure applied in the process. Several research organizations focused on potential use of pyrolytic bio- oils in wood preservation and mainly antifungal compounds were identified (Mourant et al., 2005; Yang et al., 2004; Yang, 2009).

Resins from wood and stem of guayule (Parthenium argentatum Gray) provided protection against wood destroying organisms including decay fungi, termites and marine borers (Nakayama et al., 2001). Wood particle boards impregnated with Pinus bruita bark extracts showed improved performance in decay resistance and other board characteristics were also enhanced (Nemli et al., 2006). Recently, phenolic glucosides extracted from the bark of Populus ussuriensis have been shown to possess antioxidant properties (Zhang et al., 2006; Si et al., 2011).

2.4.3. Wood extractives

Significant variations in durability of different woods are due to resistance of their heartwoods to decaying agents in/above ground contact. Western red cedar (Thuja plicata Donn ex D. Don), eastern white cedar (Thuja occidentalis L.), yellow cedar (Chamaecyparis nootkatensis (D. Don) Spach), yew (Taxus spp.), redwood (Sequoia spp.) and teak (Tectona grandis Linn.) have been extensively studied for the presence of extractives in their heartwoods.

The extractives hold both toxicity and repellency against several termite species. Wood, wood extracts and saw dusts of Neoblanocarpus heimii and Shorea ovalis trees affected termites’ survival and extracted and un-extracted wood consumption and secondary compounds in the woods were considered for such effects (Sajap and Sahri, 1983). Hashimoto et al. (1997) interrelated that poorer extractive contents reduce wood resistance against termites. Resistance of wood species against termites differs depending on several features including density, natural durability and extractive quantities and types (Akhtar, 1981; Kirker et al., 2013). Durability of non-refractory and non-durable wood species can be enhanced by utilization of extractives isolated from naturally durable species (Schultz and Nicholas, 2000; Thevenon et al., 2001).

In addition to extractives mentioned in introduction, manool, ferruginol (Scheffrahn et al., 1988), nootkatone (Zhu et al., 2001a; Maistrello et al., 2001), cedrol and alpha-cadinol (Chang et al., 2001b) have been reported. α-cadinol in heartwood extracts of Taiwania cryptomerioides exhibited greater antitermitic and antifungal activities (Chang et al., 2001a; Chang et al., 2003). Thujaplicins and thujic acid in heartwoods of western red cedar and eastern white cedar @ 0.1 to 0.3% inhibited fungal growth (Stirling et al., 2007). Thujaplicin was also implicated @ 0.5% in several multicomponent biocide systems with borate and carbon-based biocides protected woods from mold, decay and termite damage (Yang and Clausen, 2007).

Several laboratory studies have demonstrated use of extractives from durable wood species in improvement of durability of susceptible woods. Heartwood extracts of eastern white cedar (Thuja occidentalis L.) increased aspen strand panel durability (Wan et al., 2007). Heartwood extracts of bald cypress (Taxodium distichum) (Scheffrahn et al., 1988), southern

19 catalpa (Catalpa bibnonioides) (McDaniel, 1992), red louro (Sextonia rubra) (Rodrigues et al., 2011) exhibited similar properties.

Choice and no-choice feeding filter paper bioassay were dominant laboratory evaluation methods of biocide properties of wood extractives. Kadir et al. (2014) reported anti- termite activity of heartwood and bark extracts of two Malaysian trees (Madhuca utilis and Neobalanocarpus heimii) and found that both trees extracts were toxic against Coptotermes gestroi. They treated the filter paper with different concentrations of extracts in ethanol and exposed the termites to treated and untreated filter papers for 14 days and determined the LC50 for each type of extract against subterranean termites. They also analyzed the composition of each extract by using GC-MS and found that monoterpenoids and sesquiterpenoids were major groups identified in each extract.

Similar studies also have been conducted in Pakistan. Wood extracts and essential oils from Dalbergia sissoo, Pinus wallichiana and Cedrus deodara hexane, water, and acetone against Coptotermes heimi revealed C. deodara extracts more toxic to termites than D. sissoo. Same results were obtained with the use of essential oils of these timbers. Termites’ survival was 0% on un-extracted sawdust of C. deodara and P. wallichiana as compared to 40% on D. sissoo. All termites were dead after one week on all types of wood species in forcibly fed block test (Akhtar, 1981). In another study, feeding responses of Bifiditermes beesoni on wooden blocks treated with wood extracts of D. sissoo, C. deodara and P. wallichiana in the laboratory was identical as above (Akhtar and Jabeen, 1981). Response of Coptotermes heimi and Heterotermes indicola on wood powder of D. sissoo, Eucalyptus camaldulensis and Acacia arabica revealed toxic effect due to extractives on gut flagellates (Qureshi et al. 2008).

Other studies such as Sethy et al. (2005) reported on smaller blocks of rubber wood with alcoholic extracts of Dalbergia latifolia at different retention levels (0.1%, 0.2% & 0.5%) had activity against brown rot and white rot fungi. These extracts reduced the wood loss up to 15% as compared to control in which wood loss was 57 percent at maximum retention level.

Kharkwal et al. (2012) found that chloroform heartwood extracts of D. sissoo proved repellent (92%) against Microcerotermes beesonii. Hexane heartwood extracts of D. congestiflora showed had 100% inhibition of Trametes versicolor fungus. A crystalline

20 substance found in hexane extracts was characterized as (+)-3-hydroxy-9-methoxypterocarpan or (+)-Medicarpin by MS and NMR spectroscopy (Martínez-Sotres et al., 2012).

Although mechanisms of naturally durable wood are not fully understood, previous studies have shown that heartwood extractives possess distinct anti-oxidant properties. It might not be exclusively toxicity and repellency of extractives in the durable wood samples that gives the heartwoods the termites’ resistance, but extractives’ dual toxicity and antioxidant properties, or two or more factors propose their action, probably synergistically. It has been suggested that the presence of antioxidants in the heartwood may interfere with the numerous single electron redox reactions that take place in the termite gut to convert cellulose into acetate via the shikimate acid pathway (Ragon et al., 2008).

Several researchers have explored anti-oxidant ability of extractives by using natural and synthetic antioxidants agains termites and fungi. Grace (1990) prepared the fungal extracts by extraction of decayed wood using dichloromethane and cyclohexane. Following assay by adding two antioxidants (BHA &BHT) against R. flavipes, addition of BHA completely suppressed the response of termite to fungal extracts while addition of BHT had no such effect on termites but concentration dependent repellency was observed in orientation bioassay. Ragon et al. (2008) hypothesized the dual properties i.e. toxicity and antioxidant properties of heartwood extractives of durable trees. They used artificial antioxidant BHT to treat the Southern pine wood. BHT caused 100% mortality of R. flavipes at 2% retention level. It also affected C. formosanus at 4% retention level. Schultz et al. (2008) conducted some field tests by using artificial antioxidant BHA against R. flavipes on southern yellow pine sapwood wafers for three years in the field. Treated wafers have less weight loss as compared to control in laboratory and field tests. Little et al. (2010) investigated the mortality and deterrent activity of natural and synthetic antioxidants and their analogs against R. flavipes. They found that BHA, gallic acid and other antioxidants have strong activity against termites while non- oxidants have little effect on the termites except heterocyclic flavanone.

2.4.4. Plant oils

Increased interest in natural wood preservatives has also augmented the interest for testing plant oils as a wood protectant. Oils of numerous plants such as Eucalyptus globulus

(eucalyptus), Trachyspermum copticum (ajowan), Cymbopogon flexuosus (lemongrass), Anethum graveolen (dill weed), Pelargonium graveolens (geranium), Rosmarinus officinalis (rosemary), Melaleuca alternifolia (tea tree) and Thymus zygis (white thyme), crude tall, cinnamon, citrus peels, tung seeds (Vernicia fordii) and kukui plant (Aleurites moluccanus) oils have been tested against termites and decay (Clausen and Yang, 2008; González-Laredo et al., 2015). Hemp oil (Sailer and Rapp, 2001), linseed oil (Sailer and Rapp, 2001; Paajanen and Ritschkoff , 2002; Kartal et al., 2006; Temiz et al., 2013), clove oil (Ahmed et al., 2013), castor bean oil (Ahmed et al., 2014), linseed and neem oil (Fatima and Morrell, 2015) are recent addition in list of oils having potential to protect wood from termites and fungal decay and reported elsewhere. Crude tall oil (CTO), rapeseed and linseed oils tested against decay on sterilized malt agar inoculated with the fungi Coniphora puteana, Poria placenta and Coriolus versicolor showed that CTO had the best effect especially on soft rot fungi (Paajanen and Ritschkoff , 2002). Vetiver oil as wood preservative against Coptotermes formosanus showed that tunneling and wood consumption was significantly reduced as compared to control treatments (Maistrello et al., 2003). Four refined tall oils with varying contents of free fatty and resin acids by a filter paper assay against Poria placenta and Coriolus versicolor revealed that tall oils were more efficient against C. versicolor than against P. placenta (Alfredsen et al., 2004). Commercially available refined tall oil derivates have shown promising antifungal properties and exhibited the efficacy against decay fungi in laboratory tests (Temiz et al., 2008). Oil from Jatropha curcas imparted resistance in wood against Microcerotermes beesoni. (Singh and Sushilkumar, 2008). Toxicity and feeding repellency tests of Jatropha oil against Coptotermes vastator showed an increase in mortality and reduction in feeding of termites as compare to control treatments (Acda, 2009). Oils of Azadirachta indica and Ricinus communis for the improvement of resistance of Ceiba pentandra wood against Nasutitermes corniger presented some repulsive effect on termites (Paes et al., 2010) Potentially the plant oils are capable to decrease moisture and water uptake and protect woods by making a layer on their surfaces. Several studies revealed protection against lignin degradation from surfaces of woods during weathering which makes woods aesthetically acceptable for outdoor construction. Thus, immobilization of plant oil-based wood

22 preservatives and the combination of suitable extractives is a significant research direction leading to creosote replacements. 2.5. Gut microbiota of termites

Termite’s gut harbor a dense and diverse microbiota which is vital for symbiotic digestion. The main players in lower termites are unique lineages of cellulolytic ﬂagellates, however, higher termites harbor only archaea and bacteria (Brune and Dietrich, 2015). Despite having endogenous cellulases (Tokuda et al., 1999), termites are unable to digest cellulose for their own energy requirements from constitutive enzymes of gut (Nakashima et al., 2002). Hindgut symbionts assist break down of partially digested cellulose and glucose to acetate, carbon dioxide and hydrogen. Acetate from symbiotic protists is the dominant energy fuel for the termites (Breznak and Switzer, 1986). Without hindgut protists, the host starves even with a gut filled with wood (Cleveland, 1925). In addition to the protists, acetogenic bacteria within the hindgut use carbon dioxide and hydrogen from glucose digestion to produce acetate and water.

2.5.1. Symbiotic protozoans

The hindgut of lower termites is inhabited by a number of anaerobic symbiotic protozoans which mostly belong to Phylum Parabasalia (Ohkuma and Brune, 2011). Three out of six classes of Parabasalids are composed of species that are unique to the guts of lower termites (Noda et al., 2012). These protozoans help the termites in the digestion of cellulose rich diet. The first confirmation for the cellulolytic activity of these gut protozoans was reported by Yamin and Trager (1979). Gut protozoans are large enough to phagocytize the wood particles and their great motility due to numerous ﬂagella precludes washout—possibly adaptations to the termite diet and the microbial habitat in the termite gut. Several, but not all, species of lower termites harbor protozoans of the order Oxymonadida (Phylum Preaxostyla). Some lineages developed special holdfasts that attach to hindgut cuticle and obscure the small cells within the bacterial bioﬁlm (Tamschick and Radek, 2013). Molecular studies revealed that the diversity of termite gut protozoans is greater than expected (Gile et al., 2013; Tai et al., 2015). Each termite species usually has a distinctive composition of flagellate species. More than 430 protozoans’ species in the guts of 205 termite species were reported (Yamin, 1979). Total eleven protozoan species from the hindgut of R.

23 flavipes with populations exceeding 35,000 cells per individual gut were identified (Lewis and Forschler, 2004). These were Dinenympha gracilis Leidy, 1877, Pyrsonympha vertens Leidy 1877, Trichonympha agilis Leidy, 1877, Spironympha kofoidi Koidzumi, 1921, Holomastigotes elongatum Grassi, 1917, Monocercomonas sp. Grassi, 1917, Dinenympha fimbriata Kirby, 1924, Trichomonas trypanoides Dubosq and Grassé, 1924, Microjoenia fallax (Dubosq and Grassé 1924), Spirotrichonympha flagellata (Dubosq and Grassé, 1924), Pyrsonympha major Powell, 1928, Spirotrichonympha sp Morphologically, nine flagellate protozoan species in the gut of Heterotermes indicola were identified (Qureshi et al. 2012). Excellent reviews on the intestinal microbial communities of several termite species have been presented by Brune and Friedrich (2000), Ohkuma (2002, 2003, 2008), Brune and Stingl (2006), Brune (2006) and Ohkuma et al. (2006). 2.5.2. Symbiotic bacteria In lower termites, not only the flagellates but also the prokaryotes are crucial for the survival of termites. Bacterial gut microbial communities of termites comprise only a few dominant Phyla with distinctive differences among the major host groups. Over the past two decades, culture-independent molecular studies gave new light on the diversity of gut bacteria. These studies were usually based on 16S rRNA gene sequences. The bacterial 16S rRNA genes from the DNA extracted from the gut homogenates were ampliﬁed by PCR usually with universal bacterial primers. These ampliﬁed genes were sorted into groups after their cloning and sequencing. The representative phylotypes of the groups were then analyzed (Ohkuma and Brune, 2011). A comprehensive diversity of gut bacteria of the lower termite, Reticulitermes speratus, was reported by Hongoh et al. (2003a, 2003b). 16S rRNA gene sequences were reported not only in the lower termite genera Reticulitermes (Hongoh et al., 2005), Coptotermes (Shinzato et al., 2005) and Cryptotermes (Hongoh et al., 2007) but also in the higher termite genera Cubitermes (Schmitt-Wagner et al., 2003), Microcerotermes (Hongoh et al., 2005), Macrotermes (Hongoh et al., 2006), Odontotermes (Shinzato et al., 2007) and Nasutitermes (Hongoh et al., 2006; Warnecke et al., 2007). In these studies, the estimated bacterial diversity was generally several hundred phylotypes. Spirochetes were the most prevalent identified phyla and the second most dominant group was of Bacteroidetes, Firmicutes and TG1 phylum together comprising about

80% of the gut community. However feeding habits of the host can affect the bacterial communities (Ohkuma and Brune, 2010). Spirochetes - are characteristic members of lower and higher termite gut communities, however their abundance is not a general rule (Breznak and Leadbetter, 2006; Hongoh, 2011). The maximum proportion of spirochetes is found in wood-feeding termites but their numbers among fungus-cultivating and humus-feeding genera are usually low. These are mostly associated with the surface of flagellates or occur as free-swimming cells or the fiber fraction (Brune and Dietrich, 2015). They are phylogenetically highly diverse and comprise various monophyletic groups of termite-specific lineages (Ohkuma et al., 2015). Individual lineages differ in abundance between host groups (Dietrich et al., 2014; Rahman et al., 2015. Bacteroidetes are abundant in fungus-cultivating termites and contribute to the similarity of gut microbiota between termites and cockroaches (Brune and Dietrich, 2015). The major population of bacteriodetes comprise of subgroup bacteriodes in termite guts, which are obligatory anaerobic, gram negative non-pulsating rods. Many of the predominant taxa includes Alistipes, Dysgonomonas, Parabacteroides and Paludibacter. These taxa also possess a general preference for intestinal habitats as encountered in the guts of mammals (Yang et al., 2014; Pramono et al., 2015). Though, there are several family level clades that contain exclusively of representatives which are present in termites and cockroaches (Brune and Dietrich, 2015). Firmicutes are common gut bacteria associated with the hindgut cuticle of arthropods or alkaline gut compartments of higher termites. These mostly to family Lachnospiraceae and Ruminococcaceae and highly specific. These are (particularly Clostridiales) mostly abundant in fungus growing termites (Thongaram et al., 2005; Thompson et al., 2012). Proteobacteria are more abundant in family Macrotermitinae than in other termite groups. These are also present in cockroaches. Proteobacteria contain the strains, which are associated with gut flagellates such as strains of Deltaproteobacteria (Sato et al., 2009; Strassert et al., 2012; Rosenthal et al., 2013). Members of the Elusimicrobia make up a large proportion of the bacterial community in many lower termites and these also have been identified as endosymbionts of many flagellates (Brune, 2010; Dietrich et al., 2014). The fiber-associated members of Fibrobacteres and the candidate phylum TG3 are abundant in wood-feeding higher termites but have been detected in other lineages (Hongoh et

25 al., 2006; Dietrich et al., 2014; Rahman et al., 2015). Planctomycetes form large populations only in the posterior hindgut compartments of soil-feeding Termitinae (Koehler et al., 2011), but they also occur in low abundance in other groups (Rahman et al., 2015). Archaeas’ diversity is mostly found in sub-family of Termitinae of higher termites. These archaeal communities contain hydrogenotrophic and methylotrophic populations in each hindgut compartment while archaeal communities in lower termites are dominated by Methanobrevibacter species (Brune and Dietrich, 2015). Increasing interest in the ligno-cellulolytic degradation process performed by termites and their symbiotic fauna has led to a number of metagenomic and metatranscriptomic studies examining the effects of different diets on these holobionts (Scharf, 2015; Duarte et al., 2016). Different protist flagellate species are believed to be involved in separate steps of cellulose digestion (Cook and Gold, 2000; Tanaka et al., 2006). Limiting termite development through the destruction of the gut microbes is a control method that has shown crucial success (Doolittle et al., 2007). For this purpose, a number of researchers have examined how the loss of symbiotic gut flagellates could negatively affect termites’ health and a few have studied how change in bacterial community might impact termites’ success in terms of reproduction and survival (Mannesmann, 1972b). Mauldin et al. (1981) examined the feeding behavior of R. flavipes on 21 American woods and concluded that extractives of woods has negatively impacted the population of gut flagellates. Jones et al. (1983) tested extracts of six Brazilian wood species against protozoan’s of R. flavipes and found that all species extractives were protozoacide. Similarly Carter et al. (1983) examined the antitermitic material from five Brazilian woods extracted sequentially with hexane, acetone, a mixture of acetone-hexane-water and 80 % methanol. Low protozoan numbers were found in termites surviving for 4 weeks after feeding on extracts treated filter paper. Termites generally preferred untreated paper to extract-treated paper. With help of molecular biological techniques, Boucias et al. (2013) using pyrosequencing for examination of changes in gut microbiota after feeding of R. flavipes on lignin-rich and lignin-poor diets and found little change in microbial composition by diet. However, many still hold hypothesis that plant oils can also affect termites’ health due to diversity loss in gut flagellates (Fatima and Morrell, 2015).

CHAPTER THREE MATERIALS AND METHODS

Present studies are divided into six sections and each section is further comprised of different preparative steps and /or experiments.

SECTION-I

1. Selection of test woods and preparation of heartwood extractives 2. Termites collection and maintenance

SECTION-II

Toxicity, repellency, antifeedant and antioxidant activities of heartwood extractives

SECTION-III

1. Effect of heartwood extractives on gut protozoans of two termites species 2. Dynamics of termites’ guts’ bacterial community of H. indicola before and after exposure to extractives treated filter papers.

SECTION-IV

1. Choice and no-choice tests on extracted and un-extracted woods against termites 2. Termites’ bioassay on extractives treated Southern Yellow Pine and Cotton Woods by vacuum pressure treatment 3. Determination of leaching resistance of extractives impregnated woods against termites 4. Potential of combination of wood extractives with linseed oil against termites 5. Evaluations of extractives from durable woods against rot fungi (Postia placenta and Trametes versicolor)

SECTION-V

Characterization of heartwood extractives using Gas chromatography – Mass spectrometry (GC-MS) analysis

SECTION- VI

Potential of wood extractives and linseed oil as preservatives in field tests against termites attack and fungal decay

SECTION –I 3.1.1. Selection of woods and sample preparation

Previously known naturally resistant heartwoods of four tree species viz., Tectona grandis Linn (Teak), Dalbergia sissoo Roxb. ex DC (Shisham) Cedrus deodara Roxb. G. Don (Himalayan Cedar) and Pinus roxburghii Sarg. (Chir Pine) were selected for current studies (Fig. 1). Some physical properties of these woods are described in Table 3.1. Two non-durable woods, Southern yellow Pine (SYP) and cottonwood (CW) (Populus sp.) were selected due to common use, availability and size to test effectiveness of heartwood extractives. Defect free wooden logs of durable and non-durable trees except T. grandis and SYP were obtained from Timber Market located at Faisalabad, Pakistan, while Marine grade T. grandis and SYP were acquired from a supplier in the United States (McIlvain, Pittsburg, PA). Wooden logs of Pakistani woods were shipped to Forest Products Laboratory, 201 Lincoln Green Starkville Mississippi (USA) to perform experiments on Reticulitermes flavipes. After air drying for one week, wooden logs of durable and non-durable trees were cut into 19×19×19 mm blocks. For the preparation of extractives, a part of wood from durable species was converted into shavings using an electric planer (Fig. 3.1).

3.1.2. Preparation of extractives and extractive-free wood

Air dried wood shavings of durable species were extracted using 300 ml of ethanol: toluene (2:1) as solvent according to ASTM D1105-96 ʺStandard Test Method for Preparation of Extractive-Free Woodʺ with minor modifications (ASTM 2014). Shavings (12-15 gm) were added to ~20 Soxhlet apparatuses with a small pad of cotton below and above shavings in each and extraction process was run for a period of total six hours. The resulting aliquot was evaporated at reduced pressure by using rotary evaporator and extraction yield was calculated per gram of wood shavings (Ordonez et al., 2006). A total of 1000 -1200 ml of extractives of each wood was prepared and these were stored at 4oC in air tight bottles after preparation of stock solution (150 mg ml-1).

For preparation of extractive-free wood, blocks of size 19×19×19 mm were also extracted according to ASTM D1105-96 with the following modifications. Conditioned blocks (33°C, 62±3%) were numbered and weighed prior to being placed in Soxhlet apparatus and

28 extracted for six hours using 300 ml of ethanol: toluene (2:1). Blocks were then washed by dipping in alcohol to remove excess toluene and for the second time again these were extracted for six hours in ethanol (95%) alone. Ethanol extracted blocks were air dried overnight and then boiled for six hours with three 1-L of each hourly changed distilled water. Blocks were conditioned at 33°C and 62±3% R.H.

Table 3.1. Properties of durable woods selected for current study

Species Family SG* Type Information Durable reference Tectona grandis material. Class 1 (very Verbenaceae 0.55-0.66 Hardwood (Teak) durable). High levels of quinones. Commercial species in Dalbergia sissoo Pakistan, heartwood (Shisham) Fabaceae 0.62-0.82 Hardwood contains stilbenes /polyphenols, Class 1 (very durable). Widely available Cedrus deodara softwood in Pakistan, (Deodar) Pinaceae Unknown Soft wood medicinal properties, class 3 (moderately durable). Widely grown and Pinus roxburghii utilized in Pakistan, (Chir Pine) Pinaceae 0.43-0.49 Soft wood building, medicinal, stilbenes, class 4 (non- resistant). SG*= specific gravity

D. sissoo

C. deodara P. roxburghii

Heartwoods Electric planer Wood shavings Shavings in Soxhlets

Extraction Stock solutions Rotary evaporator Resulting aliquot

Fig. 3.1. Steps for preparation of heartwood extractives at Department of Sustainable Bio-products, MSU.

3.1.3. Termites collection and maintenance

All laboratories investigates on Reticulitermes flavipes were conducted in Forest Product Laboratory, Starkville, Mississippi (USA) and all tests against Heterotermes indicola were conducted jointly in Termites Research Laboratory, Department of Entomology, University of Agriculture, Faisalabad and Nuclear Institute for Food and Agriculture (NIFA), Peshawar, (Pakistan).

Workers and soldiers of R. flavipes were collected from fallen logs and dead trees at Sam D. Hamilton Noxubee National Wildlife Refuge (Starkville, Mississippi) (Fig. 3.2) and maintained on the wood from which it was collected in a laboratory at 25 °C in dark. For collection of H. indicola, foraging points were identified at Peshawar, Pakistan by installing untreated poplar (Populus sp.) stakes (4 cm wide x 2.5 cm thick x 28 cm high), which were examined after every two weeks for presence of the termites. The infested stakes were exchanged with underground monitoring stations by digging a hole in soil in a way that upper edge of station just touching soil surface. The monitoring station was comprised of five wooden poplar slices (15 cm high x 8 cm wide x 1 cm thick) wrapped in a blotting paper and held together by a rubber band, which was surrounded by a 2 mm thick plastic collar (17 cm diameter x 22 cm high). The space between poplar and plastic collar was filled with soil and exposed end of the PVC pipe was enclosed with a plastic bag to prevent water from entering the trap (Fig. 3.3). These traps were inspected every two weeks and infested bundles were exchanged with new ones. Infested bundles were carried to laboratory for testing. Collected termites were kept in glass Petri dishes (14 cm dia.) containing two pieces of moistened blotting paper (14 cm dia.) in incubators at 27 ± 2 °C and 75 ± 1 % R.H.

Collection of R. flavipes Underground monitoring stations and detection stakes for H. indicola Fig. 3.2. Methods for collection of R. flavipes and H. indicola.

SECTION-II 3.2.1. Toxicity tests of heartwood extractive treatments on filter papers against R. flavipes and H. indicola

Oven dried (60°C) and weighed Whatman No. 1 filter papers were treated with five different concentrations (1.25, 2.50, 5.0, 7.50, 10.0 mg ml-1) of each heartwood extractives. Concentrations were prepared from stock solution using ethanol- toluene as a solvent and 200µl of this solution was applied to each filter paper. Treatments were done in replicates of three along with a control treatment which was treated with ethanol: toluene alone. After treatment, filter papers were oven dried (60°C) for 12 hours and weight gain after treatment was calculated. A total of 50 termites were released into jars containing 20 grams of sand, moistened with water @ 18 % of weight of sand (= 3.6 ml water) and extractives treated filter papers; this experimental set up was maintained in an incubator at 27±2°C and 75±1% R.H. for 15 days (Fig. 3.4a). At the end of the test, termite mortality was calculated by counting the number of alive termites. Filter paper was cleaned, oven dried at 60°C for 12 hours. A vacuum desiccator was used to equilibrate the weight of filter paper after drying and weight loss was calculated. Image J software (Developed by Wayne Rasband, Bethesda, Maryland) was used to calculate the area of filter paper consumed by the termites. Corrected mortality of termites was calculated using Abbott’s Formula and data were analyzed using one way ANOVA in

Minitab 16. Median lethal concentrations (LC50) of each extractive against termites’ species were calculated by using Polo Plus software. Tukey's HSD test was used to separate means of different treatments at p<0.05.

3.2.2. Repellent and antifeedant activities of heartwood extractives of four durable woods against R. flavipes and H. indicola

The method outlined by Kadir et al. (2014) was followed to test for repellency. Whatman filter papers (9 cm diameter) were sliced into two equal halves; one-half was treated with 1 ml of each concentration of each extractives and second half was treated with solvent (Ethanol : toluene) only. After drying under a fume hood, both halves (treated and control) were rejoined using adhesive tape that was placed on the underside of the two filter paper halves. The rejoined filter paper pieces were then placed in Petri plate (diameter - 9.1 cm) and 50 active termites were released in the center of the plate (Fig. 3.4 b). Assessment of repellency

32 was performed after 1, 2, 3, 4 and 12 hour by counting the number of termites on treated and untreated filter papers. Formula used to calculate % repellency is given below:

Repellent % = (푁푐 − 푁푡)/(푁푐 + 푁푡) × 100

Where Nc is number of termites in control while Nt is a number of termites on treated filter paper.

Antifeedant indices were determined on the basis of weight loss of filter paper (Absolute coefficient of antifeedant A), which was calculated by formula as given under:

A (%) = [(KK - EE)/ (KK + EE)] x 100

Where KK is a weight loss of the filter paper in the control treatments and EE is a weight loss of the treated filter paper. All treatments which treatments were divided into four classes according to their A values (Table. 3.2.) (Dungani et al., 2012).

Table 3.2. Indicators to Evaluate Antifeedant Activity of extractives (Antifeedancy %)

Antifeedancy (%) Activity level 75 ≤ A < 100 Very strong activity 50 ≤ A < 75 Strong activity 25 ≤ A < 50 Moderate activity 0 ≤ A < 25 Minimal activity The data so obtained on repellency test were analyzed using two factors factorial design under CRD while A% was analysed using one way ANOVA in Minitab 16.

a b

Fig. 3.3. Filter paper test set up (a) and repellency test set up (b).

3.2.3. Effect of heartwood extractives on DPPH radical-scavenging activity DPPH is a stable free radical and is widely used to assess the radical-scavenging activity of antioxidant compounds. This method is based on the reduction of DPPH in methanol solution in the presence of a hydrogen-donating antioxidant due to the formation of the non- radical form DPPH-H. This transformation results in a color change from purple to yellow, which is measured spectrophotometrically at 517 nm. Method of Lu et al. (2014) was used for this assay with slight modification for the determination of DPPH (1, 1- diphenyl-2-picryl- hydrazyl) scavenging activity/anti-oxidant property of each extractive. Extractives were dissolved in methanol to make a series of concentrations (25 µg ml-1 to 800 µg ml-1). One hundred microliter of methanolic DPPH solution (2.5 µM) was added to an equal amount of extractive concentration. 200 µl of the solution was then added in each well of 96-well microtiter plate. Methanol was used as control test samples. The plate was shaken for two minutes and incubated for 20 minutes at 37°C in darkness. Yellow colour of solution was measured spectrophotometrically at 517 nm using BioTek’s Power Wave HT microplate spectrophotometer linked to a computer (Gen5™ software). Percentage of radical scavenging activity was calculated by following formula:

DPPH radical scavenging activity (%) = (퐴푏푠 푐표푛푡푟표푙 − 퐴푏푠 푠푎푚푝푙푒)/(퐴푏푠 푐표푛푡푟표푙)×100

Where Abs control is the absorbance of DPPH + methanol and Abs sample is absorbance by DPPH radical + each concentration of extractives. Butylated hydroxytoluene (BHT) and quercetin were used as the positive control and IC 50 values were calculated by using Graph pad Prism 6 software.

3.2.4. Enzymes activity in mid gut of H. indicola workers exposed to IC50s of BHT, quercetin and heartwood extractives

Filter papers were treated with IC50 of extractives and compounds (42.63, 8.17, 167.97, 28.83, 508.69 and 335.24 µg ml-1 for BHT, quercetin, T. grandis, D. sissoo, C. deodara, and P. roxburghii respectively). Method of treatment was same as described earlier in filter paper bioassay. Concentrations were prepared using ethanol:toluene as solvent. Treatments were done in replicates of three along with a control treatment, which was treated with ethanol: toluene alone. A total of 50 termites were released into jars containing 20 grams of sand, moistened with water @ 18 % of weight of sand (= 3.6 ml water), and treated filter papers and

34 maintained in an incubator at 27 ± 2 °C and 75 ± 1 % R.H. for 7 days. After 7 days termites were put into 80 % ethanol. Termites’ guts were dissected in a saline solution. Dissected guts in 80 % ethanol were stored at -20°C until used for enzyme assay. 3.2.4.1. Protocol for estrases activity

Activity of esterases was determined following procedure outlined by Nisar et al. (2015). Ten midguts (per replicate) of termite workers were homogenized in 0.1 M phosphate buffer with Yellow line IKA DI 25 Basic Homogenizer at 15000 rpm for 20 seconds. Homogenate was filtered through glass wool and was used as enzyme solution. Aliquot of un- centrifuged homogenate was incubated at 25°C with α-naphthyl acetate (0.25 mM final concentration) in a total volume of 3.0 ml of 0.1 M phosphate buffer (pH 7.0). After 30 min,

0.5 ml of Fast blue B salt (tetra-azotized o-dianisidine/ZnCl2, 1%) + sodium dodecyl sulphate (5%) was added to the incubated mixture. A red color was immediately developed that quickly changed into a fairly stable blue color, which was measured at 605 nm on a spectrophotometer (CECIL, CE2041, 2000 series). The quantity of naphthols produced was determined from standard curve of naphthol. Standard curve of α -naphthol was obtained by plotting concentrations (3-30 µmoles) of α- naphthol against absorbance at 605 nm. Specific activity of esterases was thus represented as µmoles naphthol produced per minute per mg of protein.

3.2.4.2. Protocol of glutathione S-transferase (GST) activity Ten termites’ guts were homogenized in 0.1 M sodium phosphate, pH 7.4 and centrifuged at 3000xg for 10 min at 4°C, storing supernatant as enzyme source. GST activity was measured using 1-chloro-2, 4-dinitrobenzene (CDNB) (Sigma-Aldrich) following Habig et al. (1974). Each well of spectrophotometer plate contained 300 µl of reaction mixture: 213 µl of 0.1M sodium phosphate buffer (pH 6.5), 8 µl synergist, 70µl of enzyme incubated for 10 min at 30°C, 6 µl of 50 mM CDNB and 3 µl of 100 mM GSH (Sigma-Aldrich) was added to start reaction. The change in absorbance at 340 nm was recorded for 6 min with a spectrophotometer. 3.2.4.3. Catalase (CAT) assay CAT activity was measured as described by Aebi (1984). Ten termite guts from each treatment were homogenized in 67 mM potassium phosphate buffer (pH 7) for 5 min at 0 °C. Homogenates were filtered through two layers of cheesecloth and centrifuged at 3000 g for 15

35 min. 0.5 ml of termite extracts was added to 0.5 ml 30 mM H2O2 and disappearance of hydrogen peroxide was measured at 240 nm during 3 min at 30 s intervals. Activity of the catalase was expressed as µmol decomposed H2O2 per minute per mg protein. Difference in enzyme activity in different treatments was determined by ANOVA and means were separated by Tukey’s HSD test at 5% level of significance.

Protein concentration of the sample 10 µl was determined by method of Bradford, 1976

SECTION-III

3.3.1. Effect of heartwood extractives on gut protozoans of R. flavipes and H. indicola

The method of Lewis and Forschler (2004) was used to count total gut protozoan populations. Termites were fed on filter paper treated in the same manner as described above for filter paper bioassay (3.2.1). After 15 days, termite hindguts were removed using a fine needle and forceps by gently removing the last two abdominal segments. Contents of five guts per treatment were pooled to form a single sample from each extractive concentrations. This sample was homogenized using a disposable pestle in 1.5 ml microcentrifuge tube and 250 µl of Trager U solution for each sample (Trager, 1934). Ten microliters of resulting solution was loaded onto a hemocytometer and numbers of protozoans were counted in a drop of 0.4 µl. Total protozoans population in termites’ gut from each treatment was calculated by following formula:

(푁푢푚푏푒푟 표푓 푐푒푙푙푠 푐표푢푛푡푒푑 × 푣표푙푢푚푒 표푓 푠푎푙𝑖푛푒 푠표푙푢푡𝑖표푛 𝑖푛 표푟𝑖푔𝑖푛푎푙 푠푎푚푝푙푒)

(푣표푙푢푚푒 표푓 ℎ푒푚표푐푦푡표푚푒푡푒푟 × 푛푢푚푏푒푟 표푓 푡푒푟푚𝑖푡푒푠 푝푒푟 표푟𝑖푔𝑖푛푎푙 푠푎푚푝푙푒)

Percentage reduction of protozoans was calculated by comparing total number of protozoans to numbers in control treatments (fresh termites out of colony and filter paper fed termites).

3.3.2. Changes in gut microbial community of H. indicola after feeding on extractives treated filter papers

Contrary to H. indicola, gut bacterial communities of R. flavipes have been previously described. Since R. flavipes is not recorded from Pakistan, diversity of bacterial communities disturbed by extractives was described from H. indicola. This could be first report of bacterial communities from H. indicola according to references in literature.

3.3.2.1. DNA extraction from termite gut

H. indicola were fed on filter papers, which were treated with 7.5 and 10 mg ml-1 of each extractives along with a control treatment as described in filter paper bioassay (3.2.1). After fed for 15 days, termites were surface sterilized by submersion in 70% ethanol for 1 min and then these were briefly rinsed in sterilized water before dissection. Termites were dissected aseptically using sterile fine scissors and fine-tipped forceps. Guts were removed in 0.01 M sterile phosphate buffered saline (PBS), washed in PBS and transferred to 1.5-ml microfuge tubes with 0.5 ml of PBS. Fifty guts from each treatment were pooled to make one sample. Isolated guts were gently crushed using a sterilized pestle. Total DNA was extracted with Thermo Scientific GeneJET Genomic DNA Purification Kit (K0721) according to Manufacturer’s Instruction. DNA was then quantified using a Nano Drop and diluted to 20 ng/µl prior to polymerase chain reaction (PCR). 16S rRNA genes were amplified through the commercial service (Molcare, Department of Biochemistry, University of Agriculture, Faisalabad) with the following methodology. Bacterial specific primer pair 27f (5'- AGAGTTTGATCCTGGCTCAG3') and 1492r (5'-GGTTACCTTGTTACGACTT-3') were used for 16S rRNA typing. Adaptors required for pyrosequencing were included in the primer sequences along with a 10 base pair on the reverse primer used to identify the different samples.

25 µl PCR reactions contained a final concentration of 1.5 mM MgCl2, 0.4 µM of forward and reverse primer each, 0.2 mM dNTPs, 5 ng template DNA, Taq DNA polymerase and nuclease free water to bring the volume to 25 µl. The constituted reaction was denatured at 94°C for 3 min. Cycling began with denaturing at 94°C for 45 sec, annealing at 55°C for 60 sec and extension for 1 min at 72°C and 2 min at 72°C. Steps 2-4 were repeated for a total of 35 cycles in a thermocycler. Integrity of DNA was checked on agarose gel (0.8 %), which was prepared IX TAE buffer. In 5 µl DNA sample, 1 µl 6X DNA loading dye was added. Gel was run at 80 V in 1X TAE buffer (Sambrook and Russell, 2000). After removing gel from apparatus, it was kept in ethidium bromide for 15-20 min and was documented. Samples were then amplified via emulsion PCR (emPCR). DNA library was added so that there was a ratio of 0.95 copies per bead. After emPCR, bead recovery and sequencing was done using the GS Junior Titanium emPCR Kit (Lib-L) according to Manufacturer’s Instructions. Raw data was extracted from GS Junior and imported into mothur (v. 1.32.1) according to Schloss et al. (2009). Sequence analysis followed 454 SOP (standard operating procedure) as provided online (Schloss et al.,

2009; Schloss et al., 2011), which removed sequences less than 200bp long. Unique sequences were aligned using the Silva-derived reference database (Schloss, 2009) and chimeras were removed using UCHIME. Data were then analysed for alpha and phylogenetic trees were constructed.

3.3.2.2. Phylogenetic analysis

Sequences obtained were compared with the published sequences in GenBank using Blast from NCBI (http: //www.ncbi.nlm.nih.gov/BLAST) to find matches to known bacteria taxa. As criteria for identification, a sequence similarity = 99% for identification to the species level and a sequence similarity = 97% as identification to the genus level was used. Sequence similarity < 97% with all sequences deposited in GenBank at that time signifies failure to identify (Drancourt et al., 2000). Groups of bacteria were identified by their closest match to GenBank. A neighbor-joining phylogenetic tree of aligned sequences was constructed using MEGA 6 software.

L1 L2 L3 L4 L5 L6

L1 L2 L3 L4 L5 L1 L2 L3 L4 L5 L6 L7 L8

b c

Fig. 3.4. Gel electrophoresis for DNA quality analysis (Photo sent by Molcare, University of Agriculture, Faisalabad). L1 (A, C) and L4 (B) shows ladder that served as a reference of measurement.

SECTION-IV

3.4.1. Choice and No-choice tests on extracted and un-extracted woods against R. flavipes and H. indicola

Extracted and un-extracted heartwood blocks of each durable wood species were subjected to choice and no-choice feeding tests according to slightly modified AWPA E1-13.

No-choice tests

Screw top jars were filled with 150 grams sand along with 27 ml distilled water and held for two hours to equilibrate moisture. For no-choice test, extracted and unextracted blocks were conditioned (33°C, 62±3%), weighed and placed on top of the dampened sand with one block in each jar.

Choice tests

For the choice test, each jar received one extracted and one un-extracted blocks. Both experiments were replicated five times

A total of 400 termites (396 workers and 4 soldiers) were released in each jar and jars were kept in a conditioning chamber at 27 ± 2 °C and 75 ± 1% R.H. for 28 days. At the end of 28 days, the numbers of alive termites were counted. Blocks were brushed to remove sand, conditioned for one week and re-weighed to determine weight loss. All blocks were rated visually using 0-10 scale as described in the AWPA standard (Table 3.3).

3.4.2. Vacuum Pressure treatment of extractives on Southern Yellow Pine and Cotton Wood against termites

Weighed and conditioned (33°C, 62±3% R.H.) southern yellow pine and cottonwood sapwood blocks (19×19×19 mm) were treated with different concentrations (2.5, 5 and 10 mg ml-1) of extractive of each wood species separately in vacuum pressure apparatus/chamber. For control treatments, blocks were treated with solvent only (ethanol-toluene) and/or water. Five blocks were placed in a 300 ml beaker containing the treatment solution in a vacuum- pressure chamber for one treatment. Blocks were held under vacuum for 30 min and after that

40 pressure was applied at 40 psi for 60 min. After pressure treatment, blocks were blotted dry using paper towels, weighed and re-conditioned at 33°C and 62±3% RH.

Termites’ bioassay was conducted according to AWPA E-1 with some modifications as described in 3.4.1.

a b c Fig. 3.5. Vacuum (a), pressure chamber (b) and test set up (c) according to AWPA E-1.

3.4.3. Determination of leaching resistance of extractives impregnated woods against termites

In order to determine leaching of extractives from treated blocks, procedure of AWPA E-11 was followed with some modifications. After conditioning, five blocks were submerged in 300 ml of deionized water in a 500 ml vessel and were subjected to vacuum to impregnate the blocks. Then vessel was subjected to mild agitation and water was changed after 6, 24 and 48 and thereafter at 48-hours interval. Leaching was continued for a 14 days. No- choice tests with R. flavipes and H. indicola were run according to AWPA E1. Wood weight loss and termites mortality was recorded after exposure to the termite species after 28 days.

a b Fig. 3.6. Orbital shaker for leaching (a) and test set up (b) according to AWPA E-1.

3.4.4. Wood preservative potential of extractives in combination with linseed oil

Weighed and conditioned (33°C, 62 ± 3% R.H.) southern yellow pine and cottonwood sapwood blocks (19×19×19 mm) were pressure treated with the highest concentration (10mg ml-1) of each extractive in combination with 20% boiled linseed oil. For control treatment, blocks were treated only with linseed oil in Ethanol: toluene solvent. There were five replicates for each treatment. No-choice tests with R. flavipes and H. indicola were run according to AWPA E1. Wood weight loss and termites mortality was compared after exposure to the termite species after 28 days. CW and SYP blocks (19 x19 x 19 mm) were vacuum pressure impregnated with highest concentrations of heartwood extractives alone and in combination of linseed oil (20%). There was also a control treatment, which was only treated with linseed oil. All specimens were exposed to R. flavipes and H. indicola for 28 days according to AWPA E- 1. Weight loss and mortality of termites was analyzed using one way ANOVA. Means were separated using Tukey HSD test.

3.4.5. Evaluations of durable woods and their extractives against Postia placenta and Trametes versicolor

Two soil bottle experiments were conducted according to method described in AWPA E-10 (AWPA 2014).

In first experiment, southern pine wood feeder strips were used to start soil bottles with either Postia placenta (Fr.) MJ Larsen & Lombard (Mad-698-R) or Trametes versicolor L. (Loyd) (Mad-697). SYP and CW blocks (19×19×19 mm) were treated with extractives from the four wood types at three rates: 2.5, 5 and 10 mg ml-1 and compared to untreated, water treated and solvent treated controls.

In the second experiment, southern pine feeder strips were used to start soil bottles with P. placenta, while red maple (Acer rubrum L) feeder strips were used to start T. versicolor bottles. Un-extracted blocks of the four durable woods were compared with extracted blocks in assays where hardwoods were challenged with the white rot fungus and softwoods were challenged with the brown rot fungus.

Both sets of soil bottle experiments were conducted for 12 weeks at 27°C in a 70% R.H. walk-in incubator. Blocks were weighed before and after experiments following conditioning at 27°C and 30% R.H. to calculate weight loss. Mean weight loss due to decay was modelled in Minitab to statistically test significance (ANOVA) and means were compared using Tukey’s HSD. Durability indices were calculated from each test by comparing mean weight loss of material to that of non-durable as referenced in EN 350-1 (EN 1994) using the following formula:

X* = (mean weight loss of the test specimens) / (mean weight loss of the reference material)

*X=0-0.14 (very durable), 0.15-0.29 (durable), 0.30-0.59 (moderately durable), 0.6-0.89 (slightly durable, 0.9 and greater (not durable).

SECTION-V

3.5. Characterization of heartwood extractives using Gas chromatography – Mass spectrometry (GC-MS) analysis

Analyses for the identification of components from extractives were performed by coupling gas chromatography (GC) and mass spectrometry (MS) via an Agilent 7890B GC. An Agilent 19091S-433UI HP-5ms Ultra Inert column was used. The HP-5ms capillary column was of 0°C-325°C (350°C) 30m x 250 um x 0.25um. The temperature of the gas chromatography column was programed from 50-270°C. Solvent delay was set at 3-6 minutes. The temperature of ion source in the mass spectrometer was held at 230°C and the quad temperature was 150°C. For all extracts the sample size injected was 1µL.

The Agilent 7890B GC was equipped with a split less injector at 270°C and an electron capture detector (µECD) at 250°C. Injection was done in the split less mode.

For extracts from Tectona grandis, the starting temperature was 75°C ramped to 230°C at 5°C/min and held for 80 minutes (Xie et al., 2011).

For Dalbergia sissoo, a dual ramp up was used where the starting temperature was 45°C ramped to 165°C at 4°C/min then ramped to 280°C at 4.5°C/min and held for 35 minutes (Aly et al., 2013).

For Cedrus deodara, a dual ramp up was used where the starting temperature of 70°C ramped to 200°C at 10°C/min, held for 5 minutes then ramped to 300°C at 10°C/min and held for 10 minutes (Chaudhary et al., 2011).

For Pinus roxburghii, a dual ramp up was used For Pinus roxburghii where the starting temperature of 45°C ramped to 165°C at 4°C/min then ramped to 280°C at 15°C/min and held for 9 minutes (Shah and Qadir, 2014; Hassan and Amjid, 2009).

Helium was used as the carrier gas at a constant flow rate of 1 ml/min. All mass spectra were recorded in the electron impact ionization (EI) at 70 electron volts. The mass spectrometer scanned from m/z 3-700 at a rate of 2 scans per second. An integrator automatically calculated peak area. The top five compounds taken as high percentage on the samples basis were identified using the NIST14 library.

SECTION- VI

3.6. Potential of wood extractives and linseed oil as preservatives in field tests against termites and decay

3.6.1. Evaluation of extractives as wood preservatives using ground proximity test

Ground proximity test was according to AWPA E-26 with some modifications. It was designed to expose wood test specimens to a severe challenge by the termites in a field exposure. Wooden chunks of size, 12.5×3.75×2.5 cm, were prepared from sapwood of SYP and CW boards that have not been previously treated and showed no visible appearance of mold, decay or insect attack. After conditioning (33°C, 62±3% R.H), all chunks were labeled with durable, inert and corrosion resistant tags. Weighed chunks were vacuum-impregnated with the highest concentrations of extractives alone and in combination with 20% linseed oil as well. A positive control treatment in which Cu-NAP impregnated chunks was also included among treatments.

Treatment procedure

First step before the treatment was to determine the quantity of the solution to fill void volume of the tank below height of the wood. This was calculated by finding volume of tank below height of the wood (height of wood x length x width of the tank) and then subtracting the volume of the wood to obtain the void volume around the wood.

The second step was the estimation of solution taken up by the wood (pound). This was calculated by multiplying expected uptake with the volume of wood while expected uptake was just based on previous experiences of Steve Halverson (FPL). In this way total volume needed was calculated. After adding wood in the treatment pan, it was filled with the treatment solution. It was treated using full cell vacuum- pressure with initial vacuum 27 in/Hg for 30 minutes and then the pressure was applied at 150 psi for 60 minutes. After treatment, all chunks were re- weighed to calculate gross absorption (pcf). After conditioning and re-weighing of test specimens, these were exposed to the termites at two different sites (Mississippi, USA, and Lahore, Pakistan) according to AWPA E-26 and data were taken after 6 and 12 months of exposure.

3.6.2. Evaluation of extractives as wood preservatives using field stake test

Field stake test was according to AWPA E-7 with some modifications was followed. Stakes, of size 45.7×1.09×1.09 cm, were prepared from sapwood of SYP and CW boards that have not been previously treated and showed no visible appearance of mold, decay or insect attack. After conditioning (33°C, 62 ± 3% R.H), all stakes were labeled with durable, inert and corrosion resistant tags. Weighed stakes were vacuum- impregnated with the highest concentration of extractives alone and also in combination with 20% linseed oil. There was also be positive control treatment in which stakes were impregnated with Cu-NAP. All treatment procedure was same as in E-26 and field stakes were installed according to E-7 at both sites (Pakistan and USA). Data was taken after 6 and 12 months of exposure. Obtained data was analyzed using one way ANOVA in Minitab 16 and means were separated using Tukey’s HSD test at 0.05 level of significance.

a b

Fig. 3.7. Ground proximity test (AWPA- E26) set up in USA (a) and Pakistan (b)

Table 3.3. Termite rating scheme (AWPA E-1, E-7, E-26). 10 Sound 9.5 Trace, surface nibbles permitted 9 Slight attack, up to 3% of cross sectional area affected 8 Moderate attack, 3-10 % of cross sectional area affected 7 Moderate/ severe attack, penetration, 10-30% of cross sectional area affected 6 Severe attack, 30-50% of cross sectional area affected 4 Very severe attack, 50-75 % of cross sectional area affected 0 Failure

b a

Fig. 3.8. Field stake test (AWPA- E7) set up in USA (a) and Pakistan (b).

Table 3.4. Decay rating scheme (AWPA E-10, E-7).

10 Sound No sign or evidence of decay, wood softening or discoloration caused by microorganism attack 9.5 Trace-suspect Some areas of discoloration or softening associated with superficial microorganism attack 9 Slight attack Decay and wood softening is present. up to 3% of the cross sectional area is affected 8 Moderate attack Similar to 9 but more extensive attack with 3-10 of cross sectional area affected 7 Moderate/Severe Sample has been 10-30% cross sectional area decayed attack 6 Severe Sample has been 30-50% cross sectional area decayed 4 Very severe Sample has been 50-75% cross sectional area decayed 0 Failure Sample has functionally failed. It can either be broken by hand due to decay, or the evaluation probe can penetrate through the sample

CHAPTER FOUR RESULTS

Section- I

Selection of Test Woods and Preparation of Heartwood Extractives Termites Collection and Maintenance

4.1.1. Yield of heartwood extractives from four durable woods A total of 6-12 batches of Soxhlet apparatus were run to prepare 1000-1200 ml of extractives (150 mg ml-1) from each durable wood. There were 18-20 Soxhlets in each batch, 10-12 grams of air dried shavings were filled in each Soxhlet and extraction was done for almost six hours for each batch. To calculate extraction yield, weight loss of air dried wood shavings before and after extraction was calculated. Yield was also calculated by finding the difference between weight of round bottom flask before and after rotary evaporation. Extraction yields were expressed as a percentage of dry wood shavings. Table 4.1 shows percentages of extractives contents and total weight of shavings from each wood.

Table 4.1. Yield of heartwood extractives from durable woods extracted with ethanol: toluene (2:1).

Heartwoods Number of Soxhlets Weight of shavings (g) Extraction yield (%) T. grandis 174 1958.24 5.51 D. sissoo 118 1206.71 9.11 C. deodara 120 1046.71 9.67 P. roxburghii 130 1434.5 9.40

4.1.2. Termites’ collection and maintenance

Collected R. flavipes from the dead and fallen logs of Pinus were kept in large metal buckets and maintained at 25 °C in dark. To separate termites from dead logs, moist card board was used to attract termites. H. indicola was maintained on moist cardboard in vacuum desiccators in the darkness. Fine camel hair brush was used to separate termites from cardboards and for counting.

Section- II

Toxicity, repellency, antifeedant and antioxidant activities of heartwood extractives

4.2.1. Toxicity tests of filter paper treated with heartwood extractives of four wood species against R. flavipes and H. indicola

Table 4.2.1 shows LC50s of crude heartwood extractives against R. flavipes and H. indicola. Extractives showed differential activity against termite species. Minimum LC50s for R. flavipes was given by extractives of D. sissoo (3.89 mg ml-1) while against H. indicola T. -1 grandis showed minimum LC50 (3.21 mg ml ). C. deodara appeared to be least toxic to R. flavipes while response of H. indicola was intermediate (4.80 mg ml-1) to these extractives. Heartwood extractives of P. roxburghii were least toxic to H. indicola (5.66 mg ml-1).

Table 4.2.1. Median lethal concentrations (LC50s) of filter paper treated with four type of heartwood extractives against R. flavipes and H. indicola.

2 Termite spp. Wood spp. LC50 χ Fiducial limit Regression equation (mg ml-1) CI (Y= ax + b) R. flavipes T. grandis 4.43 100.03 4.18-4.67 -221.66x + 63489 D. sissoo 3.89 81.56 3.21-3.98 -238.32x + 56079 C. deodara 7.47 34.50 6.93-8.08 -74.618x + 56876

P. roxburghii 4.60 39.65 4.12-5.01 -202.18x + 58573 H. indicola T. grandis 3.21 141.38 2.99-3.43 -24.489x + 3582.5 D. sissoo 5.54 6.48 5.22-5.86 -35.408x + 3756.2 C. deodara 4.80 37.21 4.52-5.07 -16.067x + 3521.5 P. roxburghii 5.66 56.63 5.31-6.23 -21.471x + 3602.6

spp. = species Effects of each of the wood extractives on mortality of termites, weight loss and area loss of filter paper are shown in Tables 4.2.2 to 4.2.5. The general observations in all these cases are that both termite species consumed significantly more amount of filter papers treated with solvent only as compared to filter papers treated with extractives. Both termites fed on solvent treated filter paper alone exhibited lower mortality than those fed on extractive treated filter paper. Weight loss and area loss of the filter paper were significantly reduced with increasing extractive concentrations. In case of T. grandis, 99 and 100% mortalities of R. flavipes and H. indicola were recorded @ 10 mg ml-1 of extractives, respectively. Weight loss of filter paper was more (4.2 %) by R. flavipes than H. indicola (1.0 %), however, area reduction of filter papers by two termite species was similar (12.3 and 11.9 %, respectively). Results showed a linear relationship between amount of filter paper consumption and termites’ mortality. Maximum consumption of filter paper by both termite species (37.8 and 41.7 %) was observed in control

52 treatments. A parallel trend was also observed among the death of both termite species and weight loss of filter paper (Table 4.2.2). Table 4.2.2. Mean weight loss, area loss and mortality of R. flavipes and H. indicola after toxicity test on filter paper treated with T. grandis heartwood extractives.

Termite spp. Concentration Mortality (%) Weight loss (%) Area loss (%) Control 4.0 ± 1.15c 37.8 ± 6.98a 39.1 ± 1.51a -1 15.6 ± 1.20c 31.6 ± 0.90a 28.9 ± 0.87ab

R.. flavipes 1.25 mg ml -1 c ab ab 2.5 mg ml 21.3 ± 3.18 26.7 ± 1.30 24.3 ± 1.74 -1 b ab bc 5.0 mg ml 59.0 ± 9.71 23.3 ± 1.89 23.4 ± 0.19 -1 78.3 ± 1.76ab 17.4 ± 4.96bc 17.3 ± 0.62bc

7.5 mg ml -1 a c c 10 mg ml 99.0 ± 0.57 4.2 ± 1.12 12.3 ± 0.33 F=72.15; p<0.05 F=10.31; p<0.05 F=11.24; p<0.05 Control 2.3 ± 1.20d 41.7 ± 4.30a 40.4 ± 4.78a -1 d a a 1.25 mg ml 3.6 ± 0.88 37.9 ± 3.54 38.9 ± 0.51

H. indicola -1 c b b 2.5 mg ml 59.0 ± 3.51 10.3 ± 0.66 25.9 ± 2.05 -1 b b bc 5.0 mg ml 75.3 ± 0.33 6.3 ± 0.33 20.5 ± 0.78 -1 b c bc 7.5 mg ml 80.3 ± 1.20 2.6 ± 0.33 21.3 ± 1.79 -1 a c c 10 mg ml 100 ± 0.00 1.0 ± 0.57 11.9 ± 1.39 F=70.00; p<0.05 F=10.83; p<0.05 F=22.68; p<0.05 Means ± SE sharing same letters in columns for each termite species are not significantly different from each other at p>0.05. spp. = species.

R. flavipes and H. indicola after exposure to D. sissoo heartwood extractives showed 72 and 81.3 % mortality at 10 mg ml-1 which was contrary to T. grandis heartwood extractives whose highest concentrations yielded 99 to 100 % mortality. In case of R. flavipes, non- significant difference was recorded in mortality, weight and area losses of filter papers among concentrations of extractives except 1.25 mg ml-1. However, mortality in H. indicola, weight and area losses of filter papers by these termites varied significantly among the concentrations of extractives (Table. 4.2.3).

Table 4.2.3. Mean weight loss, area loss and mortality of R. flavipes and H. indicola after toxicity tests on filter paper treated with D. sissoo heartwood extractives.

Termite spp. Concentration Mortality (%) Weight loss (%) Area loss (%) Control 4 ± 1.15b 37.8 ± 6.98a 39.1 ± 5.29 a -1 60 ± 17.3a 11.53 ± 3.29b 8.7 ± 4.17 b

R.. flavipes 1.25 mg ml -1 2.5 mg ml 50 ± 11.4a 8.7 ± 0.77 b 8.1 ± 0.74 b -1 5.0 mg ml 60 ± 12.2a 8.2 ± 2.23 b 8.0 ± 2.66 b -1 67.3 ± 12.3a 6.0 ± 1.1 b 6.2 ± 1.29 b

7.5 mg ml -1 10 mg ml 72 ± 8.0a 5.9 ± 1.8 b 6.1 ± 0.59 b F=4.63; p<0.05 F=13.11; p<0.05 F=18.30; p<0.05 Control 2.3 ± 1.20d 41.7 ± 3.37a 40.4 ± 4.46a -1 1.25 mg ml 16 ± 2.31cd 46.6 ± 1.07a 36.46 ± 3.58ab

H. indicola -1 2.5 mg ml 23.3 ± 3.33c 34.6± 1.15bc 33.32 ± 0.43ab -1 5.0 mg ml 51.3 ± 1.33b 24.1 ± 2.54c 26.83± 1.86bc -1 7.5 mg ml 66.6 ± 6.36a 5.3 ± 2.30d 18.15 ± 1.65cd -1 10 mg ml 81.3 ± 1.33a 3.9 ± 2.15d 7.21 ± 0.51d F=94.48; p<0.05 F=66.09; p<0.05 F=23.79; p<0.05 Means±SE sharing same letters in columns for each termite species are not significantly different from each other at p>0.05. spp. = species.

C. deodara heartwood extractives showed concentration dependent mortalities of both termite species. 80 and 94.6 % mortality of R. flavipes and H. indicola was recorded @ 10 mg ml-1 of extractives, respectively. Weight and area losses of filter papers were more (13.3 and 11.6 % respectively) by H. indicola than R. flavipes (0.8 and 5.8 % respectively). In both termite species, the percentage of filter paper consumed was found to be lower in extractives treated groups than in controls. Filter paper was consumed less at the highest concentration (10 mg ml-1) where there was maximum mortality of termites (Table. 4.2.4).

Table 4.2.4. Mean weight loss, area loss and mortality of R. flavipes and H. indicola after toxicity tests on filter paper treated with C. deodara heartwood extractives. Termite spp. Concentration Mortality (%) Weight loss (%) Area loss (%) Control 4 ± 1.15c 37.8 ± 6.98a 39.1 ± 5.29a -1 7.3 ± 1.33c 27.8 ± 3.11ab 32.5 ± 4.56 a

R.. flavipes 1.25 mg ml -1 2.5 mg ml 12 ± 1.15c 16.9 ± 0.73bc 15.7 ± 0.82b -1 5.0 mg ml 18 ± 5.03c 9.9 ± 4.39c 10.5 ± 1.14b -1 40.7 ± 17.9b 2.7 ± 1.70c 6.4 ± 0.79b

7.5 mg ml -1 10 mg ml 80 ± 5.03a 0.8 ± 0.06c 5.8 ± 1.03b F=13.48; p<0.05 F=15.70; p<0.05 F=22.80; p<0.05 Control 2.3 ± 0.88e 41.7 ± 3.37a 40.4 ± 4.46a -1 1.25 mg ml 10.6 ± 1.25e 37.6 ± 0.33ab 37.0 ± 1.15ab

H. indicola -1 2.5 mg ml 25.1 ± 1.62d 32.6 ± 0.33bc 30.0 ± 0.57bc -1 5.0 mg ml 51.7 ± 1.71c 28.3 ± 0.33c 26.6 ± 0.82c -1 7.5 mg ml 62.47 ± 1.35b 20.0 ± 0.57d 22.6 ± 0.88c -1 10 mg ml 94.6 ± 3.18a 13.3 ± 0.88d 11.6 ± 1.76d F=370.92; p<0.05 F=53.98; p<0.05 F=24.56; p<0.05 Means±SE sharing same letters in columns for each termite species are not significantly different from each other at p>0.05. spp. = species.

Mortalities in both R. flavipes and H. indicola exposed to P. roxburghii heartwood extractives were shown to increase with increasing concentrations. Maximum mortality of R. flavipes (82.6%) and H. indicola (80%) was found at highest extractive concentration level (10 mg ml-1). consumption of filter paper by both termite species was found to be lower in treated filter paper groups than controls (Table 4.2.5). Tables 4.2.6 to 4.2.8 show ANOVA of percent mortality of termite species, weight and area losses of filter papers treated with extractives of four woods at various concentrations. Mortality of both termite species after feeding on filter papers treated with heartwood extractives of wood species revealed that there was non-significant difference between mortalities of both species after feeding on treated filter papers. All other interactions i.e. termite species and wood species; termite species and concentrations of extractives; wood species and concentrations of extractives; termite species, wood species and Concentrations of extractives were significantly different from one another. Similar trend was observed for percent weight and area losses of filter paper after feeding of both termite species (Appendices 4.1 to 4.3).

Table 4.2.5. Mean weight loss, area loss and mortality of R. flavipes and H. indicola after toxicity tests on filter paper treated with P. roxburghii heartwood extractives. Termite spp. Concentration Mortality (%) Weight loss (%) Area loss (%) Control 4.0 ± 1.15c 38.0 ± 6.98a 39.12 ± 5.29a -1 24.6 ± 1.76c 20.0 ± 0.25b 19.3 ± 3.69b R.. flavipes 1.25 mg ml -1 2.5 mg ml 29.3 ± 3.53bc 20.7 ± 1.00b 18.7 ± 1.09b -1 5.0 mg ml 34.6 ± 8.82bc 13.6 ± 3.94bc 12.1 ± 2.62bc -1 67.3 ± 16.3ab 8.6 ± 1.03bc 3.7 ± 1.01c 7.5 mg ml -1 10 mg ml 82.6 ± 7.69a 2.9 ± 1.29c 1.5 ± 0.73c F=12.09; p<0.05 F=13.02; p<0.05 F=21.72; p<0.05 Control 2.3 ± 0.88e 41.7 ± 4.30a 40.4 ± 4.46a -1 1.25 mg ml 10.8 ± 0.16de 14.3 ± 0.65b 12.98 ± 1.16b

H. indicola -1 2.5 mg ml 19.6 ± 0.50d 13.1 ± 0.98b 9.15 ± 1.79b -1 5.0 mg ml 33.6 ± 0.52c 7.3 ± 0.90bc 8.05 ± 3.20b -1 7.5 mg ml 60 ± 1.15b 5.5 ± 0.29c 6.81 ± 0.06b -1 10 mg ml 80 ± 5.72a 3.1 ± 0.66c 5.56 ± 0.20b F=154.05;p<0.05 F=85.28; p<0.05 F=30.49; p<0.05 Means±SE sharing same letters in columns for each termite species are not significantly different from each other at p>0.05. spp. = species.

Table 4.2.6. Mean mortality (%) of R. flavipes and H. indicola after toxicity tests on filter paper treated with four heartwood extractives. R. flavipes H. indicola

Heartwoods Conc. T. grandis D. sissoo C. deodara P. roxburghii T. grandis D. sissoo C. deodara P. roxburghii Solvent 3.33 ±1.15j 3.33±1.33j 3.33±1.15j 3.33±1.33j 3.67±1.20j 3.67±1.20j 3.67±1.20j 3.67±1.20j 1.25 15.67±1.20h-j 60.0±17.3b-g 7.33±1.33ij 24.67±1.76g-j 2.33±0.88j 16.00±2.31h-j 10.60±1.25ij 10.83±0.16ij 2.5 21.33±3.18h-j 50.0±11.4c-h 12.00±1.15ij 29.33±3.53f-j 59.00±3.51b-g 28.89±4.84f-j 25.13±1.62g-j 19.73±0.50h-j 5 59.00±9.71b-g 60.0±12.2b-g 18.00±5.03h-j 34.67±8.82e-j 75.33±0.33a-d 51.33±1.33c-h 51.73±1.71c-h 33.66±0.52e-j 7.5 78.33±3.76a-c 67.3±12.3a-e 40.7±17.9d-i 67.3±16.3a-e 80.33±1.20a-c 66.67±6.36a-e 62.47±1.35b-f 60.00±1.15b-g 10 99.00±0.57a 72.00±8.00a-d 80.00±5.03a-c 82.67±7.69a-c 100.00±0.00a 81.33±1.33a-c 94.67±3.18ab 81.40±5.91a-c Means±SE sharing same letters in columns and rows are not significantly different from each other at p>0.05. Conc. = concentration (mg ml- 1).

Table 4.2.7. Mean weight loss (%) of filter papers after feeding of R. flavipes and H. indicola treated with four heartwood extractives. R. flavipes H. indicola Heartwoods Conc. T. grandis D. sissoo C. deodara P. roxburghii T. grandis D. sissoo C. deodara P. roxburghii Solvent 37.88±6.98ab 36.33±6.08a-c 37.88±6.98ab 36.33±6.08a-c 37.88±6.98ab 37.88±6.98ab 37.88±6.98ab 37.88±6.98ab 1.25 31.65±0.90a-f 11.53±3.29g-m 27.80±3.11a-h 20.07±0.25b-l 31.65±0.90a-f 46.69±1.06a 37.66±0.33ab 14.31±0.65e-m 2.5 23.36±1.30b-k 8.72±0.77h-m 16.96±0.73d-m 20.77±0.99b-l 23.36±1.30b-k 33.64±0.53a-d 32.66±0.33a-e 13.13±0.98f-m 5 26.77±1.89b-i 8.24±2.23i-m 9.95±4.39g-m 13.66±3.94e-m 26.77±1.89b-i 24.13±2.55b-j 28.33±0.33a-g 7.30±0.90j-m 7.5 17.40±4.95c-m 6.07±1.10j-m 2.72±1.70lm 8.66±1.03h-m 17.40±4.95c-m 5.37±2.31j-m 20.00±0.57b-m 5.49±0.29j-m 10 4.23±1.12k-m 5.94±1.81j-m 0.86±0.06m 2.93±1.29lm 3.60±0.35lm 3.92±2.15lm 13.33±0.88f-m 3.10±0.66lm Means±SE sharing same letters in columns and rows are not significantly different from each other at p>0.05. Conc. = concentration (mg ml- 1).

Table 4.2.8. Mean area loss (%) of filter papers after feeding of R. flavipes and H. indicola treated with four heartwood extractives. R. flavipes H. indicola Heartwoods Conc. T. grandis D. sissoo C. deodara P. roxburghii T. grandis D. sissoo C. deodara P. roxburghii Solvent 39.12±5.29a 35.21±2.11ab 39.12±5.29a 35.21±2.11ab 39.12±5.29a 39.12±5.29a 39.12±5.29a 39.12±5.29a 1.25 28.91±0.78a-d 8.74±4.17g-k 32.53±4.56a-c 19.33±3.69c-i 28.91±0.78a-d 36.46±3.58ab37.00±1.15 ab 12.98±1.16e-k 2.5 24.38±3.74a-f 8.19±0.74h-k 15.71±0.82d-k 18.70±1.09c-j 24.38±3.74a-f 31.77±1.41a-30.00±0.57c a-d 9.15±1.78g-k 5 23.44±0.19b-g 8.05±2.65h-k 10.51±1.13f-k 12.20±2.63e-k 23.44±0.19b-g 26.83±1.86a-26.66±0.88e a-e 8.04±3.20h-k 7.5 17.39±0.61c-j 6.27±1.29i-k 6.49±0.79i-k 3.75±1.01jk 17.39±0.61c-j 18.15±1.65c-22.66±0.88j b-h 6.80±0.06i-k 10 12.33±1.76e-k 6.17±0.59i-k 5.84±1.03i-k 1.51±0.73k 1.67±0.33k 7.21±0.51i-k 11.67±1.76e-k 5.56±0.20i-k Means±SE sharing same letters in columns and rows are not significantly different from each other at p>0.05. Conc. = concentration (mg ml- 1)

4.2.2. Repellent and antifeedant activities of heartwood extractives of four durable species against R. flavipes and H. indicola a) Repellent activities of wood extractives Fig. 4.2.1 shows repellent effect of T. grandis extractives on workers of R. flavipes on treated and untreated filter papers in Petri dishes. Termites moved from treated to untreated filter paper discs; at 1.25 mg ml-1, 64.25 % termites were observed on untreated filter paper after 1 hour but numbers of termites present on treated and untreated filter paper were statistically non- significant from each other during 2 to 12 hours. As concentrations increased, fewer termites were observed on treated filter paper. However, at 10 mg ml-1 concentration, mostly termites were present on untreated filter paper indicating the strong repellent activity of heartwood extractives. At highest concentration numbers of termites repelled were 98.67% at 1 hour which rose to 100% after 12 hours. Statistically there was non- significant difference in number of termites which were present on un-treated filter paper starting from 1 to 12 hours at highest concentrations. There was significant effect of concentrations on the termites repellency (p< 0.05) but time and interaction effects were non- significant (p> 0.05). Fig. 4.2.2 shows results of H. indicola workers repelled by T. grandis heartwood extractives from treated filter papers in Petri dishes. At lowest concentration (1.25 mg ml-1), there was no significant difference between the number of termites which were present on treated and un-treated filter paper. There was very less repellency of termites observed at the lowest concentration even after 12 hours. As concentrations increased fewer termites were observed on treated filter paper. Most of the termite workers were present on untreated filter paper at 10 mg ml-1 concentration indicating the strong repellent activity of heartwood extractives. At highest concentration the numbers of termites repelled were 90. 67% at 1 hour but after 12 hours 94.67% termites were present on untreated filter paper. Statistically there was non-significant effect of time on the presence of termites on treated and untreated filter paper discs as opposed to significant effect of concentrations on termites’ repellency (p< 0.05). T. grandis heartwood extractives exhibited same repellent activity level for both termites’ species at the highest concentration.

1h 2h 3h 4h 12h

120

100

40 Repellency Repellency (%) 20

0 Solvent 1.25 mg/ml 2.5 mg/ml 5 mg/ml 7.5 mg/ml 10 mg/ml 1h 2.33 64.51 86.67 88 92 98.67 2h 2.11 61.3 90.67 94.67 98.67 100 3h 0.1 56 90.67 94.67 98.67 100 4h 1.11 52 90.67 96 96 98.67 12h 1.33 60 90.33 100 100 100 Concentrations of T. grandis extractives

Fig. 4.2.1. Repellent activities of T. grandis heartwood extractives against R. flavipes.

1h 2h 3h 4h 12h 120

100

40 Repellency Repellency (%) 20

0 Solvent 1.25 mg/ml 2.5 mg/ml 5 mg/ml 7.5 mg/ml 10 mg/ml 1h 0 44 66.67 65.33 94.67 90.67 2h 0 42.22 68 76 89.33 90.67 3h 0 66.7 82.67 88 92 92 4h 0 65.3 81.33 88 100 94.67 12h 1.33 64.7 88 88 94.67 94.67

Concentrations of T. grandis extractives

Fig. 4.2.2. Repellent activities of T. grandis heartwood extractives against H. indicola.

Fig. 4.2.3 shows the results when workers of R. flavipes were repelled by D. sissoo heartwood extractives on treated filter papers in Petri dishes. At lowest concentration there was non- significant difference in number of termites present on treated and un-treated filter paper starting from 1 hour to 12 hour as in the control treatment where the half filter paper was treated with solvent (ethanol: toluene). As the concentrations increased, fewer termites were observed on treated filter papers. Most of the termites were present on untreated filter papers at 10 mg ml-1 concentration resembling strong repellent activity of T. grandis heartwood extractives. Numbers of termites repelled were 94.67% after 12 hours at the highest concentration but this repellency percentage was significantly different from other time intervals at the highest concentration. There was significant effect of concentrations on the termite’s repellency (p< 0.05). Significant difference among concentrations repelling termites was observed, however, the response was not among concentrations of D. sissoo heartwood extractives. . Fig. 4.2.4 shows that D. sissoo extractives at lowest concentration showed no significant difference in numbers of H. indicola present on treated and un-treated filter paper starting from 1 hour to 12 hour as in control treatment. As the concentrations increased fewer termites were observed on treated filter paper. However, at 10 mg ml-1, concentration most of the termites were present on untreated filter paper indicating the strong repellent activity of heartwood extractives. At highest concentration the number of termites repelled were 88 % after 12 hours but this repellency percentage was significantly different from other time intervals at highest concentration. There was significant effect of concentrations on the termite’s repellency. D. sissoo showed concentration dependent repellency against H. indicola but it was less as compare to against R. flavipes at maximum concentration.

1h 2h 3h 4h 12h

120

100

40 Repellency Repellency (%) 20

0 Solvent 1.25 mg/ml 2.5 mg/ml 5 mg/ml 7.5 mg/ml 10 mg/ml 1h 6.67 29.3 52 55.33 46.7 34.7 2h 5.33 25.3 76 50.7 50.7 48 3h 0 21.3 84 42.7 56 53.3 4h 1.11 22.7 85.33 58.7 68 69.3 12h 1.33 24 94.67 74.22 85.33 94.67 Concentrations of D. sissoo

Fig. 4.2.3. Repellent activities of D. sissoo heartwood extractives against R. flavipes.

1h 2h 3h 4h 12h

100 90 80 70 60 50 40

Repellency Repellency (%) 30 20 10 0 Solvent 1.25 mg/ml 2.5 mg/ml 5 mg/ml 7.5 mg/ml 10 mg/ml 1h 1.64 13.3 42.67 55.33 60 61.33 2h 3.34 13.3 62.67 62.67 65.3 65.3 3h 0 16.3 61.33 64 66.67 66.67 4h 1.11 22.7 62.67 62.7 68 68 12h 1.33 32 72 74.22 85.33 88 Concentrations of D. sissoo Fig. 4.2.4. Repellent activities of D. sissoo heartwood extractives against H. indicola.

Fig. 4.2.5 shows results of repellency when workers of R. flavipes were exposed to C. deodara heartwood extractives. At lowest concentration there was non-significant difference in numbers of termites present on treated and un-treated filter paper starting from 1 hour to 12 hour as in the control treatment where the half filter paper was treated with solvent (ethanol: toluene) at the lowest concentration. Termites correspondingly repelled with each increasing concentrations on treated filter paper. Repellency was >80% at 2.5 mg ml-1 at 1 hour while at 10 mg ml-1 repellency was >98% having significant difference between two extreme concentrations. All other concentrations had non-significant difference among themselves except 1.25 mg ml-1. However, at 10 mg ml-1 concentration, 98% termites were present on untreated filter paper at first hour after release and remained there up to 12 hours. Numbers of termites repelled at 12th hour were 98.67% at the highest concentration and were non- significantly different from other time intervals at the highest concentration. Non-significant difference among concentrations except 1.25 mg ml-1 for repellency was recorded (p< 0.05). Repellent activities of heartwood of C. deodara showed linear trend with the concentrations. Fig. 4.2.6 shows non-significant difference among repelled termites (H. indicola) on C. deodara treated and un-treated filter papers at 1st and up to 12th hour after release. 94.67% to 100% termites repellency 1 hour to 12 hours was non-significantly different from each other at 24th hour at highest the concentration. Extractives of P. roxburghii did not show any repellent activities against R. flavipes at lower concentrations even after 12 hours. Repellency was not observed after 12 hours at the highest concentration as well. Extractives of P. roxburghii repelled in a same manner as shown in R. flavipes but H. indicola at the highest concentration, repellency in H. indicola was less (<40%) after 12 hours (Figs. 4.2.7 and 4.2.8). Overall all extractives showed repellent activity against both species of termites except P. roxburghii. Maximum activity was exhibited by C. deodara extractives against both species. ANOVA regarding repellency of extractives showed that only interaction between resistant wood species and concentrations of extractives was significant, all other interactions were non- significantly different (Appendix. 4.4).

1h 2h 3h 4h 12h

120

100

40 Repellency Repellency (%)

0 Solvent 1.25 mg/ml 2.5 mg/ml 5 mg/ml 7.5 mg/ml 10 mg/ml 1h 6.67 65 86.67 88 92 98.67 2h 5.33 61.3 90.67 94.67 98.67 100 3h 0 56 90.67 94.67 98.67 100 4h 1.11 52 90.67 96 96 98.67 12h 1.33 60 90.33 100 100 100 Concentrations of C. deodara

Fig. 4.2.5. Repellent activities of C. deodara heartwood extractives against R. flavipes.

1h 2h 3h 4h 12h

120

100

40 Repellency Repellency (%)

0 Solvent 1.25 mg/ml 2.5 mg/ml 5 mg/ml 7.5 mg/ml 10 mg/ml 1h 6.67 54.7 53.33 85.33 86.67 94.67 2h 5.33 61.3 57.33 86.67 90.67 100 3h 0 56 90.67 86.67 93.33 100 4h 1.11 52 90.67 96 93.33 98.67 12h 1.33 60 93.33 100 100 100 Concentrations of C. deodara

Fig. 4.2.6. Repellent activities of C. deodara heartwood extractives against H. indicola.

1h 2h 3h 4h 12h

45 40 35 30 25 20

15 Repellency Repellency (%) 10 5 0 Solvent 1.25 mg/ml 2.5 mg/ml 5 mg/ml 7.5 mg/ml 10 mg/ml 1h 1.33 2.67 9.33 15.05 15.05 29 2h 0 0 9.55 17.3 9.33 28 3h 0 1.33 10.56 13.3 14.7 37 4h 1.11 0 9.58 9.33 14 37.3 12h 1.33 0 11 18.7 16 36 Concentrations of P. roxburghii

Fig. 4.2.7. Repellent activities of P. roxburghii heartwood extractives against R. flavipes.

1h 2h 3h 4h 12h

45 40 35 30 25 20 15

Repellency Repellency (%) 10 5 0 Solvent 1.25 mg/ml 2.5 mg/ml 5 mg/ml 7.5 mg/ml 10 mg/ml 1h 2.33 9 9.33 10.69 10 38.7 2h 1.33 6.67 8.56 14 9.33 38.7 3h 0 8.3 12 13.3 14.7 36 4h 1.11 6.67 6.67 9.33 14.9 37.3 12h 1.33 7.9 8 15 16 36 Concentrations of P. roxburghii

Fig. 4.2.8. Repellent activities of P. roxburghii heartwood extractives against H. indicola.

65 b) Antifeedant activities of wood extractives Scores of feeding inhibition by different concentrations of T. grandis extractives against R. flavipes and H. indicola are listed in Table 4.2.9. T. grandis heartwood extractives showed more antifeedant indices against H. indicola as compared to against R. flavipes. Activity level ascended from minimal to very strong with 77.13% antifeedant indices against R. flavipes at the highest concentrations (10 mg ml-1) while against H. indicola activity level was strongest (96.73%).

Feeding inhibition by D. sissoo heartwood extractives at different concentrations against R. flavipes and H. indicola is listed in Table 4.2.10. Activity level was strongest with 78.52% antifeedant index at the highest concentrations (10 mg ml-1) against R. flavipes. While activity level was minimal to very strong, with highest antifeedant indices (88.13 %) at the highest concentrations of extractives against H. indicola. D. sissoo heartwood extractives showed more antifeedant indices against H. indicola as compared to R. flavipes.

Inhibition of feeding C. deodara heartwood extractive treated filter papers by R. flavipes and H. indicola is listed in Table 4.2.11. Activity level scored strongest with 95.52% antifeedant index at the highest concentrations (10 mg ml-1) against R. flavipes. Activity level was minimal to moderate at the lowest and highest concentrations. Highest antifeedant index (49.91%) at the highest concentration of extractives against H. indicola was observed. C. deodara heartwood extractives showed high antifeedant indices against R. flavipes as compared to H. indicola.

Table 4.2.9. Antifeedant activity of T. grandis heartwood extractives against R. flavipes and H. indicola.

Concentrations Reticulitermes flavipes Heterotermes indicola A% Activity level A% Activity level -1 c a 1.25 mg ml 16.83±1.04 Minimal activity 58.89±2.14 Strong activity -1 c ab 2.5 mg ml 24.01±2.00 Minimal activity 72.60±1.24 Strong activity -1 c b 5.0 mg ml 29.56±2.86 Moderate activity 87.48±1.48 V. strong activity -1 b c 7.5 mg ml 58.22±5.11 Strong activity 95.18±2.76 V. strong activity -1 a d 10 mg ml 77.13±6.56 V. Strong activity 96.73±1.63 V. strong activity F-value 39.53 70.26 p-value 0.00 0.00 A% = antifeedant indices; Means± SE sharing same letters in columns for each termite species are not significantly different from each other at p>0.05.

Table 4.2.10. Antifeedant activity of D. sissoo heartwood extractives against R. flavipes and H. indicola.

Concentrations Reticulitermes flavipes Heterotermes indicola A% Activity level A% Activity level -1 b b 1.25 mg ml 53.62±0.68 Strong activity 10.16±2.12 Minimal activity -1 ab b 2.5 mg ml 61.79±2.04 Strong activity 22.40±2.41 Minimal activity -1 ab b 5.0 mg ml 74.22±6.34 Strong activity 40.64±5.83 Moderate activity -1 ab a 7.5 mg ml 68.63±1.83 Strong activity 83.39±6.86 V. Strong activity -1 a a 10 mg ml 78.52±7.42 V. Strong activity 88.13±5.80 V. Strong activity F-value 4.76 49.96 p-value 0.02 0.00 A% = antifeedant indices; Means± SE sharing same letters in columns for each termite species are not significantly different from each other at p>0.05.

Heartwood extractives of P. roxburghii inhibited consumption of treated filter papers by R. flavipes and H. indicola (Table 4.2.12) at different extractive concentrations. Against R. flavipes, the activity level was from moderate to very strong activity with the highest (80.36%) antifeedant index at the highest concentration. Activity level was minimal to strong activity starting from minimum to maximum concentrations with the highest antifeedant index (63.09%) at the highest concentration of extractives against H. indicola. P. roxburghii

67 heartwood extractives showed more antifeedant indices against R. flavipes as compared to H. indicola.

Overall ANOVA of antifeedant test revealed that heartwood extractives from durable woods showed different antifeedant activities against both termite species. Interaction between durable wood species and termite species was significantly different while all other interactions were non-significant different (Appendix 4.5 and Table 4.2.13).

Table 4.2.11. Antifeedant activity of C. deodara heartwood extractives against R. flavipes and H. indicola.

Concentrations Reticulitermes flavipes Heterotermes indicola A% Activity level A% Activity level -1 a a 1.25 mg ml 27.41±1.31 Moderate activity 2.84±0.44 Minimal activity -1 c b 2.5 mg ml 41.51±2.43 Moderate activity 9.93±0.50 Minimal activity -1 c c 5.0 mg ml 44.12±2.96 Moderate activity 16.92±0.56 Minimal activity -1 b d 7.5 mg ml 81.11±2.14 V. Strong activity 33.21±1.28 Moderate activity -1 d e 10 mg ml 95.25±0.67 V. Strong activity 49.91±2.46 Moderate activity F-value 193.5 212.30 p-value 0.00 0.00 A% = antifeedant indices; Means± SE sharing same letters in columns for each termite species are not significantly different from each other at p>0.05.

Table 4.2.12. Antifeedant activity of P. roxburghii heartwood extractives against R. flavipes and H. indicola. Concentrations Reticulitermes flavipes Heterotermes indicola A% Activity level A% Activity level -1 b d 1.25 mg ml 34.30±3.50 Moderate activity 2.04±2.04 Minimal activity -1 b cd 2.5 mg ml 35.09±2.85 Moderate activity 9.95±2.93 Minimal activity -1 b bc 5.0 mg ml 49.77±6.03 Moderate activity 27.62± 2.82 Moderate activity -1 a ab 7.5 mg ml 68.56±2.91 Strong activity 45.93±9.27 Moderate activity -1 a a 10 mg ml 80.36±1.21 V. Strong activity 63.09±6.04 Strong activity F-value 31.32 22.17 P-value 0.00 0.00 A% = antifeedant indices; Means± SE sharing same letters in columns for each termite species are not significantly different from each other at p>0.05.

Table 4.2.13. Antifeedant activity of four heartwood extractives against R. flavipes and H. indicola. Termite species Heartwoods Reticulitermes flavipes Heterotermes indicola T. grandis 41.15±6.29bc 72.27±6.02a D. sissoo 60.51±5.87a 59.32±8.44a C. deodara 59.59±5.92a 31.40±6.97c P. roxburghii 55.34±5.96ab 28.93±5.81c A% = antifeedant indices; Means± SE sharing same letters in columns for each termite species are not significantly different from each other at p>0.05.

4.2.3. Antioxidant /radical scavenging potential of heartwood extractives and their effect on mid gut enzymes of termites. 4.2.3.1. Effect of heartwood extractives on DPPH radical-scavenging activity

The radical-scavenging property and IC50s of all heartwood extractives is presented in

Fig. 4.2.9. IC50 = the concentration where 50% inhibition of the DPPH radical is obtained. -1 Minimum IC50 was shown by Quercetin (8.17 µg ml ). Heartwood extractive, D. sissoo -1 showed maximum % inhibition with lowest IC50 (28.83 µg ml ) as compared to all other three extractives. IC50 value of D. sissoo was median to those for BHT and Quercetin. Percentage inhibition in case of D. sissoo was not concentration dependent while % inhibition shown by other extractives was linearly dependent on concentrations. Among heartwood extractives, T. grandis showed highest % inhibition (89.7) but this inhibition was lower as compared to positive controls (BHT, Quercetin) at the highest concentration. D. sissoo at 100 µg ml-1 showed maximum inhibition (91.06%) which declined to 77% at maximum concentration.

4.2.3.2. Enzymes activity in the mid gut of H. indicola workers exposed to IC50 concentrations of BHT, quercetin and heartwood extractives Tables 4.2.14 to 4.2.16 shows activities of three gut enzymes of H. indicola after feeding on filter papers which were treated with IC50s of the synthetic compounds and heartwood extractives. Results showed that minimum GSTs (Glutathione S- transferases) activity (12.83 μ moles min-1 mg protein-1) was observed in workers of termites, which were exposed to quercetin. All concentrations of extractives and other compounds performed significantly different from one another (p<0.05). Extractives of T. grandis and D. sissoo non-significantly reduced the activity of GSTs but these showed significant difference from rest of extractives (Table 4.2.14). Maximum GSTs activity (54.27 μmoles min-1 mg protein-1) was found in workers of H. indicola, which were fed on C. deodara extractives treated filter paper. Comparison of esterases (ESTs) activity in midgut of H. indicola after feeding on filter paper treated with different concentrations of extractives and compounds is given in Table 4.2.15. Significantly, minimum ESTs activity (0.22 μmoles min-1 mg protein-1) was found in workers which were exposed to quercetin. Among the extractives, P. roxburghii extractives fed termites showed significant minimum activity (10 μmoles min-1 mg protein-1). This was contrary to GSTs in which D. sissoo and T. grandis showed minimum activities.

Table 4.2.16 shows activities of Catalases (CATs) in termites fed on extractives and synthetic compound treated filter papers. Results showed non-significant reduction in activities of catalases in termites, which were fed on extractives of P. roxburghii and C. deodara. As compared to control, maximum reduction in activities of Catalases (CATs) was observed in -1 termites, which were fed on Quercetin treated filter papers (9.23 µmoles H2O2 min mg protein-1).

Compound/Extractives BHT Quercetin T. grandis D. sissoo C. deodara P. roxburghii IC50 (µg ml-1) 42.63 8.17 167.97 28.83 508.69 335.24

1 5 0 B H T

Q u re c e tin

n o

i T . g ra n d is

t 1 0 0 i

b D . s is s o o i

h C . d e o d a ra

5 0 P . ro x b u rg h ii %

0 0 2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0

- 1 C o n c . o f t e s t c o m p o u n d /e x t r a c tiv e s ( u g m l )

Fig. 4.2.9. Effect of heartwood extractives on DPPH radical scavenging activity (% inhibition); Conc. = concentration.

Table 4.2.14. Mean GSTs activity of H. indicola exposed to IC50s concentrations of BHT, Quercetin and heartwood extractives.

-1 Compound/ Extractives IC50 (µg ml ) GSTs activity Grouping Control --- 77.60 ± 1.07 a BHT 42.63 23.96 ± 0.73 e Quercetin 8.17 12.83 ± 0.72 f T. grandis 167.97 29.33 ± 1.17 de D. sissoo 28.83 30.76 ± 0.54 d C. deodara 508.69 54.27 ± 1.39 b P. roxburghii 335.24 44.50 ± 1.80 c F= 365.73 p < 0.05 BHT, Butylated hydroxy toluene; GSTs, Glutathione-S-transferases (μmoles min-1 mg protein-1). Means sharing same letters in a column are not significantly different from each other at p>0.05.

Table 4.2.15. Mean ESTs activity of H. indicola exposed to IC50s concentrations of BHT, Quercetin and heartwood extractives.

-1 Compounds/ Extractives IC50 (µg ml ) ESTs activity Grouping Control --- 41.03 ± 0.89 A BHT 42.63 25.03 ± 3.24 Bc Quercetin 8.17 0.22 ± 0.01 E T. grandis 167.97 27.70 ± 3.96 B D. sissoo 28.83 16.26 ± 0.63 Cd C. deodara 508.69 32.60 ± 0.70 Ab P. roxburghii 335.24 10.70± 1.67 D F= 43.82 p <0.05 BHT, Butylated hydroxy toluene; ESTs, Esterases (µmoles min-1 mg protein-1). Means sharing same letters in a column are not significantly different from each other at p>0.05.

Table 4.2.16. Mean CATs activity of H. indicola exposed to IC50S concentrations of BHT, quercetin and heartwood extractives.

-1 Compound/ Extractives IC50 (µg ml ) CATs activity Grouping Control --- 16.41 ± 0.35 a BHT 42.63 15.07 ± 0.43 ab Quercetin 8.17 9.23 ± 1.30 c T. grandis 167.97 11.00 ± 0.52 bc D. sissoo 28.83 12.48 ± 0.74 abc C. deodara 508.69 15.36 ± 0.48 a P. roxburghii 335.24 16.53 ± 1.58 a F= 10.24 p < 0.05 -1 -1 BHT, Butylated hydroxy toluene; CAT, Catalases (µmoles H2O2 min mg protein ). Means sharing same letters in a column are not significantly different from each other at p>0.05.

SECTION-III

Effect of heartwood extractives on gut protozoans of two termite species

Variations in gut bacterial community of H. indicola after feeding on extractives treated filter paper

4.3.1. Effect of heartwood extractives on gut protozoans of R. flavipes and H. indicola. Although wood extractives elicited considerably destructive effects on gut protozoans in both termite species, complete protozoan defaunation was not shown to occur, even at the highest concentrations of extractives. Change in protozoan densities upon treatment with extractives in both species of termites was, however, dose dependent. Guts of R. flavipes and H. indicola, respectively based on individual termite in control treatment, had on an average of 60,042±1,877 and 3,530±62.4 total protozoans. Termites fed on untreated filter papers had as similar number of protozoans as in the laboratory reared termites fed on SYP and CW. Numbers of gut protozoans in R. flavipes and H. indicola workers were significantly reduced after feeding on various concentrations of heartwood extractives. In both termite species, results showed a maximum reduction (45 to 82%) in protozoans at the highest extractive concentration (10 mg ml-1) (Tables 4.3.1 - 4.3.5).

Significant dose dependent decline in total population of gut protozoan occurred in R. flavipes fed on T. grandis heartwood extractives. Termite mortality and reduction in total number of protozoans also related linearly making it apparent that death of protozoans directly influenced host survival. Maximum reduction in protozoans (39.20 %) was observed at the highest extractive concentration (10 mg ml-1) at which mortality of R. flavipes was also maximum. Extractives behaved significantly different at their respective concentration among themselves. Overall, significant differences among concentrations were also observed (F= 12.28; p<0.005). Similar results were found against H. indicola where maximum reduction at the highest concentration was 82.53%. In case of R. flavipes, control population of protozoans had non-significant difference with number at concentrations 1.25 to 5.00 mg ml-1 whereas control had non-significant difference with 1.25 mg ml-1 in H. indicola. This explains larger reduction of protozoans in H. indicola than R. flavipes (Table 4.3.1).

Table 4.3.1. Effect of T. grandis heartwood extractives on total population of gut protozoans of R. flavipes and H. indicola. Termite spp. Concentrations Protozoans termite-1 % reduction a Control 60042 ± 1877 ------1.25 mg ml-1 ab 0.00

R. flavipes 60042 ± 5048 -1 ab 2.50 mg ml 57333 ± 2003 4.50 -1 ab 5.00 mg ml 57083 ± 561 5.52 7.50 mg ml-1 bc 19.69 48217 ± 1770 -1 c 10.00 mg ml 36500 ± 2774 39.20 F = 12.28; p< 0.05 ------a Control 3530 ± 62.4 ------1.25 mg ml-1 ab 10.28

H. indicola 3167 ± 120 -1 b 2.50 mg ml 2900 ± 57.7 17.84 -1 c 5.00 mg ml 2057 ± 243 41.72 7.50 mg ml-1 d 60.36

1399 ± 138 -1 e 10.00 mg ml 623 ± 63.7 82.53 F= 72.88; p< 0.00 ------Means±SE sharing same letters in column for each termite species are not significantly different from each other at p>0.05. spp. = species.

Total population of gut protozoans and their reduction after feeding on D. sissoo heartwood extractives treated filter papers is shown in Table 4.3.2. Response of aggregate protozoans densities were considerably dose dependent. Maximum reduction in protozoans was observed at the highest extractive concentration (10 mg ml-1) where there was maximum mortality of termite. Reduction rate of flagellates at the highest concentrations (10 mg ml-1) was 45.73 and 81.51% in R. flavipes and H. indicola, respectively.

Table 4.3.2. Effect of D. sissoo heartwood extractives on total population of gut protozoans of R. flavipes and H. indicola. Termite spp. Concentrations Protozoans termite-1 % reduction a Control 60042 ± 1877 ------1 b 1.25 mg ml 44833 ± 614 25.32

R. flavipes -1 b 2.50 mg ml 44583 ± 651 25.74 -1 bc 5.00 mg ml 41750 ± 617 30.46 7.50 mg ml-1 c 36.69

38009 ± 724 -1 d 10.00 mg ml 32583 ± 583 45.73 F= 92.43; p< 0.05 ------a Control 3530 ± 62.4 ------1 a 1.25 mg ml 3200 ± 100 9.35

H. indicola -1 a 2.50 mg ml 3008 ± 104 14.78 -1 b 5.00 mg ml 2098 ± 249 40.56 -1 c 7.50 mg ml 1432 ± 106 59.43

-1 d 10.00 mg ml 653 ± 49.9 81.50 F= 75.42; p< 0.05 ------Means±SE sharing same letters in column for each termite species are not significantly different from each other at p>0.05. spp. = species.

Total population of gut protozoans and their reduction after feeding on C. deodara treated filter papers is shown in Table 4.3.3. Aggregate protozoan’s population was considerably dose dependent. The maximum reduction in protozoans was observed at the highest extractive concentration (10 mg ml-1) where there was maximum mortality of termite. The reduction rate of flagellates at the highest concentrations (10 mg ml-1) was 15.5 and 45.32% in R. flavipes and H. indicola, respectively. There was non-significant death of gut protozoans as compared to D. sissoo and T. grandis. Comparison of termites’ species showed that gut protozoans in H. indicola were affected largely by C. deodara extractives as compared to R. flavipes.

Consumption of P. roxburghii treated filter papers showed a general reduction in total population of gut protozoans (Table 4.3.4.), which was dose dependent. Maximum reduction

in protozoans was observed at the highest extractive concentration (10 mg ml-1) where there was maximum mortality of termite. The reduction rate of flagellates at the highest concentrations (10 mg ml-1) was 36.43 and 52.93 % in R. flavipes and H. indicola, respectively.

Overall, all extractives showed protozoicidal activities in both termites’ species (Table 4.3.5). Percentage reduction of protozoans was statistically analyzed using ANOVA and results revealed that interactions among termites’ species, woods and extractives concentrations were significant (Appendix 4.6).

Table 4.3.3. Effect of C. deodara heartwood extractives on total population of gut protozoans of R. flavipes and H. indicola. Termite spp. Concentrations Protozoans termite-1 % reduction

R. flavipes a Control 60042 ± 1877 ------ab 1.25 mg ml-1 56250 ± 1377 6.31 ab 2.50 mg ml-1 54500 ± 232 9.22

ab 5.00 mg ml-1 54042 ± 273 9.99 ab 7.50 mg ml-1 53333 ± 1702 11.17 b 10.00 mg ml-1 51000 ± 2152 15.05 F= 4.30; p< 0.05 ------

H. indicola a Control 3530 ± 62.4 ------a 1.25 mg ml-1 3333 ± 66.7 5.57 b 2.50 mg ml-1 2966 ± 88.2 15.96

bc 5.00 mg ml-1 2860 ± 64.7 18.96 c 7.50 mg ml-1 2540 ± 14.9 28.04 d 10.00 mg ml-1 1930 ± 83.2 45.32 F= 72.17; p< 0.05 ------Means sharing same letters in column for each termite species are not significantly different from each other at p>0.05. spp. = species.

Table 4.3.4. Effect of P. roxburghii heartwood extractives on total population of gut protozoans of R. flavipes and H. indicola. Termite spp. Concentrations Protozoans termite-1 % reduction a Control 60042 ± 1877 ------1.25 mg ml-1 ab 8.53

R. flavipes 54917 ± 1714 -1 bc 2.50 mg ml 52125 ± 1392 13.18 -1 bc 5.00 mg ml 49208 ± 1341 18.04 7.50 mg ml-1 c 20.19

47917 ± 1071 -1 d 10.00 mg ml 38167 ± 1119 36.43 F= 26.06; p< 0.05 ------a Control 3530 ± 62.4 1.25 mg ml-1 3300 ± 57.7 6.52 H. indicola ab -1 bc 2.50 mg ml 2933 ± 120 16.90 -1 c 5.00 mg ml 2794 ± 126 20.85 7.50 mg ml-1 d 38.47

2172 ± 97 -1 e 10.00 mg ml 1661 ± 87 52.93 F= 54.12; p< 0.05 ------Means±SE sharing same letters in column for each termite species are not significantly different from each other at p>0.05. spp. = species.

Table 4.3.5. Percentage reduction of total population of gut protozoans of R. flavipes and H. indicola after feeding on filter paper treated with four durable heartwood extractives.

R. flavipes H. indicola Heartwoods T. grandis D. sissoo C. deodara P. roxburghii T. grandis D. sissoo C. deodara P. roxburghii Conc. 1.25 1.83±1.83p 25.32±1.02g-k 6.31±2.29m-p 8.53±2.86l-p 10.28±1.45l-p 9.35±2.83l-p 5.57±1.89n-p 6.52±1.64m-p 2.5 4.76±3.14op 25.74±1.08g-j 11.16±0.38k-p 13.18±2.32j-p 17.84±1.83h-o 14.78±2.6i-p 15.96±2.50i-p 16.90±3.40h-o 5 4.92±0.93op 30.46±1.03f-h 9.22±2.83l-p 18.04±2.23h-o 41.72±1.44d-f 40.56±7.06d-f 18.96±1.83h-o 20.85±3.58h-l 7.5 19.69±2.95h-n 36.69±1.21e-g 9.98±0.45l-p 20.19±1.78h-m 60.36±1.62b 59.44±3.02bc 28.03±0.42f-i 38.47±2.75e-g 10 39.20±4.62d-g 45.72±0.97c-e 15.05±3.59i-p 36.42±1.87e-g 82.35±0.78a 81.50±1.42a 45.32±2.36c-e 52.93±2.46b-d Means±SE sharing same letters in columns and rows are not significantly different from each other at p>0.05. Conc. = concentration (mg ml-1).

4.3.2. Changes in gut bacterial community of H. indicola after feeding on extractives treated filter papers

This part of experiment was done only with H. indicola because identification of microbial communities of R. flavipes and effect of different diets and treatments on these communities has been investigated by number of researchers. There is no literature which shows identification of bacterial communities and effect of different treatments on these communities in H. indicola. Table 4.3.6 shows average number of sequences per sample and their obtained OTU’s (observed taxonomic units) at each treatment along with treatment groups and their percentage coverage (an estimate of how well the community was sampled). Percentage coverage was nearly 60%, which did not reveal overall composition of gut bacterial communities among the samples. OTU’s were defined at 3% dissimilarity level and 12571 sequences were generated after removing short reads and chimeras. To calculate the diversity of bacterial communities, inverse Simpson diversity estimate was used in Mothur (v. 1.32.1). Fig. 4.3.1 shows diversity of gut bacteria in H. indicola after feeding on extractives treated filter papers (10 and 7.5 mg ml-1). Diversity index shows that there was non-significant difference in diversity of bacterial communities in termites which were fed on C. deodara and P. roxburghii heartwood extractives compared to their respective controls. T. grandis and D. sissoo heartwood extractives showed some reduction in diversity as compared to control treatments in which termites were fed on solvent treated filter paper. T. grandis extractives showed dose dependent action on bacterial diversity unlike D. sissoo. We detected six bacterial phyla and a large number of uncultured and unclassiﬁed bacteria across the different treatments (Fig. 4.3.2). In all treatment groups, an unclassified bacteria followed by protobacteria and spirochetes were dominant groups. All other phyla were very small in each treatment group. Overall, examination of the abundance of major taxonomic groups in treated versus control groups did not appear to show any major differences for presence of phyla of gut bacteria.

Table 4.3.6. Number of sequences, OTUs, and percent coverage for each sample by treatment. Treatment Sample Reads analyzed Number of OTUs % coverage Control Control 1 454 90 65 Control 2 442 95 61 T. grandis 7.5 mg ml-1 615 84 45 10 mg ml-1 585 86 66 D. sissoo 7.5 mg ml-1 448 47 59 10 mg ml-1 419 53 58 C. deodara 7.5 mg ml-1 358 45 61 10 mg ml-1 339 52 52 P. roxburghii 7.5 mg ml-1 501 75 57 10 mg ml-1 481 93 63 OTU= Operational Taxonomic Units.

100

80 Control

7.5 60 10 40

20 Inverse Simpson Inverse Simpson Diversity Index

0 T. grandis D. sissoo C. deodara P. roxburghii Treatments

Fig. 4.3.1. Inverse Simpson diversity index of bacterial communities of H. indicola after feeding on treated filter paper.

Protobacteria 20 Actinobacteria

Firmicutes Number Number OTUs 15 Uncultured bacteria Fibrobacteres 10 Spirochetes Bacteroidetes 5

Treatments

Fig. 4.3.2. Major bacterial phyla identified in the guts of H. indicola separated by treatments.

The sequence similarities of isolated strains were compared with the available databases in NCBI and only 99 to 100 % similarities are tabulated (Table 4.3.7). Sequence data were aligned and evolutionary relationship of sequence information was studied by phylogenetic analyses. Overall, 5 strains of bacteria based on closest matches (100 and 99 %) in GenBank belonged to genus Enterobacter, Bacillus, Pseudomonas, Pantoea and an uncultured bacterium. Phylogenetic analysis was done using Mega 6 software (Appendices 4.10-4.15).

Table 4.3.7. Sequence similarity analysis of isolated bacterial strains from H. indicola with the available databases in NCBI. Closely related microorganisms Accession Sequence Similarity (%) Enterobacter sp. DQ11(2010) gi 307135747|GU377060.1 100 Enterobacter cloacae strain JV gi|225735021|FJ799063.1 100 Cronobacter sakazakii strain MM045 gi|1104335019|KT933253.3 99 Bacillus cereus strain BCRh5 gi|952951796|KT153601.1 100 Pseudomonas aeruginosa strain Iraq.PA-5 gi|1126523287|KX963360.1 100 Pseudomonas sp. OX5 gi|929558305|KR075080.1 100 Pantoea agglomerans strain AIMST 2 gi|334980881|JF819683.1 100 Uncultured bacterium gi|697256477|LN564018.1 99 Uncultured bacterium clone gi|148248405|EF604435.1 99

SECTION-IV

Choice and No-choice tests on extracted and un-extracted wood against termites

Termite bioassay on pressure treated Southern Yellow Pine and Cotton Wood

Determination of leaching resistance of extractives impregnated woods against termites

Wood preservative potential of extractives in combination with linseed oil

Evaluation of wood extractives against Postia placenta and Trametes versicolor

4.4.1. Choice and No-choice tests on extracted and un-extracted wood against R. flavipes and H. indicola Extractives free woods were prepared following ASTM D1105-96 and subjected to Choice and No-choice tests. Results of bioassay are presented in Tables 4.4.1 to 4.4.4. Bioassays of R. flavipes and H. indicola on un-extracted and extractive-free woods of T. grandis revealed in choice test that R. flavipes apparently ignored un-extracted block and consumed a large proportion of extracted block (>2%) while in no-choice test weight losses for extracted blocks were almost similar (2.87 %) to the choice test. In case of un-extracted blocks, consumption was <1% and 1.32% in choice and no choice tests after 28 days, respectively. There was non-significant difference in feeding of termites on extracted and un- extracted blocks of T. grandis in both types of tests. In the choice test, H. indicola also ignored the un-extracted T. grandis heartwood blocks and consumed a large part of extracted block (3.93 %) while in the no-choice test weight losses for extracted blocks were similar (5.13% in each case to choice test. In the case of un-extracted blocks, consumption was <1% under no- choice test while in case of choice test consumption was 1.33% after 28 days of exposure. At 28th day, 100% mortality of termites’ species in choice and no-choice tests occurred (Table 4.4.1).

Table 4.4.2 shows the results when extracted and extractive free blocks of D. sissoo were exposed to termites in Choice and No- choice tests. Mortality of R. flavipes was 84.5% in choice test while mortality was 85% on extracted blocks (no-choice test), having non- significant difference between each other. Mortality was 91% on un-extracted blocks in no- choice which was significantly different from extracted one. In no-choice-un-extracted test, 92% termites of H. indicola died while in no-choice test on extracted blocks, mortality was non- significantly different (74.6%) from that in choice test (77%). Consumption of extractive free D. sissoo heartwood blocks by R. flavipes was higher in both choice and no choice tests compared to un-extracted blocks. In choice tests, termites consumed 10.7% of extractive free compared to 1.9% of un-extracted blocks. Consumption was significantly different in choice and no- choice test on extracted and un-extracted heartwood blocks. In no-choice test, H. indicola generally avoided un-extracted block (1.75% weight loss) and consumed significantly a large part of extracted block (13.50%), while in the choice test weight losses in extracted

85 blocks were 3.68%. In the case of un-extracted blocks, consumption was 4.5 % which was non- significantly different from consumption in extracted blocks after 28 days. Table 4.4.1. Mean weight loss (%) for extractive free and un-extracted blocks of T. grandis and mortality of R. flavipes and H. indicola under Choice and No-choice tests. No -choice test Choice test Termite Types of Weight loss Mortality Weight loss Mortality spp. blocks (%) (%) (%) (%) Extracted 2.87±0.33a 100.00±0.00a 2.93±0.46a R. flavipes 100.00±0.00a Un-extracted 1.32±1.32a 99.60±0.24a 0.33±0.33a t-value (0.94)ns (1.63)ns (4.16)ns ------

p- value 0.39 0.17 0.06 ------Extracted a a a 5.13±0.33 86.20±1.88 3.93±0.47 a H. indicola 100.00±0.00 b a a Un-extracted 0.86±0.38 100.00±0.00 1.33±0.34 ** ns t-value (6.34)** (-7.33) (5.02) ------

p- value 0.003 0.002 0.06 ------**- Significantly different; ns = non- significant; spp. = species Table 4.4.2. Mean weight loss (%) for extractive free and un-extracted blocks of D. sissoo and mortality of R. flavipes and H. indicola under Choice and No-choice tests. No- choice test Choice test Termite Weight loss Mortality Weight loss Mortality Types of blocks spp. (%) (%) (%) (%) a a Extracted 10.73±2.08 11.80±1.07 85.45±6.73 a R. flavipes 84.58±1.28 b b Un-extracted 3.28±1.93 91.00±8.81 1.93±1.20 t-value (3.39)** (-0.40)** (2.74)** ------

p- value 0.02 0.050 0.01 ------

a a a Extracted 13.50±0.70 74.60±2.87 3.68±0.11 a H. indicola 77.00±9.75 b a a Un-extracted 1.75±0.56 92.10±7.71 4.52±0.12 ns ns t-value (13.44)** (-2.01) (-0.37) ------

p- value 0.00 0.11 0.064 ------**- Significantly different; ns = non- significant; spp. = species

Results of bioassays of R. flavipes and H. indicola on un- extracted and extractive-free woods of C. deodara are presented in Table 4.4.3. In choice test, R. flavipes expectedly ignored un-extracted blocks and consumption was 1.93% but termites consumed significantly more of extracted blocks (33.4%) while in no-choice test weight losses for extracted blocks were again significantly different from one another after 28 days. In both tests, H. indicola also ignored un-extracted C. deodara heartwood blocks and consumed more of extracted blocks; being significantly different in weight losses of extracted and un-extracted blocks under no choice and choice tests. Mortality of R. flavipes was significantly different in both choice and no- choice tests on extracted and un-extracted heartwood blocks after feeding of 28 days. There was 100% mortality of R. flavipes on un-extracted blocks in no-choice test as against 53% when termites were fed on extracted heartwood blocks of C. deodara. Mortality of R. flavipes in choice test was 64.9%. H. indicola were completely dead in no-choice test with un-extracted C. deodara heartwood blocks while in case of the extracted blocks, mortality was significantly less (52 %) which was non-significantly different from the mortality in choice test.

Table 4.4.4 shows that in the choice test, R. flavipes ignored the un-extracted blocks of P. roxburghii and consumed significantly large portion extracted block (38.4%) while in no- choice test weight loss for extractive free blocks was 39.6% which was non-significantly different to that choice test. In the case of un-extracted blocks, consumption was 1.14 % in choice test while under no choice test consumption of un-extracted wood was 1.78% by R. flavipes after 28 days. In the choice test, H. indicola also ignored the un-extracted block and consumed a large part of extracted block (44.6%) under choice test while in no-choice test weight losses for extracted blocks were similar (37.9 %) to choice test. In case of un-extracted blocks, consumption was less than 1% in no- choice test while under choice test consumption of un-extracted wood was 4.52 % after 28 days. 99% termites of both species were dead in no- choice test after feeding on un-extracted blocks while under choice test mortalities of R. flavipes and H. indicola were significantly less (25.4 and 63.41%, respectively).

Table 4.4.3. Mean weight loss (%) for extractives free and un-extracted blocks of C. deodara and mortality of R. flavipes and H. indicola under Choice and No-choice tests. No- choice test Choice test Termite Type of Weight loss Mortality Weight loss Mortality spp. blocks (%) (%) (%) (%) a a a Extracted 37.13±1.27 53.00±7.08 33.46±4.58 R. flavipes 64.91±1.27a b b b Un-extracted 0.27±0.13 100.00±0.00 1.93±1.39 t-value (27.67)** (-5.28)** (6.16)** ------

p- value 0.00 0.006 0.024 ------

a a a Extracted 38.11±1.27 52.00±9.08 33.46±5.51 H. indicola 68.11±1.29a b b b Un-extracted 0.27±0.13 100.00±0.00 1.93±2.41 t-value (27.67)** (-5.28)** (6.17)** ------

p- value 0.00 0.006 0.025 ------**- Significantly different; ns = non- significant. spp. = species.

Table 4.4.4. Mean weight loss (%) for extractive free and un-extracted blocks of P. roxburghii and mortality of R. flavipes and H. indicola under choice and no-choice tests. No choice test Choice test Termite Weight loss Mortality Weight loss Mortality spp. Type of blocks (%) (%) (%) (%) a a a Extracted 39.61±1.25 24.45±2.07 38.43±0.51 a R. flavipes 25.41±1.81 b b b Un-extracted 1.78±0.54 99.95±0.05 1.14±0.90 t-value (25.76)** (-27.30)** (28.30)** ------

p- value 0.00 0.00 0.001 ------

a a a Extracted 37.98±1.07 52.70±8.87 44.66±2.02 H. indicola 63.41±7.26b b b b Un-extracted 0.49±0.15 98.80±0.48 4.52±0.65 t-value (39.67)** (-5.028)** (21.04)** ------

p- value 0.00 0.007 0.002 ------**- Significantly different; ns = non- significant. spp. = species.

4.4.2. Bioassays of vacuum pressure treated southern yellow pine (SYP) and cotton wood (CW) against R. flavipes and H. indicola Two non-durable woods, SYP and CW, were treated with extractives of four durable wood species using vacuum-pressure application. Stock solutions of four woods extractives were further diluted to three concentrations (2.50, 5.0, 10.0 mg ml-1) to treat SYP and CW non- durable woods. These treated blocks were exposed to termites for 28 days and at the end, alive termites were counted along with weight loss of the woods.

4.4.2.1. Bioassays of pressure treated southern yellow pine (SYP) and cotton wood (CW) after 28 days of exposure to R. flavipes Mean weight loss of T. grandis in treated and un-treated SYP and CW exposed to R. flavipes along with retention and E-1 rating is presented in Table 4.4.5. Solvent and water treated SYP controls lost a weight of 25.12 and 26.54 %, respectively, with average rating of 3.6 and 4.6 while CW showed a weight loss of 36.45 and 37.54 % respectively for solvent and water treated blocks with same average rating of 1.6 for both controls. Conversely, SYP and CW treated with T. grandis extractives showed minimum weight loss (5.48 and 12.36 %, respectively) at the highest concentration (10 mg ml-1). Weight loss was found to be inversely related to extractive concentrations. Average rating at maximum retention (48.70 and 58.20 kg/m3 for SYP and CW, respectively) was 8.90 for both CW and SYP. Weight loss of SYP control treatments was statistically significant different than all other treatments. A similar trend was found in CW. T. grandis extractives were not very toxic to the termites at the lowest concentration (2.5 mg ml-1) and showed average rating of 6.0 and 5.4 for SYP and CW respectively.

Mortalities in solvent and water treated SYP and CW controls were 25.25 and 24.85 %, 32.10 and 36.20 % respectively. Mortalities were non-significantly different on SYP except maximum percent mortality (82.8) at 10 mg ml-1 whereas mortalities on CW were non- significantly different from water, solvent and 2.5 mg ml-1 concentrations (Table 4.4.5).

Table 4.4.5. Average rating, retention, mortality and weight losses of SYP and CW treated with T. grandis extractives after feeding of R. flavipes. Woods Conc. Mortality Weight loss Retention Rating (avg) (mg ml-1) (%) (mg) (%) (kg m-3) pcf

Southern b a a c Water 24.85±0.78 1192±42.1 26.54±0.81 ------4.60 b a a c Solvent 25.25±0.84 1172±22.7 25.12±0.39 ------3.60 2.5 b a a c c c Y 26.35±1.45 1024±40.2 22.90±1.3 13.47±0.24 3.04±0.36 6.00

ellow Pine b b b b b b 5.0 39.15±2.78 834.0±38.3 18.03±0.71 26.65±0.32 1.66±0.02 8.00 a c c a a a 10.0 82.8±10.1 248.0±52.9 5.48±1.16 48.70±5.7 3.04±0.36 8.90 F 27.07 92.46 82.27 28.22 28.22 13.22 p 0.00 0.00 0.00 0.00 0.00 0.00 b a a d Water 32.10±5.08 1176±76.30 37.54±2.00 ------1.6 b a a d Cotton Wood Cotton Solvent 36.20±2.29 1168±26.10 36.45±1.05 ------1.6 b a a c c c 2.5 37.95±7.08 1064±98.80 33.08±2.89 15.23±0.26 0.95±0.01 5.4 a b b b b b 5.0 70.60±7.48 630±70.20 20.01±2.23 30.19±0.47 1.88±0.02 7.8 a b b a a a 10.0 82.05±7.37 412±121.0 12.36±3.42 58.20±2.08 3.63±0.13 8.9

F 13.67 16.91 20.64 309.47 309.47 29.56 p 0.00 0.00 0.00 0.00 0.00 0.00 Means±SE sharing same letters in columns for each wood species are not significantly different from each other at p>0.05. Conc. = concentration

Mean weight loss of D. sissoo extractives treated and un-treated SYP and CW exposed to R. flavipes along with retention and E-1 rating is presented in Table 4.4.6. Maximum mortality (88.5 and 93.5 %) of termite was found at 10 mgml-1 after feeding on extractives treated SYP and CW. Highest concentrations of D. sissoo heartwood extractives showed minimum weight loss (5.9 and 12.21%, respectively) with average rating of 9.1 and 8.2 for SYP and CW, respectively. Weight loss was found to be inversely related to the extractive concentrations. Weight losses of control treatments of SYP were statistically higher than all other treatments and a similar trend was found in CW.

Mean weight loss of C. deodara treated and un-treated SYP and CW exposed to R. flavipes along with retention and E-1 rating is presented in Table 4.4.7. SYP and CW treated with C. deodara extractives showed minimum weight loss (14.42 and 15.35%, respectively) at the highest concentration (10 mg ml-1). Weight loss was found to be inversely related to the extractive concentrations. Average rating at maximum retention (53.24 and 57.01 kg m-3 for SYP and CW respectively) was 8.5 and 7.8 for SYP and CW, respectively. Weight loss of control treatments of SYP was statistically higher than all other treatments. A similar trend was found in cotton wood. C. deodara extractives were even toxic to termites at the lowest concentration (2.5 mg ml-1) and showed average rating of 6.2 and 5.8 for SYP and CW respectively. At maximum concentration (10 mg ml-1) mortality was 58 % on SYP. While mortality in control treatments for CW was 32.10 and 36.20% for water and solvent respectively. Maximum number of dead termites (89.35%) were found at the highest concentration.

Mean weight loss of P. roxburghii treated and un-treated SYP and CW exposed to R. flavipes along with retention and E-1 rating is presented in table 4.4.8. Maximum mortality (91.60 and 80.8%) of R. flavipes was found at 10 mg ml-1 after feeding on extractive treated SYP and CW, respectively. Minimum mortality was observed after feeding on woods treated with the lowest concentrations (2.50 mg ml-1) of extractives (56.4 and 51.0% for SYP and CW, respectively). SYP and CW treated with P. roxburghii heartwood extractives showed minimum weight loss (3.98 and 17.77% respectively) at the highest concentration (10 mg ml-1) with the average rating of 8.8 and 8.0 for SYP and CW respectively.

Table 4.4.6. Average rating, retention, mortality and weight losses of SYP and CW treated with D. sissoo extractives after feeding of R. flavipes. Woods Conc. Mortality (%) Weight loss Retention Rating (mg ml-1) (mg) (%) (kg m-3) pcf (avg.)

Southern Y b a a cd Water 24.85±0.78 1192±42.1 26.54±0.81 ------4.60 b a a d Solvent 25.25±0.84 1172±22.7 25.12±0.39 ------3.60 ab b b c c bc 2.5 55.3±11.3 692±136 15.16±3.07 13.79±0.14 0.86±0.00 6.40

ellow Pine ab c bc b b ab 5.0 57.5±13.8 648±140b 14.56±3.15 28.32±0.13 1.77±0.00 8.50 a c c a a a 10.0 88.50±6.25 274±38.7 5.92±0.84 55.22±0.70 3.45±0.04 9.10 F 9.80 17.95 17.23 2460.7 2460.7 19.10

p 0.00 0.00 0.00 0.00 0.00 0.00 c a a c Water 32.10±5.08 1176±76.1 37.54± 2.00 ------1.60 bc a a c Cotton Cotton Solvent 36.20±2.29 1168±26.3 36.45±1.05 ------1.60 c a a c c b 2.5 28.15±2.62 1150±19.2 36.48±0.83 15.09±0.28 0.94±0.01 4.80

Wood b b b b b ab 5.0 51.55±4.52 742±26.9 23.49±0.63 29.06±1.49 1.81±0.09 7.00 a c c a a a 10.0 93.50±5.23 392±116 12.21±3.59 61.29±1.03 3.83±0.06 8.20

F 41.82 29.12 32.45 500.50 500.50 19.14 p 0.00 0.00 0.00 0.00 0.00 0.00 Means±SE sharing same letters in columns for each wood species are not significantly different from each other at p>0.05. Conc. = concentration

Table 4.4.7. Average rating, retention, mortality and weight losses of SYP and CW treated with C. deodara extractives after feeding of R. flavipes.

Woods Conc. Mortality (%) Weight loss Retention Rating (mg ml-1) (mg) (%) (kg m-3) pcf (avg.)

Southern Y a a a bc Water 24.85±0.78 1192±42.1 26.54±0.81 ------4.60 a a a c Solvent 25.25±0.84 1172±22.7 25.12±0.39 ------3.60 a a a c c ab 2.5 47.9±13.2 894±218 19.57±4.78 13.31±0.15 0.83±0.00 6.20

ellow Pine b a a b b a 5.0 60.5±14.20 920±18 20.48±3.93 27.57±0.30 1.72±0.01 8.00 a a a a a a 10.0 58.0±10.4 666±116 14.42±2.53 53.24±0.80 3.32±0.05 8.50 F 2.95 2.48 2.56 1605.95 1605.95 11.99

p 0.04 0.07 0.07 0.00 0.00 0.00 Water d a a ------1.60 32.10±5.08 1176±76.1 37.54±2.00 b cd a a b Cotton Wood Cotton Solvent 36.20±2.29 1168±26.3 36.45±1.05 ------1.60 bc b b c c a 2.5 58.45±1.90 772±43.7 26.28±1.48 15.11±0.29 0.94±0.01 5.80 ab bc bc b b a 5.0 67.00±8.87 644±76.5 20.21±2.63 29.30±0.78 1.83±0.04 6.80 a c c a a a 10.0 89.35±7.69 466±67.0 15.35±2.24 57.01±2.23 3.56±0.13 7.80 F 15.95 26.90 24.85 239.58 239.58 18.40 p 0.00 0.00 0.00 0.00 0.00 0.00 Means±SE sharing same letters in columns for each wood species are not significantly different from each other at p>0.05. Conc. = concentration

Table 4.4.8. Average rating, retention, mortality and weight losses of SYP and CW treated with P. roxburghii extractives after feeding of R. flavipes. Woods Conc. Mortality (%) Weight loss Retention Rating (avg.) (mg) (%) (kg m-3) Pcf

(mg ml- 1)

Southern Y b a a bc Water 24.85±0.78 1192±42.1 26.54±0.81 ------4.60 b ab ab c Solvent 25.25±0.84 1172±22.7 25.12±0.39 ------3.60 ab c c c c ab 2.5 56.4±11.6 698±15b 15.61±3.60b 13.63±0.15 0.85±0.00 7.00

ellow Pine a cd cd b b a 5.0 77.3±12.5 346±16 7.61±3.59 26.89±0.25 1.68±0.01 7.80 a d d a a a 10.0 91.60±8.40 182±10 3.98±2.13 54.03±0.98 3.37±0.06 8.80 F 12.55 16.49 16.40 1197.05 1197.05 14.08

p 0.00 0.00 0.00 0.00 0.00 0.00 b a a c Water 32.10±5.08 1176±76.1 37.54±2.00 ------1.60± b a ab c Cotton Wood Cotton Solvent 36.20±2.29 1168±26.3 36.45±1.05 ------1.00 b ab ab c bc 2.5 51.0±10.7a 918±82.3 31.87±3.15 14.35±0.49c 0.89±0.03 3.20 a c bc b ab 5.0 61.45±8.21 782±56.0b 26.05±2.35 29.56±0.46b 1.84±0.02 6.80 a c c a a 10.0 80.8±10.6 500±10 17.77±3.79 56.62±0.98a 3.53±0.06 8.00 F 6.05 14.45 9.56 966.86 966.86 11.16 p 0.00 0.00 0.00 0.00 0.00 0.00 Means±SE sharing same letters in columns for each wood species are not significantly different from each other at p>0.05. Conc. = concentration

4.4.2.2. Bioassays of pressure treated southern yellow pine (SYP) and cotton wood (CW) after 28 days of exposure to H. indicola

Mean weight loss of T. grandis treated and un-treated SYP and CW exposed to H. indicola along with retention and E-1 rating is presented in Table 4.4.9. T. grandis heartwood extractives were lethal to termites at each concentration and caused 93.99 % mortality of H. indicola at maximum concentration (10 mg ml-1) after feeding on treated SYP with average rating of 9.0. A similar trend of mortality of termites was found after feeding on CW. maximum (73.3%) mortality of termites with average rating of 8.20 was recorded at maximum concentration. Average weight loss of treated and un-treated SYP and CW exposed to H. indicola was also concentration dependent. Solvent and water treated SYP controls lost 27.52 and 27.08% with average rating of 5.6 and 4.6, respectively while CW showed a weight loss of 36.20 and 32.10% for solvent and water treated blocks with average rating of 2.4. Weight losses of SYP and CW at maximum concentration were reduced up to 5.53 and 12.36 % with average rating of 9.0 and 8.2 respectively.

Mean weight loss of D. sissoo treated and un-treated SYP and CW exposed to H. indicola along with retention and E-1 rating is presented in Table 4.4.10. D. sissoo heartwood extractives were lethal to termites at each concentration and caused 84.65% and 83.35 % mortality of H. indicola at maximum concentration (10 mg ml-1) after feeding on treated SYP and CW, respectively. The average rating at the highest concentrations were 8.40 for each CW and SYP. All treatments differed significantly from each other and also from control treatments. SYP and CW treated with highest concentrations of D. sissoo extractives showed 4.76 and 9.17% weight loss, respectively. Weight loss of control treatments of SYP was statistically higher than all other treatments. A similar trend was found in CW.

Table 4.4.9. Average rating, retention, mortality and weight losses of SYP and CW treated with T. grandis extractives after feeding of H. indicola. Woods Conc. Mortality (%) Weight loss Retention Rating (avg.) (mg ml-1) (mg) (%) (kg m-3) pcf

Southern Y c a a c Water 27.08±0.85 1299.3±45.9 26.81±0.82 ------4.60 c a a c Solvent 27.52±0.92 1277.5±24.7 25.37±0.39 ------5.60b c a a c c abc 2.5 28.72±1.58 1116.2±43.8 23.13±1.34 15.68±0.92 0.98±0.05 6.80

ellow Pine b b b b b ab 5.0 42.67±3.03 909.1±41.7 18.21±0.71 30.53±2.10 1.90±0.13 8.20 a c c a a a 10.0 93.99±3.17 270.3±57.6 5.53±1.17 56.99±3.26 3.56±0.20 9.00 F 176.89 92.46 82.27 82.81 82.81 8.27

p 0.00 0.00 0.00 0.00 0.00 0.00 b a a c Water 32.10±5.08 1249.8±28.2 37.54±2.00 ------2.40 b a a c Cotton Wood Cotton Solvent 36.20±2.29 1258.3±81.4 36.45±1.05 ------2.40 b a a c c c 2.5 37.95±7.08 1138±106 31.96±2.20 16.91±0.26 1.05±0.01 4.00b 5.0 a b b b 2.13±0.05 ab 70.60±7.48 674.1±75.1 20.01±2.23 34.21±0.82 b 7.20 a b b a a a 10.0 73.3±12.2 441±130 12.36±3.42 68.09±0.97 4.25±0.06 8.20

F 7.10 16.91 22.84 1199.00 1199.00 10.84 p 0.00 0.00 0.00 0.00 0.00 0.00 Means±SE sharing same letters in columns for each wood species are not significantly different from each other at p>0.05. Conc. = concentration

Table 4.4.10. Average rating, retention, mortality and weight losses of SYP and CW treated with D. sissoo extractives after feeding of H. indicola.

Woods Conc. Mortality (%) Weight loss Retention Rating (avg.) (mg ml-1) (mg) (%) (kg m-3) Pcf

Southern Y d a a b Water 27.08±0.85 1299.3±45.9 26.81±0.82 ------4.60 d a ab b Solvent 27.52±0.92 1277.5±24.7 25.37±0.39 ------5.60 c b ab c c ab 2.5 33.50±1.08 699±138 22.61±1.29 15.16±1.18 0.94±0.07 6.60

ellow Pine b c bc b b ab 5.0 49.25±2.52 654±14b 13.29±6.72 29.65±2.13 1.85±0.13 7.20 a c c a a a 10.0 84.65±0.87 276.7±39.1 4.76±1.66 62.33±3.07 3.89±0.19 8.40 F 298.22 22.37 8.66 114.36 114.36 4.89

p 0.00 0.00 0.00 0.00 0.00 0.00 a a c Water 32.10±5.08c 1249.8±28.2 37.54±2.00 ------2.40 a a c Cotton Wood Cotton Solvent 36.20±2.29c 1258.3±81.4 36.45±1.05 ------2.40 a a c c bc 2.5 26.15±0.39c 1184.5±19.8 33.81±1.97 15.81±0.38 0.98±0.02 3.60 b b b b ab 5.0 50.70±0.38b 764.3± 27.7 24.80±3.27 29.86±1.15 1.86±0.07 6.20 c c a a a 10.0 83.35±4.65a 404±120 9.17±1.27 68.39±2.09 4.27±0.13 8.40

F 49.61 31.17 32.87 379.22 379.22 10.55 p 0.00 0.00 0.00 0.00 0.00 0.00 Means±SE sharing same letters in columns for each wood species are not significantly different from each other at p>0.05. Conc. = concentration

Mean weight loss of C. deodara treated and un-treated SYP and CW exposed to H. indicola along with retention and E-1 rating is presented in Table 4.4.11. SYP and CW treated with C. deodara extractives showed minimum weight loss (12.98 and 10.07%, respectively) at the highest concentration (10 mg ml-1). Weight loss was found to be inversely related to extractive concentrations. Average rating at maximum retention (66.54 and 63.87 kg/m3 for SYP and CW, respectively) respectively was 8.0 and 7.6. Weight loss of control treatments of SYP was statistically higher than all other treatments. A similar trend was found in CW. At maximum concentration (10 mg ml-1), mortality was 58.92 % with SYP and 92.28% termites were found dead at 10 mg ml-1 on CW. Mean weight loss of P. roxburghii treated and un-treated SYP and CW exposed to H. indicola along with retention and E-1 rating is presented in Table 4.4.12. P. roxburghii heartwood extractives were lethal to termites at each concentration and caused 100 and 81.9 % mortality of H. indicola at maximum concentration (10 mg ml-1) after feeding on treated SYP and CW, respectively. Average weight loss of treated and untreated SYP and CW exposed to H. indicola was also concentration dependent. SYP and CW treated with P. roxburghii extractives showed minimum weight loss (6.76 and 9.75%, respectively) at the highest concentration (10 mg ml-1).

Weight losses of SYP and CW treated with extractives of four woods revealed that the interaction among wood species, concentration of extractives and non-durable woods were significant (Appendix 4.7). Minimum weight loss was observed on SYP treated with D. sissoo heartwood extractives was non-significantly different from other heartwood extractives at 10 mg ml-1. Minimum weight loss of CW was also recorded after the treatment of D. sissoo extractives at the highest concentration (Table. 4.4.13). ANOVA for mortality of termites after feeding on treated SYP and CW revealed that interaction among durable wood species, concentration of extractives and non-durable woods was significant (Appendix 4.8). Maximum mortality (95.80%) of termites was observed after feeding on SYP which was treated with P. roxburghii heartwood extractives while maximum mortality (90.8 %) was found with C. deodara extractives after feeding on CW. Further, all extractives performed non-significantly different from one another at the highest concentration (Table. 4.4.14).

Table 4.4.11. Average rating, retention, mortality and weight losses of SYP and CW treated with C. deodara extractives after feeding of H. indicola. Woods Conc. Mortality (%) Weight loss Retention Rating (avg.) (mg ml-1) (mg) (%) (kg m-3) pcf

Southern Y a a a b Water 27.08±0.85 1299.3±45.9 26.81±0.82 ------4.60 a a a ab Solvent 27.52±0.92 1277.5±24.7 25.37±0.39 ------5.60 a ab ab c c ab 2.5 48.40±13.4 805±19 17.61±4.30 14.89±1.00 0.93±0.06 5.80

ellow Pine a ab ab b b ab 5.0 61.20±15.1 828±16 18.43±3.54 29.85±1.17 1.86±0.07 7.40 a b b a a a 10.0 58.52±10.5 599±10 12.98±2.28 66.54±1.49 4.15±0.09 8.00 F 2.61 6.15 4.47 459.41 459.41 3.55

p 0.00 0.00 0.01 0.00 0.00 0.02 c a a b Water 32.10±5.08 1249.8±28.2 37.54±2.00 ------2.40 c a a b Cotton Wood Cotton Solvent 36.20±2.29 1258.3±81.4 36.45±1.05 ------2.40 b b b c c a 2.5 64.30±2.09 764.3±43.3 26.02±1.47 15.91±0.76 0.99±0.04 5.80 b bc b b b a 5.0 64.70±3.58 637.6±75.8 20.01±2.60 33.17±1.09 2.07±0.06 7.40 a c c a a a 10.0 92.28±6.51 461.3±66.3 10.07±2.37 63.87±4.19 3.99±0.26 7.60

F 33.19 33.91 33.85 91.39 91.39 14.59 p 0.00 0.00 0.00 0.00 0.00 0.00 Means±SE sharing same letters in columns for each wood species are not significantly different from each other at p>0.05. Conc. = concentration

Table 4.4.12. Average rating, retention, mortality and weight losses of SYP and CW treated with P. roxburghii extractives after feeding of H. indicola.

Woods Conc. Mortality (%) Weight loss Retention Rating (avg.) (mg ml-1) (mg) (%) (kg m-3) Pcf

Southern Yellow Pine c a a b Water 27.08±0.85 1299.3±45.9 26.81±0.82 ------4.60 c a a ab Solvent 27.52±0.92 1277.5±24.7 25.37±0.39 ------5.60 c b a c c b 2.5 54.3±10.7b 712±163 22.34±3.63 15.73±0.59 0.98±0.03 4.20 ab c b b b ab 5.0 77.3±12.5 353±16b 12.48±1.80 29.72±2.71 1.85±0.16 7.40 a c b a a a 10.0 100±0.00 186±103 6.76±2.92 59.63±5.00 3.72±0.31 8.20 F 18.65 19.44 14.79 46.16 46.16 4.52

p 0.00 0.00 0.00 0.00 0.00 0.00 c a a bc Water 32.10±5.08 1249.8±28.2 37.54±2.00 ------2.40 bc a a bc Cotton Wood Cotton Solvent 36.20±2.29 1258.3±81.4 36.45±1.05 ------2.40 bc ab a c c c 2.5 46.95±6.32 939.1±84.2 39.85±4.52 14.74±1.16 0.92±0.07 2.20 ab bc a b b ab 5.0 61.45±8.21 800.0±57.3 31.28±2.84 31.02±1.60 1.93±0.10 6.40 a c b a a a 10.0 81.9±10.0 512±110 9.75±2.81 58.48±1.26 3.65±0.07 7.80

F 8.64 16.78 18.12 267.58 267.58 7.75 p 0.00 0.00 0.00 0.00 0.00 0.00 Means±SE sharing same letters in columns for each wood species are not significantly different from each other at p>0.05. Conc. = concentration

Table 4.4.13. Weight losses of SYP and CW treated with four durable wood extractives. Wood species Concentrations SYP CW

100

Solvent 25.34±0.30d-f 36.44±0.70a Water 26.68±0.54b-f 37.54±1.33a T. grandis 2.5 mg ml-1 23.00±0.88d-g 32.52±1.72a-d 5.0 mg ml-1 18.09±0.48f-j 20.01±1.48e-i 10 mg ml-1 5.50±0.77k 12.36±2.28h-k Solvent 25.43±0.24d-f 36.44±0.70a Water 24.14±2.43d-g 37.54±1.33a D. sissoo 2.5 mg ml-1 18.88±2.00f-j 35.14±1.10a-c 5.0 mg ml-1 13.92±3.51g-k 24.14±1.58d-f 10 mg ml-1 5.33±0.90k 10.69±1.87i-k Solvent 25.24±0.26d-f 36.87±0.71a Water 26.67±0.54b-f 37.54±1.33a C. deodara 2.5 mg ml-1 20.74±3.96e-h 26.14±0.98c-f 5.0 mg ml-1 19.45±2.52e-j 20.11±1.74e-i 10 mg ml-1 13.69±1.62g-k 12.71±1.77h-k Solvent 25.24±0.26d-f 36.44±0.70a Water 26.67±0.54b-f 37.54±1.33a P. roxburghii 2.5 mg ml-1 18.97±2.67f-j 35.85±2.92ab 5.0 mg ml-1 10.04±2.06jk 28.66±1.94a-e 10 mg ml-1 5.37±1.76k 13.76±2.60g-k Means±SE sharing same letters in columns for each wood species are not significantly different from each other at p>0.05.

Table 4.4.14. Mortality of termites after feeding of SYP and CW treated with four durable wood extractives. Wood species Concentrations SYP CW T. grandis Solvent 26.38±0.70k 36.20±1.53h-k

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Water 26.34±0.75k 32.10±3.38i-k 2.5 mg ml-1 27.53±1.08jk 37.95±4.72h-k 5.0 mg ml-1 40.91±2.02g-k 70.60±4.98a-f 10 mg ml-1 88.39±5.34ab 77.67±6.88a-e Solvent 27.34±1.21jk 36.20±1.53h-k Water 28.63±2.73jk 32.10±3.38i-k D. sissoo 2.5 mg ml-1 44.38±6.46f-k 27.15±1.29jk 5.0 mg ml-1 53.38±6.76e-j 51.13±2.14e-k 10 mg ml-1 86.58±3.04a-c 88.42±3.71ab Solvent 26.38±0.70k 32.63±3.59i-k Water 26.04±0.63k 32.10±3.38i-k C. deodara 2.5 mg ml-1 44.12±9.83f-k 61.37±1.65c-h 5.0 mg ml-1 60.83±9.98c-h 65.85±4.53b-g 10 mg ml-1 58.24±6.98d-i 90.82±4.77ab Solvent 26.38±0.70k 36.20±1.53h-k Water 25.96±0.65k 32.10±3.38i-k P. roxburghii 2.5 mg ml-1 55.33±7.44d-i 48.95±5.88f-k 5.0 mg ml-1 77.35±8.30a-e 61.45±5.47c-h 10 mg ml-1 95.80±4.20a 81.35±6.80a-d Means±SE sharing same letters in columns for each wood species are not significantly different from each other at p>0.05.

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4.4.3. Determination of leaching resistance of extractives impregnated woods against termites 4.4.3.1. Mean termites’ mortality and % weight loss of leached and un-leached SYP and CW after R. flavipes feeding

Mean percent termites’ mortality and weight losses of SYP and CW in the no-choice test for un-leached and leached extractives treated wood specimens after release of R. flavipes on them are shown in Table. 4.4.15. Leaching signiﬁcantly affected termites’ resistance against both SYP and CW specimens treated with C. deodara and P. roxburghii extractives. There was significant difference in feeding and mortality of R. flavipes on SYP and CW treated with C. deodara and P. roxburghii extractives. More feeding and less mortality of R. flavipes was observed on leached samples as compared to un- leached samples. Mortality of termites in case of C. deodara extractives treated un-leached SYP was 57 % while it was 24 % on leached samples. SYP and CW treated with T. grandis and D. sissoo did not show any significant difference in mortality and weight losses on leached and un-leached samples. Mean average rating and retention of SYP and CW following no-choice tests for un- leached and leached wood extractives treated specimens after feeding of R. flavipes are shown in Table 4.4.16. Results showed that there was non-significant difference in rating of T. grandis and D. sissoo treated leached or un-leached SYP and CW after feeding of R. flavipes.

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Table 4.4.15. Mean mortality (%) of R. flavipes and weight losses of SYP and CW woods after leaching and un-leaching tests of extractives.

Wood species Treatments Mean Mortality (%) Mean Weight loss (%) L UL t p L UL t p-value

Southern Y NS NS Solvent 27.10 25.25 2.79 0.07 27.38±.61 25.12±0.39 6.94 0.06 NS NS Water 22.63±0.70 24.85±0.78 -1.75 0.15 25.15±0.55 26.54±0.81 -1.68 0.16 NS NS T. grandis 76.68±1.28 82.80±10.13 -0.645 0.55 7.65±0.57 5.47±1.16 2.64 0.06 NS NS ellow Pine D. sissoo 79.61±2.72 88.50±6.24 -1.10 0.33 7.01±0.46 5.92±0.84 1.08 0.33 ** ** C. deodara 24.80±0.46 57.95±10.41 -3.07 0.03 27.90±0.64 14.41±2.52 5.68 0.00 ** ** P. roxburghii 39.28±4.04 91.60±8.40 -4.92 0.00 15.02±2.70 3.97±2.12 4.72 0.00

NS NS Solvent 38.60±0.88 32.10±5.07 1.38 0.23 28.94±1.18 37.53±2.00 ˗2.78 0.06

Cotton Wood Cotton NS ns Water 37.92±0.81 36.20±2.29 0.66 0.54 36.69±0.86 36.45±1.05 0.12 0.90 NS NS T. grandis 81.59±1.33 84.17±6.53 ˗0.44 0.68 9.68±0.63 12.35±3.41 ˗0.86 0.43 NS ** D. sissoo 83.29±7.52 93.50±5.23 ˗1.03 0.35 28.18±0.68 12.21±3.59 4.24 0.01 C. deodara 53.51±2.96 89.35±7.68 ** 0.05 30.41±1.31 15.35±2.23 ** 0.00 ˗5.48 8.38 ** ** P. roxburghii 54.30±1.74 80.80±10.54 ˗2.52 0.04 33.06±1.44 17.77±3.79 3.35 0.02 L- leached; UL-un-leached; t, t-test value; p-p-value; **- significant; NS- non-significant

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Table 4.4.16. Mean rating of leached and un-leached SYP and CW after feeding of R. flavipes. Wood Treatments Retention (kg m-3) Rating (avg.) species L UL t P Solvent ------4.40±0.40 3.60±0.97 NS 0.58 Southern 0.59 NS Control ------4.80±0.48 4.60±0.60 0.21 0.83 NS Pine T. grandis 48.69± 5.78 8.10±0.40 8.90±0.24 ˗1.72 0.16

Y NS D. sissoo 55.22±0.707 8.60±0.24 9.10±0.10 0.08 ellow ˗2.23 ** C. deodara 53.24±0.80 4.00±1.09 8.50±0.31 ˗5.03 0.00 ** P. roxburghii 54.03±0.98 6.80±0.37 8.80±0.20 ˗6.31 0.00 NS Solvent ------2.40±0.97 1.60±0.97 0.53 0.62

Cotton Wood Cotton NS Water ------3.20±0.80 2.40±0.97 0.53 0.62 ns T. grandis 58.20±2.07 7.60±0.24 8.90±0.24 ˗2.98 0.06 NS D. sissoo 61.29±1.02 7.20±0.37 8.20±0.37 ˗1.58 0.18 C. deodara 57.00±2.23 6.60±0.24 7.80±0.20 ** 0.00 ˗6.00 ** P. roxburghii 56.62±0.98 6.80±0.37 8.00±0.31 ˗3.20 0.03 L- leached; UL-un-leached; t, t-test value; p-p-value; **- significant; NS- non-significant

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4.4.3.2. Mean termites’ mortality and % weight loss of leached and un-leached SYP and CW after H. indicola feeding

Mean % mortality and weight losses of SYP and CW in no-choice test for un-leached and leached wood extractives treated specimens after feeding of H. indicola are shown in Table 4.4.17.

Leached specimens of both SYP and CW became highly susceptible to be attached by H. indicola when SYP and CW were treated with C. deodara and P. roxburghii extractives. There was significant difference in feeding and mortality of H. indicola after feeding on SYP and CW treated with C. deodara and P. roxburghii extractives. Mortalities of termites on C. deodara extractives treated un-leached and leached SYP specimens were, respectively, 92 and 51%. SYP and CW treated with T. grandis and D. sissoo did not show any significant difference in mortality and weight losses on leached and un-leached samples.

Mean average rating and retention of SYP and CW following no-choice tests for un- leached and leached wood extractives treated specimens after feeding of H. indicola are shown in Table 4.4.18.

Results showed that there was no significant difference in rating of T. grandis and D. sissoo treated SYP and CW after feeding of H. indicola. There was significant difference in average rating of C. deodara and P. roxburghii heartwood extractives treated CW and SYP after leaching.

In nutshell, extractives of C. deodara and P. roxburghii were not retained in SYP and CW following leaching procedure as evident by more feeding and less mortality of termites on them. Conversely, SYP and CW specimens treated with T. grandis and D. sissoo heartwood extractives restricted termites’ feeding after leaching. Mortalities of termites after feeding on leached specimens were also non- significantly different from un-leached samples.

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Table 4.4.17. Mean mortality (%) of H. indicola and weight losses of SYP and CW woods after leaching and un-leaching tests of extractives.

Wood Treatments Mean mortality (%) Mean weight loss (%) species L UL t p L UL t p NS NS Solvent 24.73±0.51 27.52±0.92 ˗3.47 0.35 24.44±1.05 25.37±0.39 ˗1.10 0.33 Y Water 27.10±0.75 27.08±0.85 NS 0.99 24.60±0.47 26.81±0.82 NS 0.10

Southern ellow Pine 0.01 ˗4.43 NS ** T. grandis 93.98±3.16 83.26±3.79 1.73 0.15 10.52±0.58 5.53±1.17 5.56 0.00 ** NS D. sissoo 78.32±2.53 84.65±0.87 ˗2.84 0.04 8.37±0.67 4.76±1.66 1.71 0.16

NS NS C. deodara 32.76±3.84 58.52±10.52 ˗2.52 0.06 17.17±0.81 12.97±2.27 1.69 0.16 ** NS P. roxburghii 43.73±2.93 100.00±0.00 ˗19.17 0.00 19.39±2.98 9.75±2.81 2.33 0.07 NS NS Solvent 34.95±1.03 36.20±2.29 ˗0.91 0.41 34.00±1.16 35.52±1.59 ˗4.16 0.10 Cotton Wood Cotton NS NS Water 31.84±3.24 32.10±5.07 ˗0.04 0.96 36.29±1.08 37.53±2.00 ˗0.46 0.66 NS NS T. grandis 79.58±2.57 73.30±12.20 0.50 0.63 17.91±2.08 12.36±3.41 1.20 0.29 NS ** D. sissoo 74.36±4.52 83.35±4.64 ˗1.32 0.25 14.72±1.01 9.17±1.27 3.31 0.03 C. deodara 51.50±1.58 92.28±6.50 ** 0.00 23.70±2.08 10.07±2.36 ** 0.01 ˗7.87 4.14 ** ** P. roxburghii 55.35±1.01 81.90±10.04 ˗2.68 0.05 24.79±1.15 9.75±2.81 4.25 0.01 L- leached; UL-un-leached; t, t-test value; p-p-value; **- significant; NS- non-significant

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Table 4.4.18. Mean rating of leached and un-leached SYP and CW after feeding of H. indicola. Wood Treatments Retention (kg m-3) Rating (avg) species L UL t p Solvent ------4.40±.40 5.60±.40 NS 0.07 Southern Yellow ˗2.44 NS Water ------3.20±.80 4.60±1.24 ˗2.33 0.08 ** Pine T. grandis 56.98±3.25 8.20±.20 9.00±.27 ˗3.13 0.03 NS D. sissoo 82.5±3.06 8.20±.20 8.40±.24 ˗1.00 0.37 NS C. deodara 66.53±1.49 7.00±.31 8.00±.31 ˗2.23 0.08 ** P. roxburghii 59.62±4.99 6.80±.37 8.20±.37 ˗2.74 0.05 NS Solvent ------4.40±.40 2.40±.97 1.58 0.18

Cotton Wood Cotton NS Water ------4.00±.00 2.40±.97 1.63 0.17 NS T. grandis 68.09±0.97 7.60±.24 8.20±.37 ˗2.44 0.07

NS D. sissoo 68.39±1.87 8.00±.31 8.40±.24 ˗0.78 0.44 C. deodara 63.87±4.19 5.40±.60 7.60±.24 ** 0.02 ˗3.77 ** P. roxburghii 58.48±1.25 5.60±.40 7.80±.37 ˗3.77 0.02 L- leached; UL-un-leached; t, t-test value; p-p-value; **- significant; NS- non-significant

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4.4.4. Wood preservative potential of four heartwood extractives in combination with linseed oil against R. flavipes and H. indicola

4.4.4.1. Wood preservative potential of four heartwood extractives in combination with linseed oil against R. flavipes

Mean wood weight loss, termites’ mortality and E1 rating of the test for T. grandis extractives and linseed oil combination treated and un-treated SYP and CW exposed to R. flavipes are presented in Table 4.4.19. Solvent and water treated SYP controls lost a weight of 20.56 and 20.64% with average rating of 5.6 and 6.0, respectively while CW showed a weight loss of 23.52 and 23.20% respectively for solvent and water treated blocks with the average rating of 5.0 and 5.2, respectively. Weight losses of SYP and CW in control treatments were non- significantly different from each other. SYP and CW treated with 20% linseed oil only was non-significantly different from other two control treatments in term of mortality, weight loss and average rating. Conversely, SYP and CW treated with T. grandis extractives alone and in combination with oil showed minimum weight loss. Weight losses of both CW and SYP were non-significantly different when treated with extractives alone or in combination but mortality of R. flavipes was significantly higher (in terms of number) when termite were fed on woods treated with oil and extractive combination. 100% termites’ mortality was found on SYP and CW treated with T. grandis extractive + oil combination as compared to extractive alone. Weight loss of control treatments of SYP was statistically higher than all other treatments. A similar trend of extractive activity was found on cotton wood.

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Table 4.4.19. Weight losses, termites’ mortality and rating of SYP and CW treated with T. grandis extractives and linseed oil after feeding of R. flavipes.

Wood Treatments Weight loss Weight loss Mortality Rating species (mg) (%) (%)

a b c c Solvent 908±177 20.56±3.79 21.50±1.06 5.60 So Water a b c c u 940.7±33.40 20.64±0.75 24.80±1.65 6.00

thern Yellow Pine ab b b c Oil (20%) 574±114 10.82±2.26 44.25±5.28 6.40

b a a ab Extractives 248.0±52.9 6.80±1.1 82.8±10.10 8.90

ab a a a Extractives + 167.9±13.1 4.12±0.2 100.00±0.00 9.40 oil

F; p 13.37; 0.00 69.19; 0.00 46.01; 0.00 11.92;0.0

ab a cd b Solvent 640.00±60.60 23.52±2.56 35.30±3.05 5.00

a a d b Water 702.0±21.5 23.20±0.67 27.600±0.66 5.20

Cotton Wood Cotton a ab c b Oil (20%) 778.00±61.80 20.36±1.18 44.00±1.66 6.40

bc bc a Extractives 412.00±121 12.36±3.42 82.05±7.37b 8.90

c c a a Extractives + 290.00±26.60 9.811±0.74 99.95±0.05 9.50 oil F; p 9.02; 0.00 9.80; 0.00 74.32; 0.00 28.72;0.0 Means±SE sharing same letters in columns for each wood species are not significantly different from each other at p>0.05.

Mean wood weight loss, termite mortality and E1 rating of test for D. sissoo extractives and linseed oil combination treated and un-treated SYP and CW exposed to R. flavipes are presented in Table 4.4.20. SYP and CW treated with D. sissoo extractives alone and in combination with oil showed minimum weight loss (5.92 and 6.85% which was non- significant with each other). Weight losses of both CW and SYP were non-significantly different when treated with extractives alone or in combination but mortality of R. flavipes was significantly higher (in terms of number) when termite were fed on woods treated with oil and extractive combination. 100% termites were dead after feeding on SYP and CW treated with

110

D. sissoo extractive + oil combination as compared to extractive alone raising average rating to >9. Weight loss in control treatments of SYP was statistically higher than all other treatments. A similar trend was found in cotton wood.

Table 4.4.20. Weight losses, termites’ mortality and rating of SYP and CW treated with D. sissoo extractives and linseed oil after feeding of R. flavipes.

Wood Treatments Weight loss Weight loss Mortality Rating species (mg) (%) (%)

a a c c Solvent 908±177 20.56±3.79 21.50±1.06 5.60 So Water a a c bc u 940.7±33.4 20.64±0.75 24.80±1.65 6.00

thern ab b b c Oil (20%) 574±114 10.82±2.26 44.25±5.28 6.40

Yellow Pine b b a ab Extractives 274.0±38.7 5.92±0.84 88.50±6.25 9.10

b b a a Extractives + 330.1±53.5 6.85±1.02 100.00±0.00 9.70 oil

F; p 9.82;0.00 11.95;0.00 93.59;0.00 14.89;0.00

ab a bc b Solvent 640±60.60 23.52±2.56 35.30±3.05 5.00

a a c b Water 702±21.50 23.20±0.67 27.60±0.66 5.20

Cotton Wood Cotton a ab b b Oil (20%) 778±61.80 20.36±1.18 44.00±1.66 6.40

bc bc a a Extractives 392±116 12.21±3.59 93.50±5.23 8.20

c c a a Extractives + 222 ±19.30 7.64±0.57 100.00±0.00 9.80 oil F; p 12.36; 0.00 11.66; 0.00 145.23; 0.00 24.63;0.00 Means±SE sharing same letters in columns for each wood species are not significantly different from each other at p>0.05. Mean wood weight loss, termite mortality and E1 rating of test for C. deodara extractives and linseed oil combination treated and un-treated SYP and CW exposed to R. flavipes are presented in Table 4.4.21. SYP and CW treated with 20% linseed oil was non- significantly different from other two control treatments in term of mortality, weight loss and average rating. Conversely, SYP and CW treated with C. deodara extractives alone and in combination with oil showed minimum weight loss (14.42 and 8.42 of SYP). Weight loss of

111 both CW and SYP was non-significantly different when treated with extractives alone or in combination but mortality of R. flavipes was significantly higher (in terms of number) when termite were fed on woods treated with oil and extractive combination. 95.5 and 100 % termites were dead after feeding on SYP and CW treated with C. deodara extractive + oil combination as compared to extractive alone (58%) with average rating >9. Weight loss of control treatments of SYP was statistically higher than all other treatments. A similar trend was found in cotton wood.

Table 4.4.21. Weight losses, termites’ mortality and rating of SYP and CW treated with C. deodara extractives and linseed oil after feeding of R. flavipes.

Wood Treatments Weight loss Weight loss Mortality Rating species (mg) (%) (%)

a a c c Solvent 908.00±177 20.56±3.79 21.50±1.06 5.60 So Water a a c c u 940.7±33.40 20.643±0.75 24.80±1.65 5.2

thern Yellow Pine ab ab bc b Oil (20%) 574.00±114 10.82±2.26 44.25±5.28 7.0

ab ab b b Extractives 666±116 14.42±2.53 58.0±10.4 8.5

b b a a Extractives + 406.2±53.3 8.22±1.13 95.25±4.75 9.6 oil

F; p 4.15; 0.01 5.71; 0.00 27.52; 0.00 33.72;0.00

ab a bc c Solvent 640±60.60 23.52±2.56 35.30±3.05 5.0

a a c c Water 702±21.50 23.20±0.67 27.60±0.66 5.2

Cotton Wood Cotton a ab b bc Oil (20%) 778±61.80 20.36±1.18 44.00±1.66 6.4

b b a b Extractives 466±67 15.35±2.24 86.69±9.31 7.8

b b a a Extractives + 454±24.40 14.91±1.22 100.00±0.00 9.1 oil F; p 7.92; 0.00 5.77; 0.00 74.64; 0.00 20.33;0.00 Means±SE sharing same letters in columns for each wood species are not significantly different from each other at p>0.05. Mean wood weight loss, termite mortality and E1 rating of test for P. roxburghii extractives and linseed oil combination treated and un-treated SYP and CW exposed to R.

112 flavipes are presented in Table 4.4.22. SYP and CW treated with P. roxburghii extractives alone and in combination with oil showed minimum weight loss. Weight loss of both CW and SYP was non-significantly different when treated with extractives alone or in combination but mortality of R. flavipes was significantly higher (in terms of number) when termite were fed on woods treated with oil and extractive combination. Mortality of termites was >80% after feeding on SYP and CW treated with P. roxburghii extractive + oil combination as compared to extractive alone with average rating >9. Weight loss of control treatments of SYP was statistically higher than all other treatments. A similar trend was found in cotton wood.

Table 4.4.22. Weight losses, termites’ mortality and rating of SYP and CW treated with P. roxburghii extractives and linseed oil after feeding of R. flavipes.

Wood Treatments Weight loss Weight loss Mortality Rating species (mg) (%) (%) Solvent 908±177a 20.56±3.79a 21.50±1.06c 5.6b

So a a c ab u Water 940.7±33.4 20.64±0.75 24.80±1.65 7.0

thern Oil (20%) 574±114ab 10.82±2.26b 44.25±5.28bc 6.4b

Yellow Pine Extractives 182±101b 3.98±2.13b 91.60±8.40b 8.8a Extractives + 197.3±28.60ab 11.97±0.57ab 100.00±0.00a 9.0a oil F; p 8.38;0.00 10.09;0.00 67.29;0.00 20.33;0.00 Solvent 640±60.60a 23.52±2.56a 35.30±3.05c 5.0c Water 702±21.50a 23.20±0.67a 27.60±0.66c 5.2c

Cotton Wood Cotton Oil (20%) 778±61.80a 20.36±1.18a 44.00±1.66bc 6.4bc Extractives 500±107.00a 17.77±3.79a 77.80±14.20ab 8.0b Extractives + 576±54.10a 19.51±1.19a 80.80±10.60a 8.7a oil F; p 2.61;0.06 1.25;0.32 9.28;0.00 15.33;0.00 Means±SE sharing same letters in columns for each wood species are not significantly different from each other at p>0.05.

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4.4.4.2. Wood preservative potential of four heartwood extractives in combination with linseed oil against H. indicola

Mean % weight loss of treated and un-treated SYP and CW with extractives of T. grandis eand oil exposed to H. indicola along with retention and E-1 rating is presented in table 4.4.23. Solvent and water treated SYP controls lost a weight of 22.50 and 22.41 % with the average rating of 5.6 and 5.4 respectively. Solvent and water treated CW controls lost wood weight of 25.64 and 25.28%, respectively with the average rating of 6.2 and 6.4. Mortality of H. indicola and weight losses of CW and SYP were non-significantly different from solvent and water treated controls. Maximum mortality (100 and 97 %) of H. indicola was found after feeding on extractives + oil treated SYP and CW, respectively after 28 days of exposure. SYP and CW treated with highest concentrations of T. grandis heartwood extractives and extractives + oil combination showed minimum weight loss with the highest average rating for both SYP and CW. Weight loss of control treatments of SYP was statistically higher than all other treatments and a similar trend was found in CW. Mean % mortalities, weight losses and E1 rating of SYP and CW treated with D. sissoo extractives and oil exposed to H. indicola is presented in Table 4.4.24. Mortality of H. indicola and weight losses of CW and SYP were non-significantly different from solvent and water treated controls. Maximum mortality (100 %) of H. indicola was found after feeding on extractives + oil treated SYP and CW, respectively after 28 days of exposure. SYP and CW treated with the highest concentration of D. sissoo heartwood extractives and extractives + oil combination showed minimum weight loss with the highest average rating for both SYP and CW. Weight losses of SYP and CW controls were statistically higher than all other treatments.

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Table 4.4.23. Weight losses, termites’ mortality and rating of SYP and CW treated with T. grandis extractives and linseed oil after feeding of H. indicola.

Wood Treatments Weight loss Weight loss Mortality Rating species (mg) (%) (%)

a a c bc Solvent 990±193.00 22.41±4.13 19.35±0.95 5.6 So Water a a c c u 1025.30±36.40 22.50±0.81 22.32±1.48 5.4

thern ab b b ab Oil (20%) 626.00±125.00 11.80±2.47.00 39.83±4.75 7.4

Yellow Pine b b a a Extractives 270.30±57.60 5.53±1.17 93.99±3.17 9.0

b b a a Extractives + 184.00±14.30 4.02±0.31 100.00±0.00 9.5 oil

F; p 13.35;0.00 15.64;0.00 214.08;0.00 15.20;0.00

ab a c c Solvent 697.60±66.00 25.64±2.79 31.77±2.75 6.4

a a c c Water 765.20±23.50 25.28±0.73 24.84±0.60 6.2

Cotton Wood Cotton a a c bc Oil (20%) 848±67.40 22.19±1.29 39.60±1.50 7.0

bc b b ab Extractives 441±130.0 12.36±3.42 73.30±12.2 8.2

c b a a Extractives + 316.10±29.0 10.69±0.808 97.95±2.04 8.8 oil F; p 9.36;0.000 11.57;0.00 29.52;0.00 13.46;0.00 Means±SE sharing same letters in columns for each wood species are not significantly different from each other at p>0.05.

Mean % mortalities, E1 rating and weight losses of SYP and CW treated with C. deodara heartwood extractives and oil exposed to H. indicola is presented in table 4.4.25. Mortality of H. indicola and weight losses of CW and SYP were non-significantly different from solvent and water treated controls. Maximum mortality (100 %) of H. indicola was found after feeding on extractives + oil treated SYP and CW, respectively after 28 days of exposure. SYP and CW treated with highest concentrations of C. deodara heartwood extractives and extractives + oil combination showed minimum weight loss with the highest average rating for both SYP and CW. Weight loss of SYP and CW in control treatments were statistically higher than all other treatments except 20 % oil control.

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Mean weight losses of P. roxburghii extractives and oil treated and un-treated SYP and CW exposed to H. indicola along with retention and E-1 rating is presented in Table 4.4.26. Maximum mortality (100 and 97%) of H. indicola was found after feeding on extractives + oil treated SYP and CW, respectively after 28 days of exposure. SYP and CW treated with the highest concentrations of P. roxburghii heartwood extractives and extractives + oil combination showed minimum weight loss with the highest average rating for both SYP and CW. Weight losses of control treatments were non-significantly different from one another and with other treatments except on CW which was treated with extractives + oil.

Table 4.4.24. Weight losses, termites’ mortality and rating of SYP and CW treated with D. sissoo extractives and linseed oil after feeding of H. indicola.

Wood Treatments Weight loss Weight loss Mortality Rating species (mg) (%) (%)

a a d bc Solvent 990±193.00 22.41±4.13 19.35±0.95 5.6 So Water a a d c u 1025.30±36.40 22.50±0.81 22.32±1.48 5.4

thern ab b c ab Oil (20%) 626±125.00 11.80±2.47 39.83±4.75 7.4

Yellow Pine b b b a Extractives 276.70±39.10 4.76±1.66 84.65±0.87 8.4

b b a a Extractives + 202.80±12.50 4.44±0.235 100.00±0.00 9.0 oil

F; p 13.33;0.00 15.08;0.00 258.12;0.00 12.63;0.00

ab a cd c Solvent 697.60±66.00 25.64±2.79 31.77±2.75 6.4

a a d c Water 765.20±23.50 25.28±0.73 24.84±0.60 6.2

Cotton Wood Cotton a c ab Oil (20%) 848.0±67.4 22.19±1.29 A 39.60±1.50 7.0

bc b b a Extractives 404.00±120 9.17±1.27 83.35±4.65 8.4

c b a a Extractives + 242.0±21 8.33±0.622 100.00±0.00 9.0 oil F; p 13.67;0.00 31.26;0.00 177.70;0.00 21.39;0.00 Means±SE sharing same letters in columns for each wood species are not significantly different from each other at p>0.05.

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Table 4.4.25. Weight losses, termites’ mortality and rating of SYP and CW treated with C. deodara extractives and linseed oil after feeding of H. indicola.

Wood Treatments Weight loss Weight loss Mortality Rating species (mg) (%) (%)

a c c b Solvent 990±193 22.41±4.13 19.35±0.95 5.6 So Water a c c b u 1025.3±36.4 22.50±0.81 22.32±1.48 5.4

thern ab bc bc ab Oil (20%) 626±125 11.80±2.47 39.83±4.75 7.4

Yellow Pine ab b b a Extractives 599±104 12.98±2.28 58.5±10.5 8.0

b a a a Extractives + 442.80±58.10 8.96±1.23 100.00±0.00 9.1 oil

F; p 4.86;0.00 6.53;0.00 39.98;0.00 10.77;0.00

a a bc bc Solvent 697.60±66.00 25.64±2.79 31.77±2.75 6.4

a a c c Water 765.20±23.50 25.28±0.73 24.84±0.60 6.2

Cotton Wood Cotton b ab b bc Oil (20%) 848.00±67.40 22.19±1.29 39.60±1.50 7.0

c c a b Extractives 461.30±66.30 10.07±2.37 92.28±6.51 7.6

c bc a a Extractives + 494.90±26.60 16.25±1.33 100.00±0.00 9.0 oil F; p 9.82;0.00 12.75;0.00 120.56;0.00 14.57;0.00 Means±SE sharing same letters in columns for each wood species are not significantly different from each other at p>0.05.

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Table 4.4.26. Weight losses, termites’ mortality and rating of SYP and CW treated with P. roxburghii extractives and linseed oil after feeding of H. indicola.

Wood Treatments Weight loss Weight loss Mortality Rating species (mg) (%) (%)

a a c c Solvent 990±193 22.41±4.13 19.35±0.95 5.6 So Water a a c c u 1025.30±36.4 22.50±0.81 22.32±1.48 5.4

thern ab ab b b Oil (20%) 626.00±125.00 11.80±2.47 39.83±4.75 7.4

Yellow Pine b b a ab Extractives 186.00±103.00 6.76±2.92 100.00±0.00 8.2

ab ab a a Extractives + 651.00±31.20 13.04±0.62 100.00±0.00 8.5 oil

F; p 8.82;0.00 7.36;0.00 321.99;0.00 8.57;0.00

ab a b c Solvent 697.60±66 25.64±2.79 31.77±2.75 6.4

ab a c Water 765.20±23.50 25.28±0.73 24.84±0.60b 6.2

Cotton Wood Cotton a a b b Oil (20%) 848.00±67.40 22.19±1.29 39.60±1.50 7.0

a b a ab Extractives 512.00±110.00 9.75±2.81 81.90±10.0 7.8

ab a a a Extractives + 627.80±59.00 21.26±1.30 97.00±3.00 8.9 oil F; p 3.33;0.03 10.70;0.00 43.45;0.00 12.28;0.00 Means±SE sharing same letters in columns for each wood species are not significantly different from each other at p>0.05.

Overall results showed that maximum mortality of both termite species was recorded when termite were fed on SYP and CW treated with extractives + 20% linseed oil. All heartwood extractives + oil performed non-significantly at latter treatment (Table 4.4.27). ANOVA revealed that interaction among wood species, concentrations of extractives and non- durable wood species were significantly different (Appendix 4.10). Wood weight losses of non-durable species were observed after 28 days and ANOVA test revealed that the interaction among durable wood species and concentrations of extractives were significantly different. Similarly interaction among non-durable wood species and concentrations of extractives were

118 also significantly different (Appendix 4.9). All type of heartwood extractives + oil performed non-significantly in term of weight losses of SYP and CW (Table 4.4.28). Minimum weight losses of SYP and CW were recorded when these were treated with extractive + oil but it varied non- significantly with extractive treated SYP and CW (Table. 4.4.29).

Table 4.4.27. Mortality of termites after feeding on SYP and CW treated with extractives of four durable woods and linseed oil. Heartwoods Treatments SYP CW Water 23.56±1.12f 26.21±0.62ef Solvent 20.42±0.76f 33.53±2.02ef T. grandis Oil (20%) 42.44±3.33de 41.81±1.28de Extractives + oil 100.00±0.00a 98.95±1.02a Extractives 89.79±5.45a-c 77.67±6.88c Water 23.56±1.12f 25.70±1.03ef Solvent 20.42±0.76f 32.95±2.33ef D. sissoo Oil (20%) 42.44±3.33de 41.67±1.34de Extractives + oil 100.00±0.00a 100.00±0.00a Extractives 86.58±3.04a-c 88.42±3.71a-c Water 23.56±1.12f 25.70±1.03ef Solvent 20.42±0.76f 32.95±2.33ef C. deodara Oil (20%) 42.44±3.33de 41.67±1.34de Extractives + oil 97.63±2.38ab 100.00±0.00a Extractives 58.24±6.98d 90.82±4.77a-c Water 23.56±1.12f 25.70±1.03ef Solvent 20.42±0.76f 32.95±2.33ef P. roxburghii Oil (20%) 42.44±3.33de 41.67±1.34de Extractives + oil 100.00±0.00a 87.40±7.57a-c Extractives 95.80±4.20ab 81.35±6.87bc Means±SE sharing same letters in columns and rows are not significantly different from each other at p>0.05.

119

Table 4.4.28. Comparison of weight losses of non-durable woods treated with four durable wood extractives and oil at different concentration levels. Heartwoods

T. grandis D. sissoo C. deodara P. roxburghii Water 22.90±0.51a 23.34±0.48a 23.34±0.48a 23.34±0.48a

Solvent 23.03±1.61a 22.75±1.60a 22.75±1.60a 22.75±1.60a oil 16.29±1.44b 15.74±1.50b 15.74±1.50b 15.74±1.50b

Treatments Extractives + oil 7.03±0.78d 6.80±0.46d 12.08±0.98b-d 16.44±1.02b Extractives 8.93±1.41cd 8.01±1.18cd 13.20±1.17bc 9.56±1.81cd

Means±SE sharing same letters in columns and rows are not significantly different from each other at p>0.05.

Table 4.4.29. Weight losses of SYP and CW treated with four durable wood extractives and oil at different concentration levels. Non-durable woods SYP CW Water 22.28±0.33ab 24.18±0.28a

Solvent 21.46±1.27ab 24.17±0.89a oil 11.30±0.76c 20.44±0.70b

Treatments Treatments Extractives + oil 7.63±0.59d 13.54±0.84c Extractives 7.47±0.86d 12.38±1.05c Means±SE sharing same letters in columns and rows are not significantly different from each other at p>0.05.

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4.4.5. Laboratory evaluation of wood extractives against fungi rot, Postia placenta and Trametes versicolor 4.4.5.1. Evaluation of the effect of extractives on the durability of four wood species against Postia placenta and Trametes versicolor a) Extracted vs. sound blocks and non-durable controls The first set of laboratory bioassays was to evaluate effect of extractives on durability of each of the wood species. Coniferous (C. deodara and P. roxburghii) species were compared to SYP controls and are presented with statistical results in Table 4.4.30. SYP control lost 58% due to decay by P. placenta while un-extracted P. roxburghii and C. deodara lost 22% and 8.62 %, respectively. Durability indices (DI) for P. roxburghii and C. deodara were 0.38 (moderately durable) and 0.15 (very durable), respectively. However, when extractives were leached using ASTM D1105-96 procedure, weight loss of P. roxburghii and C. deodara was increased to 35% (DI=0.6, slightly durable) and 49% (DI=0.84, slightly durable), respectively.

The two hardwood species (D. sissoo, T. grandis) were compared to CW as a non- durable control which lost 48% of its original weight when exposed to T. versicolor. Before extraction T. grandis was found to be highly durable compared to the reference material, (3% weight loss) and D. sissoo was found to be moderately durable at 17% weight loss. When extracted, T. grandis was reduced to non-durable (0.20 DI) while D. sissoo also could not be prevented and rated as slightly durable (0.67 DI) (Table 4.4.31).

121

Table 4.4.30. Mean weight losses of extracted and un-extracted softwood species exposed to P. placenta in a 12 week soil bottle assay.

Wood type N Mean Grouping S.E DI* CLASS Control SYP Un -extracted 9 58.29 a 1.05 1.00 ND C. deodara extracted 10 48.94 a b 5.12 0.84 SD P. roxburghii extracted 10 35.07 b c 9.04 0.60 SD P. roxburghii Un-extracted 10 22.28 c d 5.44 0.38 MD C. deodara Un-extracted 10 8.62 d 1.7 0.15 HD *DI=durability index as calculated in EN350-1 (ND=Non-durable, SD=slightly durable, MD=moderately durable, and HD=highly durable).

Table 4.4.31. Mean weight losses of extracted and un-extracted hardwood species exposed to T. versicolor in a 12 week soil bottle assay.

Wood type N Mean Grouping S.E DI* CLASS Control CW un extracted 10 47.607 a 1.85 1 ND D. sissoo extracted 10 31.738 b 3.5 0.67 SD D. sissoo un extracted 10 16.974 c 2.59 0.36 MD T. grandis extracted 10 9.532 c d 0.339 0.20 D T. grandis un extracted 10 2.908 d 0.164 0.06 HD *DI=durability index as calculated in EN350-1 (ND=Non-durable, SD=slightly durable, MD=moderately durable, D=durable and HD=highly durable).

b) Treatment of SYP and CW with extractives The results from these tests indicated that in standard soil bottle assay at these concentrations (2.5, 5.0, 10.0 mg ml-1), none of extractives tested improved decay resistance of SYP against P. placenta. Pair wise comparison through Tukey’s HSD revealed that extractive treatments had non-significant difference among themselves. The durable reference wood (T. grandis) did not show any improvement when used to treat southern pine test blocks suggesting that the current dosage of these extractives was below required threshold or not

122 effective against these strains of test wood decay fungi in the standard soil bottle assay (Fig 4.4.1). Similarly, none of the extractives tested produced any statistically significant weight losses at these concentrations used in this experiment when tested for T. versicolor (Fig 4.4.2). Results of Tukey’s HSD (not shown) indicated that treatments had non-significant difference among them. Durable reference (T. grandis) likewise did not show any improvement for reasons speculated above.

Fig. 4.4.1. Mean weight losses of softwood blocks treated with extractives of durable wood species at 2.5, 5 and 10 mg ml-1exposed to Postia placenta for 12 weeks in a soil bottle test.

123

Fig. 4.4.2. Mean weight losses of hardwood blocks treated with extractives of durable wood species at 2.5, 5 and 10 mg ml-1exposed to Trametes versicolor for 12 weeks in a soil bottle test.

124

SECTION-V

Characterization of heartwood extractives using Gas chromatography – Mass spectrometry (GC-MS) analysis

125

4.5. Characterization of heartwood extractives using Gas chromatography- Mass spectrometry (GC-MS) Analysis All mass spectra were recorded in electron impact ionization (EI) at 70 electron volts. The masses on spectrometer were scanned from m/z 3-700 at a rate of 2 scans per second. An integrator automatically calculated peak area. Top five compounds identified on the percentage basis of sample were identified in the NIST14 library.

4.5.1. Characterization of T. grandis heartwood extractives GC-MS analysis yielded 96 identified chemical compounds from T. grandis heartwood. Table 4.5.1 shows the retention time, molecular weights, identification quality and total percentage of top five peaked compounds in the samples of extractives of T. grandis heartwood. These major compounds comprised of approximately 64% of total chemical makeup of extractives fraction analyzed. The chromatogram of analysis of extractives of T. grandis is shown in Fig. 4.5.1. The top five compounds identified were 3-(1-hydroxyethyl)-2- methyl-1-benzofuran-5,6-dicarbonitrile, 1-(1,1-dimethylethyl)-4-phenoxy-Benzene, 2- methyl-9,10-Anthracenedione, 1-Methyl-3,4-dihydroisoquinoline and Squalene. Anthracenedione has also been identified in other studies as anthroquinoe and has been shown to have biocidal activity. Anthracenedione (anthraquinone) and squalene made up the majority of the sample, i.e., 24.03 and 28.24 %, respectively.

4.5.2. Characterization of D. sissoo heartwood extractives GC-MS analysis yielded 52 identified compounds from D. sissoo heartwood. Table 4.5.2 shows retention time, molecular weights, identification quality and total percent of top five compounds in the sample, from the solvent extract of this heartwood. These major compounds comprised 79% of the total chemical makeup of solvent fraction so analyzed. The chromatogram associated with this analysis is shown in Fig. 4.5.2. The top five compounds identified were 1, 2, 9-trimethoxy-dibenzcycloheptane a cycloalkane, Trismethoxyresveratrol, 6,8-dimethyl-Benzanthracene, 2-(3, 4-dimethoxyphenyl)-Indane-1, 3-dione, 1, 3-Diamino-8- n-butyl-5, 6-dihydrobenzoquinazoline. The most abundant compound was Trimethoxyresveratrol (40.6%). Trimethoxyresveratrol is a stilbene with published medicinal (Dias et al. 2013) and free radical scavenging activities.

126

1 2

Fig. 4.5.1. Chromatogram from GC-MS analysis of solvent extracted T. grandis.

Table 4.5.1. Five largest components from GC-MS analysis of solvent extracted T. grandis.

Sr. Compound Retention Molecular Quality Yield No. Time [min] Weight (%) 1 3-(1-Hydroxyethyl)-2-methyl- 36.53 226.07 74.00 3.53 1-benzofuran-5,6- dicarbonitrile 2 1-(1,1-dimethylethyl)-4- 39.98 226.14 72.00 3.06 phenoxy-Benzene 3 2-methyl-9,10 42.18 222.07 97.00 24.03 Anthracenedione 4 1-Methyl-3,4- 56.67 264.12 45.00 5.22 dihydroisoquinoline 5 Squalene 68.82 410.39 99.00 28.24

127

5 1 4

Fig. 4.5.2. Chromatogram from GC-MS analysis of solvent extracted D. sissoo.

Table 4.5.2. Five largest components from GC-MS analysis of solvent extracted D. sissoo. Sr. Compound Retention Molecular Quality Yield No. Time [min] Weight (%) 1 1,2,9-trimethoxy- 42.33 284.141 70 3.09 Dibenzcycloheptane 2 Trismethoxyresveratrol 44.49 270.126 64 40.6 3 6,8-dimethyl-Benzanthracene 45.44 256.125 64 11.41 4 2-(3,4-dimethoxyphenyl)- 57.58 282.089 58 7.25 Indane-1,3-dione 5 1,3-Diamino-8-n-butyl-5,6- 60.03 268.146 59 17.09 dihydrobenzoquinazoline

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4.5.3. Characterization of C. deodara heartwood extractives GC-MS analysis yielded 147 identified compounds from C. deodara heartwood. Table 4.5.3 shows the retention time, molecular weights, identification quality and percent top five compounds in the sample of cedar heartwood. These major compounds comprised of 37% of total chemical makeup of solvent fraction analyzed. The chromatogram associated with this analysis is shown in Fig. 4.5.3. The top five compounds identified were sesquiterpenes alpha- Cuprenene, beta-Himachalene oxide, Di-epi-alpha-cedrene, (Z)-alpha-Atlantone, and (E)- Atlantone. Forms of Atlantone compromised 21.08 % of the sample. This sesquiterpene has been shown to have biological activity. The five largest components from C. deodara compromised a much smaller amount of the total as compared to the other extractives analyzed. This species also yielded the largest number of identified peaks (147) partially could explain the lower toxicity of this species to R. flavipes as compared to other heartwood species tested in feeding tests.

4.5.4. Characterization of P. roxburghii heartwood extractives GC-MS analysis yielded 12 identified compounds from Pinus roxburghii heartwood. Table 4.5.4 shows retention time, molecular weight, identification quality and total percent of top five compounds in sample from the solvent extract of its heartwood. These major compounds comprised of 87% of total chemical makeup of the solvent fraction analyzed. The chromatogram associated with this analysis is shown in Fig. 4.5.4. The top five compounds identified were 1,2,3,4-tetrahydro-5,8-dimethyl-Acridin-9-amine, 2,3-dihydro-5-hydroxy-7- methoxy-2-phenyl-4H-1-Benzopyran-4-one,2,3-dihydro-5,7-dihydroxy-2-phenyl-4H-1- Benzopyran-4-one,-5-hydroxy-7-methoxy-2-phenyl-4H-1-Benzopyran-4-one, and chrysin. Forms of benzopryran constituted 51% of sample. Acridin-9-amine was also identified as 27.46% of the sample. Toxicity / biological activity of benzopryran has not been found in the literature, thus its function according to these assays can be explained as weak/strong repellent or termiticide.

129

4 1 2 3

Fig. 4.5.3. Chromatogram from GC-MS analysis of solvent extracted C. deodara.

Table 4.5.3. Five largest components from GC-MS analysis of solvent extracted C. deodara.

Sr. Compound Retention Time Molecular Quality Yield No. [min] Weight (%) 1 alpha-Cuprenene 18.785 204.188 99 3.84 2 beta-Himachalene 22.819 220.183 99 3.62 oxide 3 Di-epi-alpha- 24.197 204.188 90 8.14 cedrene 4 (Z)-alpha-Atlantone 26.545 218.167 99 3.16 5 (E)-Atlantone 28.893 218.167 99 17.92

130

3 4

Fig. 4.5.4. Chromatogram from GC-MS analysis of solvent extracted P. roxburghii.

Table 4.5.4. Five largest components from GC-MS analysis of solvent extracted P. roxburghii.

Sr. Compound Retention Molecular Quality Yield No. Time [min] Weight (%) 1 1,2,3,4-tetrahydro-5,8- 37.61 226.147 70 27.46 dimethyl-Acridin-9-amine 2 2,3-dihydro-5-hydroxy-7- 38.21 270.089 99 9.18 methoxy-2-phenyl-4H-1- Benzopyran-4-one 3 2,3-dihydro-5,7-dihydroxy- 38.69 256.074 99 24.42 2-phenyl-4H-1-Benzopyran- 4-one 4 5-hydroxy-7-methoxy-2- 39.426 268.074 99 17.41 phenyl-4H-1-Benzopyran-4- one 5 Chrysin 40.01 254.058 96 8.62

131

SECTION- VI

Potential of wood extractives and linseed oil as preservatives in field tests against termites attack and fungal decay

132

4.6. Comparison of performance of wood extractives and linseed oil as preservatives in field tests against termites and decay in USA and Pakistan 4.6.1. Average Termites infestation and Fungi Decay Rating of SYP and CW chunks under Field conditions (AWPA E26).

Table 4.6.1 shows the average rating of SYP when exposed to termites and fungi at Mississippi USA. Results showed that after 6 months of exposure all treated and un-treated specimens did not show any attack of termites and fungal decay. All chunks were completely sound with average rating of 10. After wards at the end of 12 months, solvent treated chunks got the lowest rating (9.7). SYP chunks treated with P. roxburghii extractives attracted some termites and got lowest average rating (9.6). Mud tubes were present on the chunks treated with T. grandis and D. sissoo extractives treated. All SYP chunks were found sound in case of decay after 12 months except T. grandis + oil treatment on which some areas showed discoloration or softening associated with superficial microbial infection.

Table 4.6.2 shows average rating of SYP when exposed decay and termites at Lahore, Pakistan. Results showed that after 6 months of exposure to termites and decay at Lahore, woods treated with T. grandis + oil and P. roxburghii+oil treatments showed some discoloration or softening associated with superficial microbial infection. All other treated chunks were intact. In case of termite attack, surfaces of solvent treated chunks were nibbled resembling termites feeding in the presence of mud galleries and scored the lowest average rating (9.2). Chunks treated with P. roxburghii extractives alone and in combination with linseed oil attracted some termites. SYP chunks treated with solvent only after 12 months were severely attacked by the termites with average rating of 3.8. While chunks treated with linseed oil only got moderate attack of termites. P. roxburghii extractives treated chunks attracted some termites but mud tubes of termites were found on the whole setup of chunks after 12 months. In case of decay solvent treated and chunks treated with extractives of P. roxburghii showed some discoloration due to decay.

133

Table 4.6.1. Average decay and termite resistance rating of treated SYP chunks for field tests located at MS USA. Treatments Retention 6 months average rating 12 months average rating Kg m-3 pcf decay termite decay termite Solvent 401.82 25.34 10 10 10 9.7 (A) Linseed oil 394.86 24.35 10 10 10 10 T. grandis 351.09 21.65 10 10 10 9.8 (MT) D. sissoo 362.46 22.35 10 10 10 9.8 (MT) C. deodara 363.71 22.43 10 10 10 10 P. roxburghii 362.01 22.33 10 10 10 9.6 (MT) T. grandis + oil 388.64 23.97 10 10 9.9 10 D. sissoo + oil 389.19 24.00 10 10 10 10 C. deodara +oil 403.73 24.94 10 10 10 10 (MT) P.roxburghii+oil 372.98 23.00 10 10 10 9.9 [A (MT)] Cu-NAP 0.64 0.04 10 10 10 10

A= presence of active termites; MT= Presence of mud tubes on blocks; Cu-NAP = Copper Naphthenate; MS= Mississppi

134

Table 4.6.2. Average decay and termite resistance rating of treated SYP chunks for field tests located at Lahore Pakistan. Treatments Retention 6 months average rating 12 months average rating Kg m-3 pcf decay termite decay termite Solvent 340.11 20.98 9.8 9.2 9.6 3.8 Linseed oil 398.14 24.55 10 9.7 9.6 8.4 T. grandis 349.74 21.57 10 10 10 9.6 D. sissoo 367.97 22.69 9.9 10 9.7 9.4 C. deodara 364.74 22.50 10 9.9 10 9.8 P. roxburghii 345.35 21.30 9.7 9.5 9.7 8.6 T. grandis + oil 372.55 22.98 9.8 10 9.8 9.9 D. sissoo + oil 372.02 22.94 10 10 10 10 C. deodara +oil 375.74 23.17 10 9.8 9.9 9.9 P.roxburghii+oil 374.25 23.08 9.6 9.5 9.6 9.7 Cu-NAP 0.64 0.04 10 9.9 9.8 9.6 Cu-NAP = Copper Naphthenate Table 4.6.3 shows the average rating of CW when exposed to termites and fungi at Mississippi, USA. All treated and untreated chunks of CW did not show any sign of termites’ infestation and fungi infection after 6 months of exposure. After 12 months, there was again no sign of attack of decay on CW and all chunks were sound. Mud tubes were found on chunks and number of termites foraged on chunks. P. roxburghii and T. grandis extractives treated chunks attracted the termites after 12 months and these chunks were surface nibbled by them as well. Active termites were also found on some chunks treated with C. deodara extractive with average rating of 9.2. There were no attack signs of termites and decay on the CW chunks treated with extractives in combination with linseed oil. Chunks treated with copper naphthenate (a positive control) were completely unharmed sound after 12 months.

Table 4.6.4 shows the average rating of CW when exposed decay and termites at Lahore, Pakistan. After 6 months of exposure to termites and decay, chunks which were treated with solvent and those which were treated with extractives of P. roxburghii extractives got the lowest rating for both termites and decay. Surface nibbling by termites on CW chunks of

135 positive control was observed. After 12 months again solvent treated and chunks treated with extractives of P. roxburghii got lowest rating (9.1 and 9.3 respectively). Termites completely destroyed CW chunks treated with solvent only (average rating 1.2) after 12 months. Linseed oil treated chunks also got severe attack of termites (average rating 6.4). C. deodara and P. roxburghii extractive treated CW chunks were severely attacked by termites with average rating of 6.4 and 5.3, respectively. There was also surface nibbling by the termites on the positive control.

Table 4.6.3. Average decay and termite resistance rating of treated CW chunks for field tests located at MS USA. Treatments Retention 6 months average rating 12 months average rating Kg m-3 pcf decay termites decay termites Solvent 497.74 30.70 10 10 10 9.8 Linseed oil 485.97 28.31 10 10 10 10 T. grandis 408.02 25.16 10 10 10 9.2 (A) D. sissoo 471.78 29.10 10 10 10 10 (MT) C. deodara 469.93 28.98 10 10 10 9.2 (A) P. roxburghii 453.51 27.97 10 10 10 9.2 [(MT) A] T. grandis + oil 487.57 30.07 10 10 10 10 D. sissoo + oil 506.29 31.22 10 10 10 10 C. deodara +oil 524.03 32.33 10 10 10 9.5 P.roxburghii+oil 456.12 28.13 10 10 10 9.9 [(MT) A] Cu-NAP 0.64 0.04 10 10 10 10 A= presence of active termites; MT= Presence of mud tubes on blocks; Cu-NAP = Copper Naphthenate

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Table 4.6.4. Average decay and termite resistance rating of treated CW chunks for field tests located at Lahore Pakistan. Treatments Retention 6 months average rating 12 months average rating Kg m-3 pcf decay termite decay termite solvent 470.62 29.46 9.6 8.5 9.1 1.2 Linseed oil 420.21 25.62 9.8 9.3 9.8 6.4 T. grandis 465.92 28.74 10 9.7 10 9.5 D. sissoo 434.71 26.81 9.9 9.5 9.9 8.2 C. deodara 522.0 32.19 10 9.7 9.8 6.4 P. roxburghii 489.42 30.18 9.8 9.1 9.3 5.3 T. grandis + oil 441.68 27.24 9.9 10 9.9 8.3 D. sissoo + oil 452.33 29.90 10 10 10 9.3 C. deodara +oil 489.75 30.82 10 9.8 9.8 8.6 P.roxburghii+oil 478.17 29.49 9.6 8.9 9.6 8.3 Cu-NAP 0.64 0.04 9.7 9.9 9.7 9.2 Cu-NAP = Copper Naphthenate.

Table 4.6.5 shows average rating of heartwoods exposed to termites and decay after 6 and 12 months at both sites. There was neither termites’ infestation and fungi decay after 6 and 12 months on all heartwood chunks exposed to biological agents at Mississippi. Heartwood of P. roxburghii attracted some termites after 12 months (at both the sites). There was slight attack of termites and decay (9.2) after exposure of chunks at Lahore, Pakistan. T. grandis and D. sissoo were completely sound after 6 and 12 months, while heartwood chunks of C. deodara and P. roxburghii attracted some termites after 12 months period.

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Table 4.6.5. Average decay and termite resistance rating of heartwoods field chunks located at (MS) USA and (Lahore) Pakistan. Location Heartwoods 6 months average rating 12 months average rating decay termite decay termite T. grandis 10 10 10 10 (MT)

SA SA D. sissoo 10 10 10 10

C. deodara 10 10 10 10 P. roxburghii 10 10 10 9.4 [A (MT)] T. grandis 10 10 9.8 10 Pakistan D. sissoo 10 10 9.4 10 C. deodara 9.9 9.8 9.5 9.8

P. roxburghii 9.7 9.7 9.5 9.2 MT= Presence of mud tubes on samples; MS, Mississippi

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4.6.2. Average Decay and Termite Rating of SYP and CW for Field Stakes (AWPA E7).

Table 4.6.6 shows average rating of SYP stakes when exposed to termites and fungi decay at Mississippi, USA. All stakes were completely sound after 6 months i.e. there were no signs of attack of termites and decay; after 12 months some treated and un-treated stakes showed some discoloration due to decay. Termites were also attracted to the stakes treated with extractives of C. deodara and P. roxburghii as well as to positive control.

Table 4.6.7 shows average rating of SYP stakes which were exposed to termites and fungi decay at Lahore, Pakistan. Results showed that after 6 months of exposure signs of attack of decay on all the stakes except those treated with linseed oil + extractives of D. sissoo and T. grandis after 6 months of exposure scoring 9.2 level for both treatments. Solvent treated SYP stakes got lowest average rating for termites’ exposure after 6 months. C. deodara and P. roxburghii extractives treated stakes also attracted some termites. After 12 months exposure period, there was moderate to slight attack of decay on the stakes treated with solvent, P. roxburghii and C. deodara extractives. Termite attack was very severe on the stakes which were treated with solvent and 20 % linseed oil with average rating of 2.2 and 4.0, respectively after 12 months. Stakes treated with C. deodara extractives and positive control was severely attacked by termites scoring 4.2.

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Table 4.6.6. Average decay and termite resistance rating of treated SYP stakes for field tests located at MS USA. Treatments Retention 6 months average rating 12 months average rating Kg m-3 pcf decay termite decay termite Solvent 524.19 32.33 9.8 10 9.5 10 Linseed oil 489.02 30.19 10 10 9.9 10 T. grandis 501.27 30.92 10 10 9.9 10 D. sissoo 479.83 29.59 10 10 9.8 10 C. deodara 506.29 31.23 10 10 9.9 9.0 P. roxburghii 529.07 32.63 10 10 9.8 9.4 T. grandis + oil 498.42 30.74 10 10 9.9 9.9 D. sissoo + oil 517.18 31.90 10 10 9.9 10 C. deodara +oil 484.46 29.88 10 10 9.7 9.6 P.roxburghii+oil 518.16 31.96 10 10 10 9.4 Cu-NAP 0.64 0.04 10 10 9.6 9.4 Cu-NAP = Copper Naphthenate; MS = Mississippi

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Table 4.6.7. Average decay and termite resistance rating of treated SYP satkes for field tests located at Lahore Pakistan. Treatments Retention 6 months average rating 12 months average rating Kg m-3 pcf decay termite decay termite solvent 501.39 30.92 9.4 7.8 8.1 2.2 Linseed oil 506.15 31.22 10 8.7 9.8 4.0 T. grandis 506.88 31.26 9.8 9.1 9.8 8.7 D. sissoo 488.99 30.16 9.8 8.2 9.5 7.6 C. deodara 475.78 29.34 9.5 8.0 8.8 4.2 P. roxburghii 484.28 29.87 9.9 9.9 8.9 9.2 T. grandis + oil 515.26 31.78 9.2 10 9.2 10 D. sissoo + oil 535.63 33.03 9.2 10 9.2 10 C. deodara +oil 535.75 33.04 9.7 10 9.0 10 P.roxburghii+oil 524.46 32.35 9.6 10 8.6 7.3 Cu-NAP 0.64 0.04 10 10 9.5 4.2 Cu-NAP = Copper Naphthenate;

Table 4.6.8 shows average rating of CW stakes exposed to termites and decay at Mississippi, USA. After 6 month exposure there was no sign of attack of termites and decay after 6 months. Lowest rating was observed in solvent treated stakes (9.1) after 12 months. Stakes treated with extractives of C. deodara had minimum score of 9.6.

Table 4.6.9 shows average rating of CW stakes for termites’ infestation and fungi decay at Lahore, Pakistan. Stakes treated with solvent only were moderately attacked by termites after 6 months of exposure. These stakes also showed some signs of discoloration due to decay. Lowest decay rating was observed for solvent and linseed control after 12 months. Stakes treated with P. roxburghii + oil were also decayed with average rating of 8.6. Positive control and solvent control stakes were completely destroyed by termites with 0 rating (Appendix. 5). Linseed oil control and stakes treated with P. roxburghii extractives + oil were also moderately attacked by the termites after 12 months of exposure.

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Table 4.6.8. Average decay and termite resistance rating of treated CW stakes for field tests located at MS USA. Treatments Retention 6 months average rating 12 months average rating Kg m-3 pcf decay termite decay termite solvent 595.14 36.14 10 10 9.1 9.4 Linseed oil 570.03 35.16 10 10 9.9 10 T. grandis 587.59 34.39 10 10 9.4 10 D. sissoo 564.69 34.83 10 10 9.9 10 C. deodara 580.72 35.82 10 10 7.6 9 P. roxburghii 571.49 35.25 10 10 9.6 10 T. grandis + oil 580.48 35.80 10 10 9.9 10 D. sissoo + oil 579.27 35.73 9.9 10 10 10 C. deodara +oil 536.57 33.09 10 10 10 10 P.roxburghii+oil 546.95 33.73 10 10 9.9 9.6 Cu-NAP 0.64 0.04 10 10 9.2 10 Cu-NAP = Copper Naphthenate; MS = Mississippi

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Table 4.6.9. Average decay and termite resistance rating of treated CW stakes for field tests located at Lahore Pakistan.

Treatments Retention 6 months average rating 12 months average rating Kg m-3 pcf Decay termite Decay termite solvent 576.24 35.54 9.2 8 7.9 0.0 Linseed oil 571.25 35.23 10 9.3 9.6 7.4 T. grandis 565.64 34.89 9.8 9.4 10 8.0 D. sissoo 564.90 33.48 9.9 8.8 8.9 6.6 C. deodara 527.43 32.53 9.9 8.4 8.6 4.4 P. roxburghii 580.65 35.81 9.9 9.1 8.4 7.5 T. grandis + oil 584.14 36.03 10 10 9.6 9.7 D. sissoo + oil 584.43 36.04 9.5 10 9.2 9.3 C. deodara +oil 518.57 31.98 9.7 10 9.2 9.5 P.roxburghii+oil 494.11 30.47 9.6 10 8.6 8.7 Cu-NAP 0.64 0.04 10 10 9.0 0.0 Cu-NAP = Copper Naphthenate

Table 4.6.10 shows average rating of heartwood (HW) stakes exposed to termites and fungi decay at both sites (Mississippi, Lahore). Heartwoods were not attacked by the termites at Mississippi but P. roxburghii and C. deodara were moderately attacked by the termites after 6 months exposure at Lahore, Pakistan site. Heartwood of P. roxburghii was nibbled on surface by the termites and all other were sound after 12 months at Mississippi. There was no attack of decay at both sites after 6 months. P. roxburghii was severely attacked by the termites with average rating of 3.7 at Lahore site after 12 months exposure. C. deodara and P. roxburghii were also severely infected by fungi decay at this site with average rating of 3.2 and 5, respectively.

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Table 4.6.10. Average decay and termite resistance rating of heartwoods field stakes located at (MS) USA and (Lahore) Pakistan. Location HW species 6 months average rating 12 months average rating Decay termite Decay termite T. grandis 10 10 10 10 D. sissoo 10 10 10 10

USA C. deodara 10 10 10 10

P. roxburghii 10 10 9.7 9.6

T. grandis 10 10 9.2 9.7 Pakistan D. sissoo 10 9.2 8.4 7.8 C. deodara 10 8.9 3.2 8.2

P. roxburghii 10 8.8 5 3.7 MS = Mississippi Over all P. roxburghii and C. deodara extractives treated blocks and stakes attracted termites at both sites. Control treatments comprising of solvent, oil treated blocks and stakes at Lahore site (Pakistan) were completely destroyed by the termites (H. indicola) and decayed after a lapse of time period of 12 months. Linseed oil treated stakes of SYP and CW showed discoloration with average rating of 9.5 due to fungal decay at USA. P. roxburghii extractive stakes were partially or completely deteriorated by the fungi at both sites.

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CHAPTER FIVE DISCUSSIONS

Naturally resistant woods possess physical and chemical attributes that are difficult to manipulate by weathering and biological agents and in this way, woods are said to be resistant to these agents. Heartwood extractives are the most considered chemicals against which termites are challenged to ascertain their usefulness in diverse environments. Present studies were carried out at two different sites and with two different species of termites. Ecologically isolated sources of extractives appeared to be applicable at more than one places. Extractives from T. grandis, D. sissoo, C. deodara, and P. roxburghii have shown promise to alleviate termites’ damage and fungi decay on Southern Yellow Pine and Cotton Woods. Termites are important pest of wood in USA and especially lower termites are nuisance in most of other countries as well (Schultz and Nicolas, 2002). This leads to reason for choosing Heterotermes indicola in Pakistan in the experiments to equilibrate results with Reticulitermes flavipes in USA. Anticipated mechanisms of action of extractives on termites were toxicity, antifeedant, and antioxidant. These have been demonstrated in the present studies. Active fraction from extractives, on the basis of percentage, were isolated and characterized. A unique but not uncommon property of these extractives that these can affect intestinal flora in hindgut was also exhibited. Important assays on H. indicola included determination of enzymes activities in midguts of H. indicola workers exposed to IC50 concentrations of BHT, quercetin and heartwood extractives and variations in gut bacterial community of H. indicola after feeding on extractives treated filter papers using 16SrRNA. To present literature, this is first report of above mentioned assays on H. indicola.

5.1. Toxicity bioassay Filter papers treated with four types of durable wood extractives fed to R. flavipes and H. indicola revealed that T. grandis and D. sissoo extractives were the more toxic to H. indicola and R. flavipes, respectively. C. deodara and P. roxburghii extractives were the least toxic to

R. flavipes H. indicola, respectively on the basis of LC50 values. Results of filter paper bioassays were similar to Raya-González et al. (2013) who found concentration dependent mortality and feeding rate of dry wood termites using aqueous

145 extracts of Enterolobium cyclocarpum. They found a six-fold reduction in consumption of treated filter paper (10 mg ml-1) compared to control treatment (0 mg ml-1). Results were also in confirmation with Dungani et al. (2012) who tested the effectiveness of various extractives of T. grandis heartwood by using discs of filter papers for impregnation against attack from subterranean termite, Coptotermes curvignathus, and had, in general way, the best results for extractions in acetone: water (9:1) at concentrations of 2-10%. Paper discs treated with ethanol, chloroform and acetone extractives of Tectona grandis heartwood under no-choice feeding tests showed feeding deterrent activity and toxicity to the subterranean termite, Reticulitermes speratus (Ismayati et al., 2016). These and similar results reported elsewhere have confirmed dose-dependent mortality of termites after feeding on extractives treated filter papers against Incisitermes marginipennis, Coptotermes gestroi and Heterotermes indicola (Raya-González et al., 2013; Se Golpayegani et al., 2014; Kadir et al., 2014, 2015; Hassan et al., 2016). Concentrations dependent mortality of H. indicola and R. flavipes after feeding on filter paper treated with heartwood extractives of D. sissoo has earlier been demonstrated (Hassan et al., 2016). Although the origin and growth characteristics of wood were not considered, extraction yield was calculated per gram of wood shavings (Ordonez et al., 2006) and mean T. grandis extractive content was 5.51 %. While in another study, Lukmandaru (2011) found 1.9-2.7% average ethyl acetate-soluble extractive and 1.8-3.7% n- hexane soluble contents from heartwood of T. grandis. Bhat et al. (2010) found 9.69 to 13.12% extractives in the outer and inner portion of heartwood of T. grandis using ethanol as solvent. Yield of C. deodara extractives was 8.15 and 3.3% after extraction with ethanol and hexane, respectively, as reported by Manzoor et al. (2016). Mean extractive contents (%) for D. sissoo, C. deodara, and P. roxburghii were 9.11, 9.67 and 7.40%, respectively. (Table 4.1). Extractives so far found lethal to termites had quinones and tectoquinones in T. grandis (Sandermann and Simatupang, 1966), which is not toxic in natural condition; merely deters the termites (Lukmandaru and Takahashi, 2008). Other extractives such as D. sissoo had stilbenes which are potential source of wood durability (Schultz and Nicholas, 2000). C. deodara extractives caused >80% mortality of both the termites similar to Manzoor et al. (2016). They found toxic potential of this wood extractives against H. indicola and

146 hexane extracts were very toxic to this termite and caused 66% mortality. The mortality difference is might be due to difference in choice of solvent used for extraction. Regarding potential of direct toxicity of P. roxburghii, results are in confirmation with Rasib and Aihetasham (2016) who obtained 90.6% mortality of Coptotermes heimi workers @ 30% extract solution and the lowest (20.5%) @ 2%. Significant difference in bait consumption between high (0.14g) and low concentrations (0.42g) of extracts was also observed. Similarly, Kadir et al. (2014) reported anti-termite activity of heartwood and bark extracts of two Malaysian trees (Madhuca utilis and Neobalanocarpus heimii). Both the tree extracts were toxic against Coptotermes gestroi on filter paper with different concentrations of extracts in ethanol after 14 days post exposure period. LC50s were in range of those obtained in the present results.

5.2. Repellent and antifeedant activities of extractives Extractives of all durable species showed repellent activities against R. flavipes and H. indicola except P. roxburghii. Repellency percentages increased with increasing concentrations of extractives excluding D. sissoo, which could not show concentration dependent repellency. Maximum repellency was observed on filter papers, which were treated with C. deodara extractives even at lower concentrations against both the termite species. At highest concentration (10 mg ml-1), T. grandis extractives showed more repellent activity towards R. flavipes (100%) as compared to H. indicola (94%) starting from 1 hour to 12 hour. Similarly, D. sissoo extractives were more repellent to R. flavipes as compared to H. indicola after 12 hours of exposure but repellency was concentration dependent against H. indicola. P. roxburghii extractives did not show any repellant activity against both the termites even at higher concentrations (10 mg ml-1) unlike Rasib and Aihetasham (2016) who found repellency to termites at 10-30% concentrations, which was obvious as most of the termites were present on untreated filter paper indicating repellency. Our results are in confirmation with Lukmandaru and Takahashi (2008) who found several distasteful chemicals in the bark and heartwood of T. grandis repellent to Reticultermes speratus. Anthraquinone (24%) was the major chemical responsible for repellency to Cryptotermes brevis after GC-MS characterization (Wolcott, 1947). Several other authors have also reported that quinones have repellent and toxic properties against termites (Ganapty et al.,

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2004; Dungani et al., 2012). Similarly, Sa et al. (2009) found no repellent activities of secondary metabolites extracted from heartwood of Myracrodruon urundeuva against Nasutitermes corniger. In contrast with other studies, D. sissoo extractives were more repellent to both the termite species than other heartwood extractives such as Madhuca utilis (39 to 59%) and Neobalanocarpus heimii (9 to 68%) at the highest concentrations, but D. sissoo extractives showed lower activity compared to bark extractives of both species (Kadir et al. 2014). Kharkwal et al. (2014) extracted repellent compounds in chloroform from D. sissoo that showed 93% repellency against Microcerotermes beesonii. C. deodara showed 96-98 % repellent activities at each concentrations of extractives except 1.25 mg ml-1 against both the termites unlike Manzoor et al. (2016) who found 60 and 20% repellency of these extractives when hexane and ethanol were used as solvents for extraction. All heartwood extractives showed antifeedant activities against both the termite species. T. grandis showed more activity against H. indicola as compared to R. flavipes. The antifeedant activity of extractives obtained from T. grandis and D. sissoo were in range of antifeedant concentrations of an earlier study with T. grandis on Coptotermes curvignathus. These results suggest that concentration of extractive is a critical factor for termite mortality and antifeedancy (Dungani et al., 2012). This is most likely due to the concentration of phenolic compounds, which are strong antifeedants and act as natural protectants for the living trees (Morimoto et al., 2006; Ates et al., 2015). C. deodara and P. roxburghii showed very low antifeedant activity against H. indicola as compared to R. flavipes. Ohmura et al. (2000) found anti-feeding in Coptotermes formosanus caused by flavonoids from cedar oil. The impregnated filter paper with two isolated flavonoids, Ceastillen D and Castillen E, deterred termites in concentration dependent manner. Repellent and antifeedant effects may results in starvation of termites. Biochemical changes in starved termites have suggested a possible link with mortality in few earlier studies. These changes were inhibition of native digestive enzymes in termites’ gut.

5.3. Antioxidant activities of wood extractives Although mechanisms of naturally durable wood are not fully understood, previous studies have shown that heartwood extractives possess distinct antioxidant properties. It might not be exclusively toxicity and repellency of extractives in the durable wood sample that gives

148 heartwood termite resistance, but the extractives’ dual toxicity and antioxidant properties acting synergistically could affect termites’ survival (Little et al., 2010). It has been suggested that the presence of antioxidants in heartwood may interfere with the numerous single electron redox reaction that take places in termites’ gut when cellulose is converted to acetate via the shikimate acid pathway (Ragon et al., 2008). Majority of active compounds identified in heartwood extractives of wood species in this study are classified as polyphenols, which contain many powerful antioxidants. These phenols can also act as enzyme and metabolism inhibitors i.e., these can bind to proteins, acting as nutritional protein precipitating agents, thus reducing their digestibility (Torres et al., 2003). Flavonoids are characterized as natural antioxidants and have received much attention due to their role in the neutralization or scavenging of free radicals (Prakash and Gupta, 2009). Well known antioxidant properties of extractives in termite’s resistant heartwoods, along with the extractives’ inherent toxicity affect subterranean termites. Grace (1990) found that antioxidants affected termites’ behavior. Butylated hydroxy toluene (BHT), a synthetic antioxidant, was used to treat wood and was found to be feeding deterrent that might have elevated mortality of the termites as later proved in modified bioassays (Ragon et al., 2008; Schultz et al., 2008). Natural and synthetic antioxidants that are benign to humans had feeding deterrence and mortality properties to R. flavipes (Little et al., 2010). Maximum antioxidant activity was observed in D. sissoo extractives with the lowest -1 IC50 (28.83 µg ml ) among the evaluated heartwoods. GC-MS results also confirm that this heartwood contains majority of phenolic compounds, which have very strong antioxidant -1 activity irrespective of the concentrations. BHT showed higher IC50 (42.63 µg ml ) as compared to D. sissoo extractives, which are thus supposed to have high potential to scavenge -1 DPPH radicals as compared to BHT. Quercetin showed the lowest IC50 (8.17 µg ml ). Concentration dependent inhibition activity was observed in three other heartwood extractives. T. grandis at the highest concentration showed maximum % inhibition statistically non- significant to BHT and quercetin. Activities of gut enzymes i.e. esterases and glutathione-s-transferases were significantly reduced after feeding on filter papers treated with extractives and synthetic antioxidants (BHT and quercetin). There are reports in the literature which correlate the total phenolics content of a plant extract with its antioxidant activity and their effect on enzymes

149 inhibition (Skerget et al., 2005; Coruh et al., 2007; Kolawole et al., 2009). Our results are in confirmation with Tang et al. (2008) who found reduction in glutathione-s-transferases activities in O. formosanus and R. chinensis after treatment with quercetin and tannic acids.

5.4. Effect of wood extractives on gut microbiota of termites Wood extractives elicited considerably destructive effects on gut protozoans in both the termite species; complete protozoan defaunation was not shown to occur at the highest concentrations of extractives. Lewis and Forschler (2004) morphologically a total of eleven species of protozoans from the hindgut of R. flavipes were observed (Appendix. 6). There were nine different species of protozoans in the hindgut of H. indicola like Qureshi et al. (2012) who has also reported similar number species from the guts of this termite (Appendix. 7). Reduction in protozoan densities in both the termites species was, however, dose dependent. In control treatments, a single termite gut contained an average of 60,042±1,877 and 3,530±62.4 total protozoans for R. flavipes and H. indicola, respectively. Termite fed on untreated filter paper had similar number of protozoans as in the laboratory reared termites feeding on SYP and CW woods. Number of protozoans in R. flavipes workers were significantly reduced after feeding on the various concentrations of the four types of heartwood extractives when compared from control treatment. In both the termite species, results showed a maximum reduction in protozoans at the highest extractive concentration (10 mg ml-1), which has also shown maximum termite mortality. Weight loss and percent consumption of filter paper was reduced when maximum reduction in protozoans compared to control treatment was observed (Jones et al., 1983). Effect of extractives on termites and their flagellate fauna was quite possibly due to chemical constituents of wood (Mauldin et al., 1981; Breznak, 1982). Different concentrations of extractives can affect the gut flagellates prior to the death of their host and death of protozoans than leads to mortality of termites. This appears to be true and in agreement with Qureshi et al. (2014). In control treatments, the number of protozoans in single R. flavipes worker on an average was similar to the number of protozoans reported by Lewis and Forschler (2004). Our approximation of total protozoan populations, however, was greater than those typically described from the workers of R. flavipes, which range from 40,083 ± 3,643 (Mannesmann, 1969) to 32,320 (Mauldin et al., 1981), 31,120 ± 8,405 (Howard, 1984), and 14,642 ± 3,395 (Cook and Gold, 1999). Thus, an individual R. flavipes contained 17 times

150 more protozoa than a single H. indicola in untreated replicates. The difference in protozoan density may have inferences for termite control using wood extractives. Loss of small number of protozoa in H. indicola may cause relatively more stress due to the comparatively greater loss of cellulose digestive ability compared with matching loss of protozoa from R. flavipes. This assumption may be tested where loss of protozoa from total population compared with negligible reduction in number and consumption rate of food can be variable for experiments with different termites’ species. Such small loss of protozoans in R. flavipes may not affect cellulose digestion severely as has already been proposed by Kard (2000). In earlier studies, complete defaunation requires more than 40 days starvation period (Hu et al., 2011). In the present studies, total duration of bioassay for effect of extractives on protozoans was 15 days. Maximum reduction of protozoans in R. flavipes and H. indicola was 45 and 82%. Complete defaunation was not observed, which implies that either dose dependency of extractives or kinds of protozoa in two geographically isolated termites species may have accounted for this effect. This difference may also accounted for nature of chemical constituents of heartwood extractives which can have acute or chronic toxicity on termites. Dose dependency of gut protozoans was observed by Fatima and Morrell (2015). With the increasing concentration of plant oils there was more reduction in Trichonympha and Trichomitopsis protozoans in guts of Zootermopsis augusticollis. Different extractives performed differently in reducing in number of protozoans similar to the study of Doolittle et al. (2007). They found that neem extracts were capable of reducing the population of P. grassii and spirochetes unlike the extracts of gleditschia and capsaicin. It was observed among all wood extractives tested that D. sissoo heartwood extractives were the most toxic to R. flavipes and exposure resulted in reduced protist numbers. Tests with H. indicola showed that T. grandis heartwood extractives were more toxic to flagellate population as compared to three other extractives. Qureshi et al. (2012) found complete elimination of gut protozoans in H. indicola after feeding on heartwood of D. sissoo and Eucalyptus cammeldulensis. This potentiates present results that heartwood extracts of D. sissoo have potential as protozoicidal compounds. Further, Qureshi et al. (2016) reported benzene-ethanol extractives of D. sissoo were also very toxic to gut flagellates of H. indicola and C. heimi.

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Our findings were different from Mannesmann (1972a) who found total elimination of gut protozoans after feeding of R. virginicus on red spruce. It indicates different interaction of termites and wood species far from both used in the present studies. Termites’ gut bacteria are not generally important in lower termites and literature profusely advocate role of protozoa for energy metabolism. Termites’ gut bacteria in R. flavipes are previously reported and their role in biology of termites has been determined to an extent that prove their usefulness (Fisher et al., 2007). Bacterial flora in H. indicola is not known yet; making a case in the present study to identify their diversity. Overall, using 16S rRNA, four strains of bacteria up to Genus level based on the basis of closest matches (100 and 99 %) in GenBank were identified and a fifth one was unclassified bacteria. These four strains belonged to Genera, Enterobacter, Bacillus, Pseudomonas and Pantoea spreading in six bacterial phyla. A large number of uncultured and unclassiﬁed bacteria from gut of H. indicola across different treatments was the dominant group, which was followed by Protobacteria and spirochetes. Overall, examination of the abundance of major taxonomic groups in treated versus control groups did not appear to show the major differences for presence of phyla of gut bacteria. Little is known about direct contact of extractives with bacterial flora, though plant secondary metabolites such as phenolic, alkaloids and essential oils can act as inhibitors against some members of gut microbiome (Reichling, 2010).

5.5. Effect of removal of extractives from durable woods Hardwood blocks (19x19x19 mm) exposed to R. flavipes and H. indicola without extraction were non-significantly attacked by the termites. There was decrease in resistance to termites after extraction. Mortality of termites and weight losses of durable woods were significantly different before and after extraction except in T. grandis (Appendix. 1-2). Weight losses in T. grandis blocks and mortality of both the termites on them were non-significantly different before and after extraction. This may indicate that the blocks are still durable to termite attack even after extraction. It could also be due to the fact that extractives embedded in the cell wall could not be removed by this solvent. This durability could also be attributed to other factors such as high densities and hardness of wood for that they are able to remain durable even after extractive removal. Wood density affects the permeability of wood hence solvents used could not be able to remove all the extractives. This is in support with Peralta et

152 al. (2003) who deduced that wood density was recognized as a factor of natural resistance of wood to termites. Kirker et al. (2013) also concluded that the natural resistance of wood could be attributed to hardness, extractive contents and specific gravity of wood. Solubility of the extractives is another factor which affects the removal of extractive from wood. The supporting results were found by Taylor et al. (2006) who observed that most extractives in the heartwood of T. plicata and C. nootkatensis were methanol soluble and their removal reduced the durability of those wood pieces to fungal decay and termite attack. Since resistance of durable wood samples reduced with extraction, this confirms that most of their heartwood extractives responsible for termite resistance were reduced. Several researchers found that removal of extractives from wood decreased its resistance against termites and fungi. Removal of extractives from lesser known Albizia malacophylla, a Kenyan wood species, significantly lowered heartwood dimensional stability, termite and fungus resistance (Kibet et al., 2013). Results are also in confirmation with Hwang et al. (2007) who found that resistance of wood decreased after extraction of wood with an increase in mass loss. Addition of chemicals may aid action of extractives of the heartwood. Action of DBF or DDAC wood preservatives contributed to enhance resistance against termite attack (Hwang et al., 2006). Wood plastic composites (WPCs) made with extracted wood flour tended to have lower fungal durability than un-extracted wood WPCs (Kim et al., 2009). Cupressus lusitanica heartwood blocks were subjected to toluene/ethanol extraction and then exposed to the brown rot fungus (Postia placenta). Results showed a strong degradation of extracted wood blocks whereas un-extracted blocks were resistant to decay (Mohareb et al., 2010). Kirker et al. (2013) found similar results on extractive-free and un-extracted wood species; which highlight the importance of extractives on the durability by looking at weight loss from termites and wood decay testing. Removal of extractives from the durable woods of Milicia excelsa, Albizia coriaria and Markhamia lutea increased their vulnerability to termites (Syofuna et al., 2012).

5.6. Efficiency of heartwood extractives and plant oils as wood preservatives The extractive treated SYP and CW showed resistance against termites. All four extractives were effective against the termites. Maximum weight losses were observed on solvent and water treated cottonwood and southern yellow pine with lower percentage

153 mortality of both termites. Weight losses of SYP and CW treated with solvent and water were 25 and 24%, 36 and 37%, respectively with minimum average rating. T. grandis extractives at highest concentration (10 mg ml-1) caused 82 % mortality of R. flavipes while 93 and 73 % H. indicola were dead after feeding on SYP and CW treated with T. grandis extractives. Weight losses of both non-durable woods were also reduced at highest concentration with average rating > 8. Results are in confirmation with several authors who reported that T. grandis extractive can improve the durability of non- durable woods. Extractives of tropical timber species such Bonsamdua (Distemonanthus benthamianus) could be employed to preserve their low durability counterparts but Bonsamdua extract improved their durability more than that of teak (Asamoah et al., 2011). Lukmandaru and Ogiyama (2005) found that unsaponifiable fraction of T. grandis heartwood extractives was the major part and exhibited strong activity both against shrimps and termite, Reticulitermes speratus. Lukmandaru and Takahashi (2008) reported that sap and heartwood of T. grandis both exhibited antitermitic activities but tree age was very important factor that affected the natural durability. Brocco et al. (2017) evaluated the preserving potential of heartwood extractives of T. grandis against termite, Nasutitermes corniger. They found that T. grandis extractives treatment imparted resistance to non-durable wood by changing mass loss and time to death of tested termites. When termites were fed on SYP and CW treated with D. sissoo heartwood extractives, mortality of R. flavipes was 88 and 93%, respectively at 10 mg ml-1 while mortality of H. indicola was 84 and 83% after feeding on treated SYP and CW. Weight loss and average rating of both the woods after termite feeding at maximum concentrations were significantly different from all other treatments. Treatment of SYP and CW with C. deodara extractives also caused reduction in weight loss of both the woods at maximum concentration as well as maximum mortality of termites. Maximum mortality of R. flavipes was 89% after feeding on CW treated with these extractives. Treatment of SYP and CW with P. roxburghii extractives also caused reduction in weight loss due to termites feeding and increased mortalities of both the termites. All termites (H. indicola) were dead after feeding on SYP treated with these extractives with the weight loss of 6.76 % and average rating of 8.2 whereas 91% termites (R. flavipes) were dead after feeding on SYP treated with these extractives at maximum concentrations. Our results are in confirmation with many studies. Akhtar (1981) studied effects of wood, wood extracts, and essential oils from three wood species (D. sissoo, P. wallichiana, and C. deodara)

154 against Coptotermes heimi and found that wood extracts in hexane, water, and acetone of C. deodara were more toxic to termites, but our results are not in confirmation in case of D. sissoo. Same results were obtained with the use of essential oils of these timbers. On un-extracted sawdust of C. deodara and P. roxburghii, termite survival was 0% while on D. sissoo sawdust survival was up to 40% (Akhtar (1981). Similar results were found by Akhtar and Jabeen (1981) on the feeding responses of Bifiditermes beesoni on wood and wood extracts of three species i.e. D. sissoo, C. deodara, P. wallichiana in the laboratory. Our results are not in confirmation with Qureshi et al. (2008) who found that D. sissoo wood powder was moderately toxic to Coptotermes heimi and Heterotermes indicola. At the concentrations evaluated in E10 test, extractives of T. grandis, D. sissoo, Cedrus deodara and Pinus roxburghii had none of the protective effects on non-durable wood species such as SYP and CW woods (Mankowski et al., 2016). Plant oils possess fungicidal constituents but also these can protect wood against decaying fungi by reduction of moisture content, i.e. by creating hydrophobicity. There are a number of investigations, which reveal the possible use of plant oils for wood protection due to these repellent properties. Many researchers have suggested that plant oil can be a potential wood preservative alone or in combination with some organic biocides such as wood extractives. In laboratory experiments, wood specimens of SYP and CW were treated with 10 mg ml-1 of each durable wood extractives+ 20% linseed oil. Results showed that 90 -100% termites were dead after 28 days of exposure. At maximum concentrations, all types of extractives showed non-significant difference in terms of mortalities of termites and weight losses of SYP and CW.

5.7. GC –MS Characterization The compounds identified from extractives of T. grandis; 3-(1-Hydroxyethyl)-2- methyl-1-benzofuran-5,6-dicarbonitrile,1-(1,1-dimethylethyl)-4-phenoxy-Benzene,2-methyl- 9,10 Anthracenedione, 1-Methyl-3,4-dihydroisoquinoline and Squalene were different in chemical nature and active portion on the basis of percentage were not shared among extractives. Difference in biological activities is evident from dissimilar nature of compounds present in wood extractives. Termiticide activity of anthracenedione and squalene from extractives of T. grandis are shared by Lukmandaru and Ogiyama (2005), Kartal et al. (2006),

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Gori et al. (2009), Lukmandaru et al. (2009), Bhat et al. (2010) and Xie et al. (2011). Anthracenedione (anthraquinone) and squalene made up majority of the sample sharing 24.03 and 28.24%, respectively. Trimethoxyresveratrol (40.6% of total) was abundant compound in D. sissoo, which is a stilbene with published medicinal and free radical scavenging activities (Shang et al., 2009; Dias et al., 2013). It also exhibits a potent antimicrobial and antibacterial activities as reported by Taylor et al. (2014). Stilbenes are regarded as potential sources of heartwood durability (Schultz and Nicholas, 2000) and are present in many other naturally durable woods such as Pinus sylvestris L, Eucalyptus Sideroxylon. (Hart and Hillis, 1974; Hart and Shrimpton, 1979). Resveratrol has been found to be biologically active and toxic to insects in previous studies (Aly et al., 2013; Nascimento et al., 2013). The sesquiterpene, (E)-Atlantone) identified from C. deodara extractives has been shown to have biological activity (Chaudhary et al., 2011; Bacci et al., 2015). Cuprenene and cedrene have been found associated with frontal gland secretions of soldier termites and are secreted as defensive chemical that repels the attacker (Maistrello et al., 2001; Piskorski et al., 2009). Forms of benzopryran from the extractives of P. roxburghii compromised 51% of the sample. Duchowicz et al. (2009) examined the antifeedant activities of benzopyran against Spodoptera litura. Acridin-9-amine was not found in literature to be toxic or biologically active. Another component from P. roxburghii was chrysin, which has been classified as a feeding stimulant for insects (Morimoto et al., 2003). Role of this chemical for stimulating termites’ feeding on treated blocks may be investigated in future termites research program.

5.8. Field studies Oil and extractive combination has protected the woods from termites’ damage. At the end of 12 months solvent treated chunks got the lowest scoring (9.7). Extractive treated SYP chunks treated with P. roxburghii extractives attracted some termites and got the lowest average scoring (9.6) all other chunks treated with other treatments were sound at Mississippi site. At Lahore site, SYP chunks treated with solvent only got severe attack of termites with average rating of 3.8 (Appendix. 4 B) while chunks treated with linseed oil (20%) got moderate attack of termites. P. roxburghii extractive treated chunks attracted some termites. Similar

156 situations of termites’ infestation and consequent rating were observed on CW. Stakes treated with P. roxburghii +oil were also found decayed with average rating of 8.6. Positive control (Cu Nap) and solvent control stakes were completely destroyed by the termites with 0 rating at Lahore site while SYP treated with oil and extractive got rating ranging from 9.4 to 9.6. At Mississippi site (USA), termite did not hit chunks and stakes after 6 months’ time period but at Lahore site both treated and un-treated CW and SYP were slightly attacked by termites. Hydrophobicity of oils has been exploited by several researchers in combination with natural of synthetic biocides. Lyon et al. (2007) used linseed oil and boron to develop low toxic wood treatment against C. formosanus and R. santonensis. Treatment had good potential as low toxic wood preservative with concurrent increase in boron retention of 30% and decrease of weight losses after exposure to termites. Field application of linseed has been reported in several investigations. Teru et al. (2004) found boiled and maleinised linseed oil as promising product for the protection of wood against decay. Edlund (2004) tested some alternative for wood preservations including linseed oil in field stake and ground proximity tests. Treatment with linseed oil at high retention gave better resistance against decay but not against discoloring fungi. Several other oils have also been scrutinized for potential as wood preservative including hemp, Eucalyptus globulus (eucalyptus), Trachyspermum copticum (ajowan), Cymbopogon flexuosus (lemongrass), Anethum graveolen (dill weed), Pelargonium graveolens (geranium), Rosmarinus officinalis (rosemary), Melaleuca alternifolia (tea tree) and Thymus zygis (white thyme), crude tall, cinnamon, citrus peels, tung seeds (Vernicia fordii) and kukui plant (Aleurites moluccanus) (Sailer et al., 2000; Paajanen and Ritschkoﬀ , 2002; Kartal et al., 2006; Clausen and Yang, 2008; Temiz et al., 2013; González-Laredo et al., 2015), clove oil (Ahmed et al., 2013), castor bean oil (Ahmed et al., 2014), linseed and neem oil (Fatima and Morrell, 2015) against termites and decay.

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Future guidelines for research on wood extractives 1. Antitermite activities of some individual components from extractives of T. grandis are known, techniques for isolation of extractives from other woods may be are yet to be develop, and later testing of individual components for anittermite activities can be carried out. 2. Chemical stability of the known extractives components may be conducted in laboratory and field tests. 3. Extractives have shown non-significant effect of gut bacteria before and after application. Techniques for determining bacterial density may be standardized as diversity remained intact in the present studies. Possibly, potent species may have reduced in numbers to perform cellulose digestion completely.

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CHAPTER SIX SUMMARY

Importance of termites for causing losses worth millions of dollars to woods and their products is well recognized and documented. Concurrently efforts to curtail these damages are also mentioned; several countries spend huge money to protect woods from termites. In the wake of growing concern and public awareness about use of synthetic preservatives on woods, natural chemicals are becoming centre of research interest. The aim of screening of heartwood extractives from durable woods and their usefulness as wood preservative / termiticide was followed in these studies, results of which have been presented. Pakistan harbors thirteen species of termites from a pool of fifty as pests of woodworks in constructions in rural and urban areas causing economic losses in huge amount. Hence efforts have been made in the past and are still being followed to check and control the progression of termites in order to save the buildings and the precious household items. Commercial termiticides being employed currently are potential environmental contaminants and even carcinogenic. Anticipated safe termiticides from natural sources can replace synthetic antitermitic compounds. There are several wood species in Pakistan which are naturally durable/ resistant to termites’ attack and fungal infection / degradation. This durability is mostly considered due to buildup of extractives in the heartwood. So the present studies have been carried out with a view to find out anti-termite agents from locally found resistant heartwoods. For this purpose, four previously known durable woods, viz., Tectona grandis, Dalbergia sissoo, Cedrus deodara and Pinus roxburghii commonly available in Pakistan were selected. To test the effectiveness of their extractives, two subterranean termites species were selected, i.e. Reticulitermes flavipes which is common structural pest throughout United States and Heterotermes indicola, which usually damages household structures and woods in Pakistan. Toxicity of extractives and its confirmation in various bioassays, later identification of components in extractives of each durable wood species, finally finding mechanisms of actions in the laboratory tests were bases of experiments under controlled conditions. These experiments culminated while observing protection of susceptible woods treated with extractives in USA and Pakistan.

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Mortality of two termites’ species in the toxicity bioassay on filter papers treated with extractives in different concentrations revealed non-significant differences among the concentrations but also among extractives of durable wood species at low concentrations. These differences in mortalities were non-significant at maximum concentrations of all extractives, exhibiting 70-80 % dead individuals of termites. Consumption of filter papers treated with heartwood extractives was also significantly reduced at the highest concentrations. All the extractives showed repellent activities against both the termites except P. roxburghii extractives. Among all heartwood extractives, C. deodara showed maximum repellent activity against both the termite species. Antioxidant activities of extractives were also proved in the present results. These extractives, however, reduced GSTs and ESTs in termites’ gut. D. sissoo -1 heartwood extractive had maximum inhibition with lowest IC50 (28.83 µg ml ) as compared to other three extractives. Its IC50 value was non-significantly different from BHT but significantly different from quercetin. Inhibition (%) in case of D. sissoo was not concentration dependent as were with other extractives. Activities of GSTs and ESTs were significantly reduced in guts of workers of H. indicola fed on filter papers treated with extractives and positive controls (BHT, Quercetin). However, all extractives did not significantly reduce CAT activity. Regarding toxicity on gut protozoa, D. sissoo and T. grandis were effective and reduced maximum number of protozoans. Maximum protozoans’ reduction corresponded with mortality at various concentrations especially at the highest level (10 mg ml-1). Molecular technique was also used to ascertain bacterial communities in H. indicola and this is first report on its bacterial flora, followed by observation on effect of extractive treatment on these gut bacteria. In all treatment groups, the dominant group was unclassified bacteria followed by Protobacteria and spirochetes. All other phyla were very small in each treatment group including the control. Overall, examination of the abundance of major taxonomic groups in treated versus control groups did not appear to show any major differences in presence of phyla of gut bacteria. Role of extractives in protection against termites was further tested by removing these extractives and offering extracted and un-extracted durable woods to termites in choice and no choice tests. T. grandis showed non-significant difference in feeding of R. flavipes on extracted and un-extracted blocks and there was non-significant difference of mortality of both the termites in two type of tests. Generally both the termite species ignored un-extracted heartwood

160 blocks of all woods and preferred extracted wood specimens. Mortality of both the termite species was significantly different on extracted and un-extracted blocks for three other durable woods except D. sissoo after feeding of H. indicola. Application of extractives on the selected susceptible woods of SYP (Southern Yellow Pine) and CW (Cotton wood) clearly improved their durability. In transfer durability tests, minimum weight loss was observed on D. sissoo extractives SYP woods, however, it was non- significantly different from other heartwoods extractives at 10 mg ml-1. Minimum weight loss in CW was also recorded after the treatment of D. sissoo extractives at the highest concentration. A significant maximum mortality (100 %) of termites (R. flavipes) was observed after feeding on P. roxburghii heartwood extractives SYP contrary to maximum mortality (92 %) C. deodara extractives treated CW woods at lower concentrations. Moreover, all extractives performed non-significantly different from one another at the highest concentration. The leaching test showed that extractives of C. deodara and P. roxburghii did not retain in SYP and CW following the leaching procedure. Leached specimens of SYP and CW treated with durable wood extractives had more feeding rate and less termites’ mortality whereas T. grandis and D. sissoo heartwood extractives treated SYP and CW specimens did not show any increase in feeding by termites after leaching. Mortalities of termites after feeding on leached specimens were also non- significantly different from un-leached samples.

Application of combination of extractives with linseed oil (10mg ml-1 + 20 %) on SYP and CW woods showed maximum mortality (>90%) of both the termite species. Similarly, minimum weight losses of SYP and CW were recoded when these were treated with extractive + oil but it varied non- significantly with only extractives treated SYP and CW woods. Effectiveness of extractives was also extended by determining their effect on fungi decay. Results of laboratory tests against decay at the same rates of extractives of T. grandis, D. sissoo, C. deodara and P. roxburghii yielded no protective coverage on non-durable woods of SYP and CW against wood decay fungi (Postia placenta and Trametes versicolor) in E-10 soil bottle assay.

GC-MS analyses yielded 96, 52, 147 and 12 compounds from T. grandis, D. sissoo, C. deodara and P. roxburghii, respectively. GC-MS analysis showed high percentage of 2-

161 methyl-9,10 Anthracenedione and Squalene, Trismethoxy resveratrol and 1,3-Diamino-8-n- butyl-5,6-dihydrobenzoquinazoline, (E)-Atlantone and Di-epi-alpha-cedrene, 1,2,3,4- tetrahydro-5,8-dimethyl-Acridin-9-amine and 2,3-dihydro-5,7-dihydroxy-2-phenyl-4H-1- Benzopyran-4-one respectively T. grandis, D. sissoo, C. deodara and P. roxburghii extractives. 2-3 compounds proportionally high in each extractive type have previously been reported as fungicide or insecticide. Conclusively, field trials of susceptible woods treated with extractives/extractive + oil were pursued. P. roxburghii and C. deodara extractives treated blocks and stakes attracted termites at both sites. Control treatments comprising of solvent, oil treated blocks and stakes at Lahore site (Pakistan) were completely destroyed by the termites (H. indicola) and decayed after a lapse of time period of 12 months. Linseed oil treated stakes of SYP and CW showed discoloration with average rating of 9.5 due to fungal decay at USA. P. roxburghii extractive stakes were partially or completely deteriorated by the fungi at both the sites. In nutshell, at this time, i.e., up till submission of thesis (02.10.2107), extractives treated stakes were not infested with termites. Thus, from results obtained at the moment, potential of extractives from Tectona grandis, Dalbergia sissoo, Cedrus deodara and Pinus roxburghii as wood preservative has been demonstrated.

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APPENDICES

Appendix 1. Extractive free (a) and un- extracted (b) heartwood blocks after feeding of R. flavipes under choice and No-choice test

a b a b

Tectona grandis Dalbergia sissoo

a b a b

Cedrus deodara Pinus roxburghii

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Appendix 2. Heartwood blocks after feeding of H. indicola on extracted free (a) and un- extracted (b) woods under choice and No-choice tests.

a b a b Tectona grandis Dalbergia sissoo

a b a b

Cedrus deodara Pinus roxburghii

Appendix 3. Treatment plant used during Vacuum pressure impregnation of wood at FPL, USA.

195

Appendix 4. Galleries of termites on ground proximity test after 12 months (a), Solvent treated chunks attacked by termites (b), Heartwoods and Positive control (c) at Pakistan site.

196

Appendix 5. Heartwoods, SYP and CW treated with solvent attacked by termites at Pakistani site in field stake test after 12 months.

197

Appendix 6. Protozoans species found in guts of R. flavipes

a b c d

e f g h

198

Appendix 7. Protozoans species found in guts of H. indicola.

199

Appendix 4.1. ANOVA for % Mortality of termites after feeding on treated filter paper.

200

Appendix 4.1. ANOVA for % Mortality of termites after feeding on treated filter paper.

Source DF Seq SS Adj SS Adj MS F p Termite 1 64.5 64.5 64.5 0.55 0.459 Heartwoods 3 5898.3 5898.3 1966.1 16.87 0.000 Concentrations 5 112940.0 112940.0 22588.0 193.83 0.000 Termite*Heartwoods 3 3581.9 3581.9 1194.0 10.25 0.000 Termite*Concentrations 5 2732.6 2732.6 546.5 4.69 0.001 Heartwoods*Concentrations 15 8385.7 8385.7 559.0 4.80 0.000 Termite*Heartwoods*Concentrations 15 3767.9 3767.9 251.2 2.16 0.013 Error 96 11187.1 11187.1 116.5 Total 143 148558.0

Appendix 4.2. ANOVA for % weight loss of filter paper after feeding of termites for 15 days

Source DF Seq SS Adj SS Adj F p MS Termite 1 1002.62 1002.62 1002.62 30.65 0.000 Heartwoods 3 1436.95 1436.95 478.98 14.64 0.000 Concentrations 5 16704.24 16704.24 3340.85 102.14 0.000 Termite*Heartwoods 3 1866.47 1866.47 622.16 19.02 0.000 Termite*Concentrations 5 384.23 384.23 76.85 2.35 0.047 Heartwoods*Concentrations 15 1078.36 1078.36 71.89 2.20 0.011 Termite*Heartwoods*Concentrations 15 1842.93 1842.93 122.86 3.76 0.000 Error 96 3139.94 3139.94 32.71 Total 143 27455.74

Appendix 4.3. ANOVA for % area loss of filter paper after feeding of termites for 15 days

Source DF Seq SS Adj SS Adj F p MS Termite 1 962.97 962.97 962.97 47.06 0.000 Heartwoods 3 1917.09 1917.09 639.03 31.23 0.000 Concentrations 5 14590.45 14590.45 2918.09 142.62 0.000 Termite*Heartwoods 3 1783.92 1783.92 594.64 29.06 0.000 Termite*Concentrations 5 329.33 329.33 65.87 3.22 0.010 Heartwoods*Concentrations 15 761.90 761.90 50.79 2.48 0.004 Termite*Heartwoods*Concentrations 15 1302.05 1302.05 86.80 4.24 0.000 Error 96 1964.20 1964.20 20.46 Total 143 23611.90

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Appendix 4.4. ANOVA for Repellent activities of four heartwood extractives against termites

Source DF Seq SS Adj SS Adj MS F p termite 1 1463 1602 1602 4.27 0.039 Heartwoods 3 429177 430086 143362 381.86 0.000 Time 4 7019 6723 1681 4.48 0.001 Concentration 5 421239 421346 84269 224.46 0.000 termite*Heartwoods 3 2019 2025 675 1.80 0.147 termite*Time 4 1004 1016 254 0.68 0.609 termite*Concentration 5 2945 2913 583 1.55 0.172 Heartwoods*Time 12 4227 4230 352 0.94 0.508 Heartwoods*Concentration 15 112183 112210 7481 19.93 0.000 Time*Concentration 20 8381 8387 419 1.12 0.328 termite*Heartwoods*Time 12 2690 2694 225 0.60 0.844 termite*Heartwoods*Concentration 15 3435 3436 229 0.61 0.868 Heartwoods*Time*Concentration 60 7633 7634 127 0.34 1.000 termite*Time*Concentration 20 2552 2539 127 0.34 0.997 termite*Heartwoods*Time* 60 6552 6552 109 0.29 1.000 Concentration Error 479 179833 179833 375 Total 718 1192353

Appendix 4.5. ANOVA for antifeedant activity of four heart wood extractives against R. flavipes and H. indicola

Source DF Seq SS Adj SS Adj MS F p Termite 1 1140.7 1140.7 1140.7 4.73 0.033 Heartwood 3 6629.3 6629.3 2209.8 9.16 0.000 Conc. 4 48260.3 48260.3 12065.1 49.99 0.000 Termite*Heartwood 3 17323.1 17323.1 5774.4 23.93 0.000 Termite*Conc. 4 358.0 358.0 89.5 0.37 0.829 Heartwood*Conc. 12 550.5 550.5 45.9 0.19 0.999 Termite*Heartwood*Conc. 12 1784.6 1784.6 148.7 0.62 0.822 Error 80 19306.5 19306.5 241.3 Total 119 95352.9

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Appendix 4.6. ANOVA for % reduction of protozoans in guts of termites after feeding on filter paper treated with heartwood extractives.

Source DF Seq SS Adj SS Adj MS F p Termite 1 6102.3 6102.3 6102.3 326.04 0.000 Heartwoods 3 6660.5 6660.5 2220.2 118.62 0.000 Concentrations 4 25028.0 25028.0 6257.0 334.31 0.000 Termite*Heartwoods 3 2101.9 2101.9 700.6 37.43 0.000 Termite*Concentrations 4 4960.7 4960.7 1240.2 66.26 0.000 Heartwoods*Concentrations 12 2844.9 2844.9 237.1 12.67 0.000 Termite*Heartwoods*Concentrations 12 905.2 905.2 75.4 4.03 0.000 Error 80 1497.3 1497.3 18.7 Total 119 50100.8

Appendix 4.7. ANOVA for % weight losses of SYP and CW treated with four heartwood extractives after feeding of R. flavipes and H. indicola

Source DF MS F p Termite species 1 0.01 0.00 0.98 Wood Type 1 9024.49 301.63 0.00 Heartwoods 3 11.82 0.39 0.757 Cencentrations 4 6683.09 223.37 0.000 Termite species*Wood Type 1 12.63 0.42 0.516 Termite species*Heartwoods 3 55.60 1.86 0.137 Termite species*Cencentrations 4 37.89 1.27 0.283 Wood Type*Heartwoods 3 289.20 9.67 0.000 Wood Type*Cencentrations 4 185.77 6.21 0.000 Heartwoods*Cencentrations 12 45.56 1.52 0.114 Termite species*Wood Type*Heartwoods 3 6.83 0.23 0.877 Termite species*Wood Type* Cencentrations 4 22.91 0.77 0.548 Wood Type*Heartwoods*Cencentrations 12 76.18 2.55 0.003 Termite species*Heartwoods* Cencentrations 12 25.98 0.87 0.580 Termite species*Wood Type* 12 22.09 0.74 0.713 Heartwoods*Cencentrations Error 320 29.92 Total 399

203

Appendix 4.8. ANOVA for % mortalities of termites after feeding on SYP and CW treated with four heartwood extractives.

Source DF Seq SS Adj Adj F p SS MS Termite species 1 11.4 11.4 11.4 0.05 0.825 Wood Type 1 1851.2 1851.2 1851.2 7.91 0.005 Heartwoods 3 3459.0 3459.0 1153.0 4.93 0.002 Cencentrations 4 163882.5 163882.5 40970.6 175.02 0.000 Termite species*Wood Type 1 79.2 79.2 79.2 0.34 0.561 Termite species*Heartwoods 3 543.5 543.5 181.2 0.77 0.509 Termite species*Cencentrations 4 235.3 235.3 58.8 0.25 0.909 Wood Type*Heartwoods 3 5139.7 5139.7 1713.2 7.32 0.000 Wood Type*Cencentrations 4 701.5 701.5 175.4 0.75 0.559 Heartwoods*Cencentrations 12 9334.8 9334.8 777.9 3.32 0.000 Termite species*Wood 3 229.8 229.8 76.6 0.33 0.806 Type*Heartwoods Termite species*Wood Type* 4 353.5 353.5 88.4 0.38 0.825 Cencentrations Wood 12 10939.4 10939.4 911.6 3.89 0.000 Type*Heartwoods*Cencentrations Termite species*Heartwoods* 12 955.7 955.7 79.6 0.34 0.981 Cencentrations Termite species*Wood Type* 12 715.6 715.6 59.6 0.25 0.995 Heartwoods*Cencentrations Error 320 74908.3 74908.3 234.1 Total 399 273340.4

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Appendix 4.9. ANOVA for % weight losses of SYP and CW treated with four heartwood extractives + linseed oil after feeding of R. flavipes and H. indicola.

Source DF Seq SS Adj SS Adj MS F p termite 1 14.67 14.67 14.67 0.59 0.00 wood type 1 2414.96 2414.96 2414.96 97.79 0.441 heartwood 3 410.52 410.52 136.84 5.54 0.000 conc 4 13102.09 13102.09 3275.52 132.64 0.001 termite*wood type 1 7.34 7.34 7.34 0.30 0.000 termite*heartwood 3 24.10 24.10 8.03 0.33 0.586 termite*conc 4 158.90 158.90 39.73 1.61 0.807 wood type*heartwood 3 86.58 86.58 28.86 1.17 0.172 wood type*conc 4 656.26 656.26 164.07 6.64 0.322 heartwood*conc 12 1177.01 1177.01 98.08 3.97 0.000 termite*wood type*heartwood 3 23.33 23.33 7.78 0.31 0.000 termite*wood type*conc 4 102.70 102.70 25.68 1.04 0.815 wood type*heartwood*conc 12 266.02 266.02 22.17 0.90 0.387 termite*heartwood*conc 12 32.65 32.65 2.72 0.11 0.550 termite*wood 12 74.26 74.26 6.19 0.25 1.000 type*heartwood*conc Error 320 7902.15 7902.15 24.69 0.995 Total 399 26453.55

Appendix 4.10. ANOVA for % mortalities of termites after feeding on SYP and CW treated with four heartwood extractives + linseed oil.

Source DF Seq SS Adj SS Adj MS F p termite 1 204.1 204.1 204.1 2.07 0.151 wood type 1 713.3 713.3 713.3 7.24 0.008 heartwood 3 433.5 433.5 144.5 1.47 0.224 conc 4 363915.8 363915.8 90979.0 923.27 0.000 termite*wood type 1 94.0 94.0 94.0 0.95 0.330 termite*heartwood 3 228.7 228.7 76.2 0.77 0.509 termite*conc 4 689.2 689.2 172.3 1.75 0.139 wood type*heartwood 3 2097.8 2097.8 699.3 7.10 0.000 wood type*conc 4 2847.9 2847.9 712.0 7.23 0.000 heartwood*conc 12 2520.7 2520.7 210.1 2.13 0.015 termite*wood type*heartwood 3 147.9 147.9 49.3 0.50 0.682 termite*wood type*conc 4 351.2 351.2 87.8 0.89 0.469 wood type*heartwood*conc 12 5597.8 5597.8 466.5 4.73 0.000 termite*heartwood*conc 12 518.8 518.8 43.2 0.44 0.947 termite*wood 12 708.8 708.8 59.1 0.60 0.843 type*heartwood*conc Error 320 31532.7 31532.7 98.5 Total 399 412602.1

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Appendix 4.11. 16S rRNA sequence of Enterobacter cloacae (HE-1) isolated from the gut of H. indicola (a) and its evolutionary relationships with taxa (b). (a)

Sample_HE-1 GGCCTACACA TGCAAGTCGA ACGGTAGCAC AGAGAGCTTG CTCTCGGGTG 50 Sample_HE-1 ACGAGTGGCG GACGGGTGAG TAATGTCTGG GAAACTGCCT GATGGAGGGG 100 Sample_HE-1 GATAACTACT GGAAACGGTA GCTAATACCG CATAACGTCG CAAGACCAAA 150 Sample_HE-1 GAGGGGGACC TTCGGGCCTC TTGCCATCAG ATGTGCCCAG ATGGGATTAG 200 Sample_HE-1 CTAGTAGGTG GGGTAACGGC TCACCTAGGC GACGATCCCT AGCTGGTCTG 250 Sample_HE-1 AGAGGATGAC CAGCCACACT GGAACTGAGA CACGGTCCAG ACTCCTACGG 300 Sample_HE-1 GAGGCAGCAG TGGGGAATAT TGCACAATGG GCGCAAGCCT GATGCAGCCA 350 Sample_HE-1 TGCCGCGTGT ATGAAGAAGG CCTTCGGGTT GTAAAGTACT TTCAGCGGGG 400 Sample_HE-1 AGGAAGGTGT TGTGGTTAAT AACCACAGCA ATTGACGTTA CCCGCAGAAG 450 Sample_HE-1 AAGCACCGGC TAACTCCGTG CCAGCAGCCG CGGTAATACG GAGGGTGCAA 500 Sample_HE-1 GCGTTAATCG GAATTACTGG GCGTAAAGCG CACGCAGGCG GTCTGTCAAG 550 Sample_HE-1 TCGGATGTGA AATCCCCGGG CTCAACCTGG GAACTGCATT CGAAACTGGC 600 Sample_HE-1 AGGCTGGAGT CTTGTAGAGG GGGGTAGAAT TCCAGGTGTA GCGGTGAAAT 650 Sample_HE-1 GCGTAGAGAT CTGGAGGAAT ACCGGTGGCG AAGGCGGCCC CCTGGACAAA 700 Sample_HE-1 GACTGACGCT CAGGTGCGAA AGCGTGGGGA GCAAACAGGA 740

(b)

Leclercia adecarboxylata strain CIP 82.92 Bacterium SFS2-2-1 Enterobacter kobei Enterobacter ludwigii strain EN-119 Bacterium MC-BAC-8 Klebsiella sp. JT42 Uncultured Enterobacter sp. clone F4oct. Uncultured Enterobacter sp. clone F5feb. Enterobacter cloacae strain PHK1 Bacterium SNISO F2 Group I Enterobacter cloacae subsp. dissolvens Enterobacter sp. BAB-3152 Enterobacter cloacae strain OS5.9 Enterobacter cloacae strain OS5.7 Methanogenic archaeon strain S2 Cronobacter sakazakii strain MM045 Enterobacter sp. DQ11 Enterobacter cloacae strain JV Sample HE-1 Enterobacter cloacae strain PW 113 Enterobacter cloacae strain PM 84 Enterobacter cloacae strain PS 57

Enterobacter cloacae strain FW 46 Group II Enterobacter cloacae strain FS 4 Enterobacter cloacae strain FM 20 Enterobacter cloacae strain LCR70 Enterobacter ludwigii strain EN-119 Escherichia coli (outgroup)

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The evolutionary history was inferred using the Neighbor-Joining method. The optimal tree with the sum of branch length = 0.18917710 is shown. The evolutionary distances were computed using the p-distance method and are in the units of the number of base differences per site. The analysis involved 28 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 559 positions in the final dataset. Evolutionary analyses were conducted in MEGA6.

Appendix 4.12. 16S rRNA sequence of Bacillus cereus (HE-2) isolated from the gut of H. indicola (a) and its evolutionary relationships with taxa (b). (a)

Sample_HE-2 AATCAAAAAA TCCTTTTTTC AAGAAGAAGT GCCAATAAGC AGTCGAGCGA 50 Sample_HE-2 TGGATTAGAG CTTGCTCTTA TGAAGTTAGC GGCGGACGGG TGAGTAACAC 100 Sample_HE-2 GTGGGTAACC TGCCCATAAG ACTGGGATAA CTCCGGGAAA CCGGGGCTAA 150 Sample_HE-2 TACCGGATAA CATTTTGAAC CGCATGGTTC GAAATTGAAA GGCGGCTTCG 200 Sample_HE-2 GCTGTCACTT ATGGATGGAC CCGCGTCGCA TTAGCTAGTT GGTGAGGTAA 250 Sample_HE-2 CGGCTCACCA AGGCAACGAT GCGTAGCCGA CCTGAGAGGG TGATCGGCCA 300 Sample_HE-2 CACTGGGACT GAGACACGGC CCAGACTCCT ACGGGAGGCA GCAGTAGGGA 350 Sample_HE-2 ATCTTCCGCA ATGGACGAAA GTCTGACGGA GCAACGCCGC GTGAGTGATG 400 Sample_HE-2 AAGGCTTTCG GGTCGTAAAA CTCTGTTGTT AGGGAAGAAC AAGTGCTAGT 450 Sample_HE-2 TGAATAAGCT GGCACCTTGA CGGTACCTAA CCAGAAAGCC ACGGCTAACT 500 Sample_HE-2 ACGTGCCAGC AGCCGCGGTA ATACGTAGGT GGCAAGCGTT ATCCGGAATT 550 Sample_HE-2 ATTGGGCGTA AAGCGCGCGC AGGTGGTTTC TTAAGTCTGA TGTGAAAGCC 600 Sample_HE-2 CACGGCTCAA CCGTGGAGGG TCATTGGAAA CTGGGAGACT TGAGTGCAGA 650 Sample_HE-2 AGAGGAAAGT GGAATTCCAT GTGTAGCGGT GAAATGCGTA GAGATATGGA 700 Sample_HE-2 GGAACACCAG TGGCGAAGGC GACTTTCTGG TCTGTAACTG ACACTGAGGC 750 Sample_HE-2 GCGAAAGCGT GGGGAGCAAA CAGGATTAGA TACCCTGGTA GTCCACGCCG 800 Sample_HE-2 TAAACGATGA GTGCTAAGTG TTAGAGGGTT TCCGCCCTTT AGTGCTGAAG 850 Sample_HE-2 TTAACGCATT AAGCACTCCG CCTGGGGAGT ACGGCCGCAA GGCTGAAACT 900 Sample_HE-2 CAAAGGAATT GACGGGGGCC CGCACAAGCG GTGGAGCATG TGGTTTAATT 950 Sample_HE-2 CGAAGCAACG CGAAGAACCT TACCAGGTCT TGACATCCTC TGACAACCCT 1000 Sample_HE-2 AGAGATAGGG CTTCTCCTTC GGGAGCAGAG TGACAGGTGG TGCATGGTTG 1050 Sample_HE-2 TCGTCAGCTC GTGTCGTGAG ATGTTGGGTT AAGTCCCGCA ACGAGCGCAA 1100 Sample_HE-2 CCCTTGATCT TAGTTGCCAT CATTAAGTTG GGCACTCTAA GGTGACTGCC 1150 Sample_HE-2 GGTGACAAAC CGGAGGAAGG TGGGGATGAC GTCAAATCAT CATGCCCCTT 1200 Sample_HE-2 ATGACCTGGG CTACACACGT GCTACAATGG ACGGTACAAA GAGCTGCAAG 1250 Sample_HE-2 ACCGCGAGGT GGAGCTAATC TCATAAAACC GTTCTCAGTT CGGATTGTAG 1300 Sample_HE-2 GCTGCAACTC GCCTACATGA AGCTGGAATC GCTAGTAATC GCGGATCAGC 1350 Sample_HE-2 ATGCCGCGTG ATACGTCCCG GGCTGTACAC ACGCCCGTCA CACCACGAGA 1400 Sample_HE-2 GTTGTACACC CGAAGTCGTT GGGTTACTTT TTGAGCAGCC GCTAGTGGAC 1450 Sample_HE-2 GATGATTGGG TGAGCGTAAC AGGGGTAACC GTAA 1484

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(b)

Bacillus cereus strain BCRh5 Bacillus cereus strain BCRh6 Bacillus cereus strain GXBC-1 Bacillus cereus strain A16 Bacillus anthracis strain IHB B 18197 Bacillus cereus strain IHB Bacillus sp. G1-24 Bacillus sp. 82344

Bacillus sp. strain EPG3 Group I Bacillus toyonensis strain Bacillus sp. AZ-1 Uncultured bacterium clone EGSB 200 Uncultured bacterium clone EGSB 200 5-2 Uncultured bacterium clone 11 K 10 Bacillus cereus strain WG38 Bacillus anthracis sample HE 2 Escherichia coli (outgroup) The evolutionary history was inferred using the Neighbor-Joining method. The optimal tree with the sum of branch length = 0.27094474 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (100 replicates) are shown next to the branches. The evolutionary distances were computed using the p-distance method and are in the units of the number of base differences per site. The analysis involved 18 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 561 positions in the final dataset. Evolutionary analyses were conducted in MEGA6.

Appendix 4.13. 16S rRNA sequence of Pseudomonas aeruginosa (HE-7) isolated from the gut of H. indicola (a) and its evolutionary relationships with taxa (b).

(a)

Sample_HE-7 CCTCACGCTA TCAGATGAGC CTAGGTCGGA TTAGCTAGTT GGTGGGGTAA 50 Sample_HE-7 AGGCCTACCA AGGCGACGAT CCGTAACTGG TCTGAGAGGA TGATCAGTCA 100 Sample_HE-7 CACTGGAACT GAGACACGGT CCAGACTCCT ACGGGAGGCA GCAGTGGGGA 150 Sample_HE-7 ATATTGGACA ATGGGCGAAA GCCTGATCCA GCCATGCCGC GTGTGTGAAG 200 Sample_HE-7 AAGGTCTTCG GATTGTAAAG CACTTTAAGT TGGGAGGAAG GGCAGTAAGT 250 Sample_HE-7 TAATACCTTG CTGTTTTGAC GTTACCAACA GAATAAGCAC CGGCTAACTT 300 Sample_HE-7 CGTGCCAGCA GCCGCGGTAA TACGAAGGGT GCAAGCGTTA ATCGGAATTA 350 Sample_HE-7 CTGGGCGTAA AGCGCGCGTA GGTGGTTCAG CAAGTTGGAT GTGAAATCCC 400 Sample_HE-7 CGGGCTCAAC CTGGGAACTG CATCCAAAAC TACTGAGCTA GAGTACGGTA 450 Sample_HE-7 GAGGGTGGTG GAATTTCCTG TGTAGCGGTG AAATGCGTAG ATATAGGAAG 500 Sample_HE-7 GAACACCAGT GGCGAAGGCG ACCACCTGGA CTGATACTGA CACTGAGGTG 550 Sample_HE-7 CGAAAGCGTG GGGAGCAAAC AGGATTAGAT ACCCTGGTAG TCCACGCCGT 600 Sample_HE-7 AAACGATGTC GACTAGCCGT TGGGATCCTT GAGATCTTAG TGGCGCAGCT 650 Sample_HE-7 AACGCGATAA GTCGACCGCC TGGGGAGTAC GGCCGCAAGG TTAAAACTCA 700 Sample_HE-7 AATGAATTGA CGGGGGCCCG CACAAGCGGT GGAGCATGTG GTTTAATTCG 750

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Sample_HE-7 AAGCAACGCG AAGAACCTTA CCTGGCCTTG ACATGCTGAG AACTTTCCAG 800 Sample_HE-7 AGATGGATTG GTGCCTTCGG GAACTCAGAC ACAGGTGCTG CATGGCTGTC 850 Sample_HE-7 GTCAGCTCGT GTCGTGAGAT GTTGGGTTAA GTCCCGTAAC GAGCGCAACC 900 Sample_HE-7 CTTGTCCTTA GTTACCAGCA CCTCGGGTGG GCACTCT 937

(b)

Pseudomonas sp. strain SAUF034 Pseudomonas sp. strain SAUF033 Bacterium GX10 Bacterium GX8 Bacterium AW5 Bacterium AW1(2013) strain AW1 Uncultured Pseudomonas sp. clone C0 4 Pseudomonas sp. KC31 Pseudomonas sp. BAB-3476 Pseudomonas sp. BAB-3474 Pseudomonas sp. BAB-3358 Uncultured bacterium clone nck128f02c1 Uncultured bacterium clone nck120b03c1 Uncultured bacterium clone nck60b12c1 Uncultured bacterium clone nck15d03c1 Group I Uncultured bacterium clone nck15a11c1 Uncultured bacterium clone nck13b03c1 Uncultured bacterium clone nck15f12c1 Uncultured bacterium clone nck15e02c1 Uncultured bacterium clone nck63e07c1 Pseudomonas aeruginosa strain Iraq.PA-5 1 Pseudomonas aeruginosa strain PIB30 Pseudomonas aeruginosa strain PIB32 Pseudomonas aeruginosa strain PIB31 Pseudomonas sp. strain SAUF159 Pseudomonas sp. strain SAUF153 Pseudomonas sp. strain SAUF087 Pseudomonas aeruginosa strain bmb1 Pseudomonas aeruginosa strain S3 sample HE 7

Escherichia coli (outgroup) The evolutionary history was inferred using the Neighbor-Joining method. The optimal tree with the sum of branch length = 0.04358354 is shown. The evolutionary distances were computed using the p-distance method and are in the units of the number of base differences per site. The analysis involved 31 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 413 positions in the final dataset. Evolutionary analyses were conducted in MEGA6.

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Appendix 4.14. 16S rRNA sequence of Pantoea agglomerans (HE-8) isolated from the gut of H. indicola (a) and its evolutionary relationships with taxa (b). (a)

Sample_HE-8 CAGTGGACGG ACGGGTGATT CGTGTCTGGG AAGCCGCCTG AAGGAGGAGG 50 Sample_HE-8 ATAACTACTG AAAACGGTAG CTAATACCGC ATACCTTCGC CCGACCGGTT 100 Sample_HE-8 AGGGGGACCT TCAGGGCTCT TGCCGTCACA TGTGCCCGGA TGGGATTAGC 150 Sample_HE-8 TTTTACGTGG GGTAACGGCT CACCTTCGCC ACGATCCCTA TCTGGACTGA 200 Sample_HE-8 AACGATGACC GTGATCACTG GAACTGAGAC TGAGTCCCGA CTCCTACTCC 250 Sample_HE-8 AGGCAGCAGT GGGGAATATT GCTCAATGGG CGCGAGCCAG ATGCAGACAT 300 Sample_HE-8 GCCGCCTGTA TGAAAAAGGC CTTCGGGTTG TAATGTACTT TCTCCGGGGA 350 Sample_HE-8 TGAAGGGAAT AAAGTGAATA CCTTTGCGCT TTGACCTTGC CCGCACAAAA 400 Sample_HE-8 AGCACCGGCT AACTCCGTGC CCGCACCCGC GGCAATAGTA AGGGTGCAGG 450 Sample_HE-8 CGTTAATCTG AATTACTGGT CGTGAAGCGC ACGCGCGCGG ATTGTTTTGT 500 Sample_HE-8 CTGATGTGAA TTCCCCGGCC TCCGGCTGGG AACTGCATCT GATACTGGCA 550 Sample_HE-8 CTCTTGAATC TCGTAGAGGA GGATGAAATT CCAAGTGTAC CGGTGAAATG 600 Sample_HE-8 CATATACATC TAGATGAGTA CCGAGTGGCG ATGGCGGCCG 640

(b)

Enterobacter sp. W25 Gamma proteobacterium Tree5 2o Pantoea agglomerans strain AIMST 9.Ac.9 Pantoea stewartii strain IAC/BECa-050 Cronobacter sakazakii strain KYU70 Uncultured Hafnia sp. Serratia sp. H62 Serratia sp. R46

Uncultured Shigella sp. clone COG-55 Group I Citrobacter sp. KC-1 Enterobacter cancerogenus strain ZR043 Enterobacter sp. ENKS2 Enterobacter sp. VJ-7 Morganella sp. NiVa101 Erwinia rhapontici Ewingella americana strain RC188 Uncultured organism clone ELU0040-T218-S-NIPCRAMgANa 000026 Pantoea agglomerans strain AIMST 2.A.sub4L Group II sample HE 8 Escherichia coli The evolutionary history was inferred using the Neighbor-Joining method. The optimal tree with the sum of branch length = 1.29529913 is shown. The evolutionary distances were computed using the p-distance method and are in the units of the number of base differences per site. The analysis involved 20 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 409 positions in the final dataset. Evolutionary analyses were conducted in MEGA6.

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Appendix 4.15. 16S rRNA sequence of Un-cultured (HE-12) isolated from the gut of H. indicola (a) and its evolutionary relationships with taxa (b). (a)

Sample_HE-12 CTGGTCTGAG AGGATGACCA GCCACACTGG AACTGAGACA CGGTCCAGAC 50 Sample_HE-12 TCCTACGGGA GGCAGCAGTG GGGAATATTG CACAATGGGC GCAAGCCTGA 100 Sample_HE-12 TGCAGCCATG CCGCGTGTAT GAAGAAGGCC TTCGGGTTGT AAAGTACTTT 150 Sample_HE-12 CAGCGGGGAG GAAGGTGTTG TGGTTAATAA CCGCAGCAAT TGACGTTACC 200 Sample_HE-12 CGCAGAAGAA GCACCGGCTA ACTCCGTGCC AGCAGCCGCG GTAATACGGA 250 Sample_HE-12 GGGTGCAAGC GTTAATCGGA ATTACTGGGC GTAAAGCGCA CGCAGGCGGT 300 Sample_HE-12 CTGTCAAGTC GGATGTGAAA TCCCCGGGCT CAACCTGGGA ACTGCATTCG 350 Sample_HE-12 AAACTGGCAG GCTTGAGTCT CGTAGAGGGG GGTAGAATTC CAGGTGTAGC 400 Sample_HE-12 GGTGAAATGC GTAGAGATCT GGAGGAATAC CGGTGGCGAA GGCGGCCCCC 450 Sample_HE-12 TGGACGAAGA CTGACGCTCA GGTGCGAAAG CGTGGGGAGC AAACAGGATT 500 Sample_HE-12 AGATACCCTG GTAGTCCACG CCGTAAACGA TGTCTATTTG GAGGTTGTTC 550 Sample_HE-12 CCTTGAGGAG TGGCTTCCGG AGCTAACGCG TTAAATAGAC CGCCTGGGGA 600 Sample_HE-12 GTACGGCCGC AAGGTTAAAA CTCAAATGAA TTGACGGGGG CCCGCACAAG 650 Sample_HE-12 CGGTGGAGCA TGTGGTTTAA TTCGATGCAA CGCGAAGAAC CTTACCTGGT 700 Sample_HE-12 CTTGACATCC ACAGAACTTT CCAGAGATGG ATTGGTGCCT TCGGGAACTG 750 Sample_HE-12 TGAGACAGGT GCTGCATGGC TGTCGTCAGC TCGTGTTGTG AAATGTTGGG 800 Sample_HE-12 TTAAGTCCCG CAACGAGCGC AACCCTTATC CTTTGTTGCC 840

(b)

Uncultured bacterium clone 16saw18-01d10.w2k926r sample HE 12 Uncultured bacterium clone 16saw43-2a08.q1k Uncultured bacterium clone 16saw43-1f10.p1k Uncultured bacterium clone 16saw43-2b12.q1k Uncultured bacterium clone 16saw43-1f01.q1k Group I Uncultured bacterium clone 16saw44-1e11.q1kb Uncultured bacterium clone 16saw14-01e04.w2k926r Uncultured bacterium clone 16saw14-01f11.w2k926r Uncultured bacterium clone 16saw45-1e12.p1k Uncultured bacterium Uncultured bacterium clone 16saw49-2h12.w2k Escherichia coli (outgroup)

The evolutionary history was inferred using the Neighbor-Joining method. The optimal tree with the sum of branch length = 0.16617211 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (100 replicates) are shown next to the branches. The evolutionary distances were computed using the p-distance method and are in the units of the number of base differences per site. The analysis involved 13 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 337 positions in the final dataset. Evolutionary analyses were conducted in MEGA6.

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