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1 PHYLOGEOGRAPHY, INTERSPECIFIC COMPETITION, AND CONTROL OF Coptotermes formosanus AND Coptotermes gestroi ( ISOPTERA: RHINOTERMITIDAE ) IN TAIWAN By HOU FENG LI A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSIT Y OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2009
2 2009 Hou Feng Li
3 To my parents for their unconditional love and support
4 ACKNOWLE DGMENTS I sincerel y thank my mentor, Dr. Nan Yao Su for guiding me on the scientific road for the past five years He gave me so much freedom trust and financial support o n my research and helped me to become a better writer. H e deli vered great value s a nd philosophy of science which equipped me for life It is my honor to inherit his intelligent genes. I would like to express my gratitude to Dr. Rudolf Scheffrahn for teaching and supporting me on termite taxonomy research He shared his knowledge, specim ens, and reference s with me and offered many value suggestions on my work It is always nice to enjoy the beauty of termites with him. I also thank another two committee members, Dr. William H. Kern, Jr. and Dr. Samira Daroub, who delivered excellent e ntom ology and p edology courses for the e ssentia l training and reviewed this dissertation I learned how to be a good instructor by observing their instruction s I profusely thank Dr. Robin Giblin Davis, the and his research team inclu ding Dr. Natsumi Kanzaki, Dr. Weimin Ye, Dr. D o rota Porazinska and Barbra Center in help ing and encouraging me on phylogenetic research. Dr. Giblin Davis also reviewed many of my manuscripts and offered precious advice I am thank ful for the support from my colleagues I especially thank Paul Ban and Ron ald Pepin w ho prepared experimental materials for me and expertly assist ed my work It is hard to imagine how I finish ed all these works without Ronald and Paul I gi ve my gratitude to Dr. Thomas Chouvenc, my five year officemate, for his patience in listening to my idea s, discuss ing my works and offer ing s olutions to various problems. I also thank Paul Bardunias for reviewing my manuscripts and Sergio Gallo and Angelica Moncada for t heir assistance on experiments
5 I am grateful for the environment provided to me in the Fort Lauderdale Research and Education Center, and the Department of Entomology and Nematology. I especially thank Sarah Kern and Deb orah Hall for their assistance with my paper work in the past five year s, since the very first day. I thank my friends including Ericka Helmick, Dr. Jan Kanzaki, Teresa Ferreira, Brian Bahder, Dr. Seemanti Chakrabarti, and Khayalethu Ntushe lo for their support and friendship. I also thank the help from Dr. Monica Elliot, Dr. Nigel Harrison, Dr. Forrest Howard, Dr. Kimberly Moore, Mikhail Ry abin, Ian Maguire, Bill Latham, Cherie Cook, Sue Shapiro, and Veronica Woodard I am thank ful for encou ragement and assistance provided by many professors and friends from Taiwan. I especially t hank Dr. How Jing Lee who led me into the world of entomology in 1995 I thank Dr. Wen Jer Wu, Dr. Err Lieh Hsu, Dr. Yau I Chu Dr. Huang Chin Ji, Ai Chi Lin and Y a Wen Teng (National Taiwan University) for their helpful correspondence, assistance, and relaying of important historical references I also thank Dr. Yen Chiu Lan (Leader University), Jing Fu Tsai (National Chung Hsing University), and Chun Chun Chang (Kenti ng National Park Headqua r ter) Reta Chen (Rentokil Ding Sharn) for their as sistances in termite collection, and Prima Chien (Taiwan Environmental Pest Management Association) for her assistance in delivering questionnaires. Lastly, I would like to thank my mother, father brother and all my family in Taoyuan for their understanding and support of my wholehearted concentration on entomological research. The greatest thanks belong to my wife Rou Ling, for her steadfast love and continued support for my life and work along the way
6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 8 LIST OF FI GURES ................................ ................................ ................................ .......... 9 ABSTRACT ................................ ................................ ................................ ................... 10 CHAPTER 1 TOKUICHI SHIRAKI, MASAMITSU OSHIMA, AND THE DISCOVERY OF Coptotermes formosanus AND Coptotermes ge stroi IN TAIWAN .......................... 12 Introduction ................................ ................................ ................................ ............. 12 Termite Research of Shiraki and Oshima in Taiwan ................................ ............... 13 Early Life of Tokuichi Shiraki and Masamitsu Oshima ................................ ...... 13 Controversial Issues in the Identification of Coptotermes spp. in Taiwan ......... 14 Shiraki ................................ ....................... 18 Current Taxonomic Research of C. formosanus and C. gestroi .............................. 20 Coptotermes gestroi in Taiwan ................................ ................................ ......... 20 Discovery of Syntypes of C. formosanus ................................ .......................... 21 Difficulty and Solution on Identification of Copt otermes spp. ............................ 22 2 PHYLOGEOGRAPHY OF Coptotermes formosanus AND Coptotermes gestroi IN TAIWAN ................................ ................................ ................................ ............. 30 Introduction ................................ ................................ ................................ ............. 30 Materials and Methods ................................ ................................ ............................ 31 Termite S amples a nd DNA Extraction ................................ .............................. 31 PCR and P urificat ion ................................ ................................ ........................ 32 Sequencing and P hylogenetic I nferences ................................ ........................ 32 Results ................................ ................................ ................................ .................... 33 Dist ribution and D ispersal F light S eason ................................ .......................... 33 Genetic A nalysis ................................ ................................ ............................... 34 Phylogenetic A nalysis and P opulation S tructure ................................ .............. 35 Discussion ................................ ................................ ................................ .............. 36 Distribution ................................ ................................ ................................ ....... 36 Phylogeography ................................ ................................ ............................... 37 Origin of C. formosanus and C. gestroi ................................ ............................ 39
7 3 INTERSPECIFIC COMPETITION AND TERRITORY DEFENSE MECHANISMS OF Coptotermes formosanus AND Coptotermes gestroi ................................ ........ 48 Introduction ................................ ................................ ................................ ............. 48 Materials and Methods ................................ ................................ ............................ 50 Termite Species ................................ ................................ ............................... 50 Petri dish Bioassay ................................ ................................ ........................... 50 Foraging Arena Bioassay ................................ ................................ ................. 51 Results ................................ ................................ ................................ .................... 52 Petri dish Bioassay ................................ ................................ ........................... 52 Tunnel Interception and Blockage ................................ ................................ .... 53 Discussion ................................ ................................ ................................ .............. 54 Interspecific Competition ................................ ................................ .................. 54 Territory Defense Mechanism ................................ ................................ .......... 55 Territo ry Dynamic Equilibrium ................................ ................................ .......... 56 4 TERMITE PESTS AND THEIR CONTROL IN TAIWAN ................................ ......... 60 Introduction ................................ ................................ ................................ ............. 60 Materials and Methods ................................ ................................ ............................ 61 Results and Discussion ................................ ................................ ........................... 62 Questionnaire and Registered Pest Control Companies ................................ .. 62 Termite Pests and Infested Buildings ................................ ............................... 63 Termiticide ................................ ................................ ................................ ........ 64 Busine ss Revenue of Termite Control Industry ................................ ................ 65 APPENDIX A Questionnaire for quantifying termite damage and the control measures used in Taiwan ................................ ................................ ................................ .................... 68 B Translatted Questionnaire ................................ ................................ ....................... 72 LIST OF REFERENCES ................................ ................................ ............................... 74 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 82
8 LIST OF TABLES Table page 2 1 Termite species, localities, and GenBank accession numbers ........................... 41 2 2 PCR and sequencing prim ers used in the present study ................................ .... 43 2 3 Distance matrix and number of difference of nucleotides of combined mitochondrial COII, 12S rRNA, and 16S rRNA gene sequences ........................ 44 3 1 The tunnel interception sequences between C. gestroi and C. formosanus ....... 58 3 2 Tunnel blockages induced by tunnel interception of C. gestroi and C. formosanus ................................ ................................ ................................ ......... 58 4 1 Distribution of licensed PCOs and termite control cases in Taiwan .................... 66 4 2 Type, cost, and use of termiti cide in Taiwan ................................ ....................... 67
9 LIST OF FIGURES Figure page 1 1 Tokuichi Shiraki a nd Masamitsu Oshima ................................ ............................ 24 1 2 Coptotermes formosanus drawn by Oshima in 1909 ................................ .......... 25 1 3 Coptotermes spp. collection sites in early 1900s and in current study .............. 26 1 4 Soldier head capsules of Coptotermes spp ................................ ........................ 27 1 5 Syntypes of C. formosanus ................................ ................................ ................ 28 1 6 Winged imagoes of C. formosanus and C. gestroi ................................ ............. 29 2 1 The distribution of C. formosanus and C. gestroi in Taiwan and Florida ............ 45 2 2 The 10001st Bayesian tree inferred from COII gene sequences ........................ 46 2 3 The 10001st Bayesian tree inferred from combined COII, 12S rRNA, and 16S rRNA gene sequences ................................ ................................ ....................... 47 3 1 Time courses study of tunnel interceptions and consequent tunnel blockages in the foraging arena bioassay ................................ ................................ ............ 59
10 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirem ents for the Degree of Doctor of Philosophy PHYLOGEOGRAPHY, INTERSPECIFIC COMPETITION, AND CONTROL OF Coptotermes formosanus AND Coptotermes gestroi ( ISOPTERA: RHINOTERMITIDAE ) IN TAIWAN By Hou Feng Li December 2009 Chair: Nan Yao Su Major: Entomology a nd Nematology Tokuichi Shiraki and Masamitsu Oshima were the first entomologists who studied termite s in Taiwan in the early 1900s The identification of Coptotermes species in Taiwan and southern Japan was a controversial issue that involved several Japan ese entomologists as well as European authorities of termite taxonomy The key point of the debate was whether the common termite pest species in Taiwan was a new species Coptotermes formosanus Shiraki or C. gestroi (Wasmann) of southeast Asia Recent e v idence indicat ed that both species existed in Taiwan at the time Coptoterme s formosanus was found to be distributed throughout the island whereas C. gestroi was limited to the south. I speculated that entomologists involved the debate in the early 1900s h ad collected or obtained Coptotermes samples from different localities of Taiwan and Japan. These collections might include either one of t he two species or both species; hence each researcher drew different conclusions. By analyzing partial mitochondrial sequences of COII, 12S rRNA, and 16S rRNA, I found that most Taiwanese C. formosanus populations were closely related to Japanese and some Chinese populations, and that Taiwanese C. gestroi populations were most closely
11 related to those from the Philippin es and Hawaii rather than populations from Thailand, Malaysia, Singapore and Indonesia. The intraspecific variation of C. formosanus was 0.7 0.8% for three genes among seven Taiwanese populations whereas all Taiwanese C. gestroi populations had identical s equences. The results support that Taiwan may be a center of origin for C. formosanus but it is a recent introduction site for C. gestroi The distributions of the two species currently overlap in Taiwan and interspecific competition is likely to occur The results of the foraging arena bioassay supported the long held assumption that interspecific competition is an important regulatory factor in maintaining termite colonial territories. Tunnel interception of the two species resulted in minor fights and then both species quickly buried the connected tunnel at multiple locations, which prevented f urther combat and heavy losses. Termite cadavers resu lting from agonistic behavior are a stimulus for tunnel block ing and a deterrent for reopening these blockage s. I speculate that tunnel interception s would offer information o n conditions of neighboring colonies in the field If both neighboring colonies are active, the agonistic interactions resulting from tunnel interception would delineate the colonial boarder If one colony is dead or weak, the healthy colony could invade the vacated territory quickly through the existing tunnel system. Of the 17 named termite species found in Taiwan, C. formosanus and C. gestroi were the most destructive and responsible for > 87% of termite structural infestation s More than 75% of infested buildings were residential premises. The liquid termiticide, containing the active ingredient fipronil accounted for baiting accounted for T he revenue of termite control industry in 2008 was estimated as
12 CHAPTER 1 TO KUICHI SHIRAKI, MASA MITSU OSHI MA AND THE DISCOVERY OF COPTOTERMES FORMOSAN US AND COPTOTERMES GEST ROI IN TAIWAN Introduction After the First Sino Japanese War between the Qing Dynasty of China and the Meiji period of Japan, Taiwan became a Japanese territory in 1895 under Treaty of Shimonoseki till the end o f WWII in 1945. Over this fifty year period, the main economic policy of the Japanese government was to develop industry in Japan and agriculture in Taiwan. As a result of this policy, many outstanding Japanese agricultural experts began to work in Taiwan, res ulting in rapid modernization. Entomolog y was one of most important subjects in the era due to the need for crop protection, the prevention of insect borne diseases, and urban pest management. Since most of Japan has a temperate climate and termites are primarily subtropical /tropical pest s Jap anese construction techniques were not adapted for termite prevention Thus, construction built in Taiwan during the early Japanese colonial period suffered from severe termite damage. Termite control became a priority of the Governor of Taiwan in the earl y 1900s. In 1907, both Tokuichi Shiraki (1882 1970) ( Figure 1 1A) and Masamitsu Oshima (1884 1965) ( Figure 1 1B) started to work in Taiwan and became the leading figures in termite research on the island. Their major control target was known at the time co now know to be Coptotermes species. There was controversy over the identification and naming of the Taiwanese Coptotermes species between 1909 and 1912. Many influential Japanese entomologists and European termite ex perts were involved in this debate. O ne hundred years after the controversy I reviewed this historical story, clarified the
13 controversial issues based on the current study, and offer ed solutions to avoid similar problems in the future. Termite Research of Shirak i and Oshima in Taiwan Early Life of Tokuichi Shiraki and Masamitsu Oshima Tokuichi Shiraki ( Figure 1 1A) was born in Hakodate, Hokkaido, Japan on 9 March 1882 (Issiki 1971). He was the eldest of six children in a wealthy family (Ou 2006). His fathe r was a president of the Hakodate Normal School (now Hokkaido University of Education, Hakodate) and his mother was an elementary school teacher. Shiraki moved around Japan with his family as his father moved through a series of jobs. He stayed in Hakodate until he was 8 years old, and then spent the next two years in Tokyo and Kumamoto. In 1892, he moved back to Hokkaido and went to middle and high school in Sapporo. He planned to enter medical school in Tokyo after high school, but when his father passed away in 1899, he lost the financial support. Instead, Shiraki entered Sapporo Agriculture College (now Hokkaido University) with a full scholarship in 1900, and he taught math in a night school as a part time job to support his family. In college, he studi ed taxonomy of Orthoptera and Dermaptera under Shounen Matsumura (1872 1960). Matsumura was the most influential entomologist in Japan at the time. Shiraki earned his B.S. degree in July 1906 and then worked in the same coll e ge as an assistant professor fo r a year. Shiraki had to teach courses not related to entomology such as mineralogy, forest zoology and agricultural product processing, and he was dissatisfied with the appointment Meanwhile Shunji Miyao (1868 1937), chair of the Bureau of Productive In dustries, Government General of Taiwan invited him to work in Taiwan, and his mentor Matsumura recommended him to take the job. In 1907 July,
14 Shiraki began his 40 years career in Taiwan. His first position was the director of the Insect Department of the Agriculture Experimental Station, Taipei, Taiwan. The background of Masamitsu Oshima ( Figure 1 ways. Oshima was born in Sapporo, Hokkaido, Japan on 21 June 1884 (Hasegawa 1967) His father was also a well known educator who had been a professor and high school president. Oshima is also the eldest son of his family. Oshima moved around went to elementary school in Sapporo and Kyoto, middle sc hool in Nara, high school in Tokyo, and then entered Tokyo Imperial University in 1904 (Hasegawa 1967). In July 1907, his uncle, Hannpei Nagao (1865 1936), the chair of the Bureau of Civil Engineering, Government General of Taiwan, invited him to conduct t ermite inspections in Taiwan. In July 1908, Oshima earned his B.S. degree in zoology and officially started to work in the Bureau of Civil Engineering on termite control. Controversial Issues in the Identification of Coptotermes spp. in Taiwan Shiraki and Oshima cooperated on termite control and taxonomy during the first two years of their careers in Taiwan. Shiraki (1909) briefly described C. formosanus Shiraki article included desc riptions of the morphology of several castes, including the male and female imago, nymph, soldier, worker, and queen, for which no figure was offered. The authority, type locality, and type specimen of C. formosanus was not mentioned either The species na me was mentioned twice but incorrectly spelled as C a ptotermes formosanus [ sic ] and Coptotermes formosanus [ sic ]. In the same year, based on morphological characteristics of C. formosanus This included drawings of the dealate,
15 soldier, worker, fore and hind wings ( Figure 1 2), and pictures of nests. Twelve collection locations in Taiwan and the Penghu islands were also described ( Figure 1 3A). Oshima vividly and comprehensively described damage caused by C. formosanus its tunneling behavior, and soldier defensive behaviors such as head banging and secreting from the fontanel le In the following year, Oshima (1910a) questioned the validity of several termite species named by Shir aki (1909), including C. formosanus Oshima (1910a) emphasized that there was no personal animosity between Shiraki and himself, and they worked together and shared references, books, and termite samples with each other. He had no wish to offend Shiraki, b ut he believed that the correct identification of termite species was a key requirement for their control. Oshima mentioned that the soldiers of C. formosanus superficially resembled those of C. gestroi described by Haviland in 1898 (Oshima 1910b, 1911). O shima did not compare the imagoes of these two species, probably due to absence of imago description of C. gestroi (Wasmann 1896, Haviland 1898). Since Taiwan was geographically close to s outheast Asia where C. gestroi occur r ed and no significant morpholo gical difference was found, Oshima proposed that C. formosanus was a junior synonym of C. gestroi Oshima also provided 12 collection locations of C. gestroi/ formosanus in Taiwan and the Penghu islands (Oshima 1911) ( Figure 1 3A). In addition to Shiraki a nd Oshima, several other Japanese researchers such as Munemoto Yano (1883 1970) of Forestry Experimental Station, Tokyo, Shozaburo Watas (1862 1929) and his student, Sanji Hozawa (1885 1947) of the Zoological Institute, Science College, Tokyo Imperial Uni versity also became involved in
16 identification of Coptotermes spp. of Taiwan and southern Japan. Yano (1911) mentioned that Watas sent Japanese specimens to a German termite taxonomist, Eric Wasmann (1859 1931) for identification. Wasmann believed that th ese samples were C. gestroi which he named in 1896 opinion. Yano mentioned that the description of C. gestroi by Wasmann was too vague to differentiate it from other Coptotermes spp. Hence, Yano further c ompared Japanese/ Taiwanese Coptotermes samples with the description of C. gestroi of Haviland (1898). He thought the two species were similar, but that their soldiers could be distinguished by the ratio of head width and length. The Japanese/ Taiwanese so ldier samples had elongated heads (1.5 mm in length and 1.2 mm in width) while C. gestroi as described by Haviland (1898) had more circular heads (1.4 mm in length and 1.3 mm in width). Additionally, Yano sent the Japanese and Taiwanese Coptotermes samples to a Swedish termite taxonomist, Nils Holmgren (1877 1954) through Chiyomatsu Ishikawa (1861 1935) of Tokyo Imperial University and another German termite researcher, Karl Escherich (1871 spec ies, Coptotermes formosae Holmgren (Holmgren 1911) and compared it with other Coptotermes measurements, Yano concluded that the Coptotermes sp. collected in Japan and Taiwan was not C. gestroi Yan nomenclature, C. formosae, because Holmgren was a recognized authority on termite C. formosanus (Shiraki 1909) in which no figures were offered, the format was informal, and the description was in
17 C. formosanus based on the publication format and language used for description instead of the rules of zoological nome nclature and the content of the work. Oshima listed the original description of C. formosae (Holmgren 1911) in The Third Official Report on Termites (Oshima 1912) to show that Holmgren only offered five measurements of alates which was less informati ve tha n the first description of C. formosanus (Shiraki 19 09 ), and no picture was offered by Holmgren either. Oshima (1912) was torn between the conflicting opinions of the two internationally recognized authorities on termite taxonomy, Wasmann and Holmgren. He agreed with a there was a difference between Japanese / Taiwanese samples and C. gestroi in the ratio of head w idth and head length of solider. However, he thought it was insufficient to name a new species (herein C. form osanus ) because the difference might be due to the intraspecific variation. This was the major reason why he doubted the validity of C. formosanus (Oshima 1910b and 1911). To solve this controversial issue, Oshima collected more samples and measured head length and width with a more accurate method based on a clear definition. He had seven soldiers from two locations in southern Japan and 11 soldiers from three locations in Taipei, Taiwan (Oshima 1912, Figure 1 3A). He removed the soldier heads and placed them on a glass plate in a natural position, and measured the distance between medium base of the labrum to hindmost margin of the head capsule. The data was rounded to two decimal places. Oshim a found the head length (1.66 1.72 mm) of Japanese/ Taiwanese were much longer than that of C. gestroi (1.4 mm ) as described by Haviland 1898 No significant difference was found between Japanese and Taiwanese samples (Taipei only), and the variation of these samples in head length
18 was only 0.03 mm. Based on the new measurements, Oshima confidently believed Coptotermes samples collected in Japan and Taiwan were not C. gestroi It is worthy of note that the head length of soldiers reported by Oshima in 1910b and 1911 was 1.5 mm but it increased to 1.66 1.72 mm when he made another measurement in 1912. Oshima attrib uted the difference to different measuring methods. In addition, Oshima thought that the alate of C. formosae described by Holmgren (1911) was similar to C. formosanus described by Shiraki (1909). Since C. for mosanus was described first, C. formosae should be a junior synonymy. After The Third Official Report on Termites was published by Oshima in 1912, most termite researchers such as Holmgren (1913), Watas, and Hozawa (1915) accpt ed his opinion that C. formo sanus as the only Coptotermes sp in southern Ja pan and Taiwan. Shiraki s C areers in Taiwan Although Shiraki only conducted termite research during his first 5 years in Taiwan (1907 1912), six of 17 currently named termite species in Taiwan we re described by him. These include: C. formosanus O dontotermes formosanus (Shiraki) Pericapritermes nitobei (Shiraki), Neotermes koshunensis (Shiraki) (Shiraki 1909), Nasutitermes parvonasutus (Shiraki), and Na. takasagoensis (Shiraki) (Nawa 1911). Shira ki himself mentioned that a failure in the maintenance of termite colonies in his laboratory caused him to cease further experiments on termites (Ou 2006). Following on termites, he focused his studies on agricultural pests. In 1909, Shi raki introduced Vedalia beetles, Rodolia cardinalis (Mulsant), a natural enemy of cottony cushion scales, Icerya purchasi Maskell, into Taiwan for biological control, and he achieved success within three year s (Chu 2005). He earned his Ph. D. degree from Ho kkaido Imperial University in 1917 based on the research of a rice pest, the yellow
19 stem borer, Scirpophaga incertulas (Walker), in Taiwan. Shiraki worked in Taiwan for almost 40 years until 1947. He served as director of the Applied Zoology Division (prev iously the Insect Department) in the Agriculture Research Institute (previously the Agricultural Experimental Station) for 35 years (1907 1942). Shiraki was also involved in the establishment of the Plant Quarantine Station, Taiwan Governor Museum, and the curriculums of Entomology and Sericulture, Taihoku Imperial University. He was a founder of the Society of Natural Sciences and the Society of Entomology in Taiwan and served it s president for several years. Shiraki was the leading figure in the developme nt of Entomology in Taiwan (Chu 2005) Oshima focused on termite research for around ten years (1907 1917) and served as an official in the Civil Engineering Bureau and then the chair of the Zoology Department, Institute of Science Oshima published over 2 0 articles related to termites in his life time and described over 40 termite species. To date, four Taiwanese termite species were named by Oshima, including Sinocapritermes mushae (Oshi m a a nd Maki), R eticulitermes flaviceps (Oshima) Glyptotermes fuscus ( Oshima) and Incisitermes inamurae (Oshima). Oshima did a comprehensive termite biology study in Taiwan including taxonomy, ecology, and control which was published in six official reports, as well as the Philippine Journal of Science, and Japanese zoologic al magazines. His research started with Japanese and Taiwanese termites, and then extended to termite fauna of Hong Kong, Singapore, and the Philippines, where he named 13 Philippine termite species ( Snyder and Francia 1960). A bout 1917, Oshima changed his research subject to vertebrates including fishes, birds, and snakes. He earned his Ph. D. degree on the taxonomy of freshwater fishes of Taiwan in 1920 and returned to Japan in 1924.
20 Current Taxonomic Research of C. formosanus and C. gestroi Coptotermes ge stroi in Taiwan After 1912, C. formosanus was believed the only Coptotermes species in Taiwan. Coptotermes gestroi in Taiwan had not been mentioned until 2003. Based on soldier morphology, the new record of C. gestroi in Taiwan was proposed again (Tsai and Chen 2003). During 2005 and 200 9 Coptotermes spp. samples were collected by the author and local pest control operators from every county in Taiwan both in urban areas and natural environments. Li et al. (2009 a ) confirmed that C. gestroi is present in so uthern Taiwan by using mitochondrial gene sequences and morphology In total, 220 Coptotermes samples including museum specimens were identified and used for mapping their distribution ( Figure 1 3B). Coptotermes spp. were mostly collected in lowland area ( < 500m). Coptotermes formosanus was distributed throughout th e island of Taiwan, while C. gestroi was only collected in the tropical zone. Oshima (1909, 1911) collected Coptotermes samples at 12 places in Taiwan ( Figure 1 3A), and seven of them were in the tropical zone where both C. gestroi and C. formosanus can be currently collected. The drawing of a dark brownish dealate ( Figure 1 2A, Oshima 1909) resembled C. gestroi instead of a light brown yellowish dealate of C. formosanus The drawing of soldier he ad capsule ( Figure 1 2B and Figure 1 4A, Oshima 1909) was somewhat rounded and also similar to C. gestroi ( Figure 1 4C) and its head length was recorded as 1.5 mm. Three years later in 1912 Oshima collected Coptotermes samples only in northern Taiwan (Tai pei city) ( Figure 1 3A) and in Japan, where only C. formosanus is currently found. The image of soldier head capsule ( Figure 1 4B, Oshima 1912) was elongated and similar to C. formosanus ( Figure 1 4D). The head length s of soldier s reported by Osh ima was 1. 66 1.72 mm in 1912. Soldiers
21 collected in 1909 and 1911 might include both C. formosanus and C. gestroi ; hence the average of their head length was shorter than that of C. formosanus collected only from Taipei in 1912. Oshima (1912) emphasized twice there was only one Coptotermes sp. in Taiwan. However, no effort was made to prove all the Coptotermes samples in Taiwan were the same species (Oshima 1909, 1910b, 1911). Based on this false assumption, Coptotermes samples collected at many places in Taiwan wer e lumped together and represented each morphological measurement as a mean of the two species. When Holmgren and Wasmann identified Japanese/ Taiwanese Coptotermes samples as C. formosae and C. gestroi respectively (Yano 1911), Oshima and Yano were confus ed but they did not suspect there might be two Coptotermes spp. in Taiwan. Shiraki, Oshima, Yano (Oshima 1909, 1910b, 1911, 1912; Shiraki 1909, Yano 1911) drew their conclusions based on termite samples collected in different places in Taiwan and Japan, wh ich may be the source of the controversy in early 1900s. Discovery of Syntypes of C. formosanus In order to investigate the controversial issue caused by identification of C. formosanus and C. gestroi in the early 1900s, the museum specimens preserved in s ix major insect collections in Taiwan were examined to search for the collect ions used by Shiraki and Oshima: Taiwan Agriculture Research Institute Insect and Mite Collection Wufeng, Taichung Taiwan, ROC (TARI) Taiwan Forestry Research Institute, Insect Collection, Taipei Taiwan, ROC (TFRI) National Museum of Natural Science, Taichung Taiwan, ROC National Taiwan University, Department of Entomology Insect Collection, Taipei Taiwan, ROC
22 National Chung Hsing University, Department of Entomology, Insect Co llection, Taichung, Taiwan, ROC National Pingtung University of Science and Technology, Plant Protection Department Insect Collection Neipu, Pingtung Taiwan, ROC A set of specimens ( Figure 1 Coptotermes formosanus as found in T ARI where Shiraki worked between 1907 and 1942. The autograph on the label ( Figure 1 ( Figure 1 5C) offered by Wen Jer Wu (National Taiwan University). Termites soldiers preserved in vials were identified as C. formosanus with two setae on each side of fontanelle ( Figure 1 5D) (Scheffrahn and Su 1990) but no collection information of these soldiers was found. Three alate samples were labeled, but all of them were damaged to some degre e. The best preserved sample ( Figure 1 5E) was collected on June 23 rd 41 th unknown to m e The other two severely damaged samples were collected by Inao Nitobe (1883 tant, on April 23 rd 41 th year of Meiji era (1908) from Pingtung county, Gangkou research station of TFRI ( Figure 1 3A). One of Nit surviving samples has only wings, and the other one only head and thorax remained, wh ich were not sufficient to be iden tified to species Shiraki might have used these samples collected in 1908 for description of C. formosanus in 1909. Hence, these collections should be assigned as syntypes. There is no doubt that Shiraki obtained some real C. formosanus samples for his in itial description and naming of C. formosanus Difficulty and Solution on Identification of Coptotermes spp. Soldiers and winged imagoes are the two main castes used for termite identification and taxonomy. Soldie rs of Coptotermes spp. can be collected year round
23 but they are superficially similar. To differentiate the soldiers of C. gestroi and C. formosanus required a microscopic examination. Coptotermes formosanus soldiers have two pairs of setae near the rim of the fontanelle, while in C. gestroi one pair originated around the fontanelle. Imagoes offer more differentiable characters for species identification The dark brownish head, pronotum, and dorsal abdomen of C. gestroi gives it a much darker appearance t han C. formosanus in general ( Scheffrahn and Su 2007 ) The winged imagoes of C. formosanus are larger than those of C. gestroi (Figure 1 6) However, imagoes are only present in mature colonies seasonally for a short period of time. Even in the dispersal f light sea s on, imagoes only dwell in specific area s of nests. Rarely were both imagoes and soldiers collected from the same colony; hence intraspecific imagoes and soldiers mi ght be described as two species such as C. gestroi and C. havilandi Holmgren (Ki rton and Brown 2003). The first description of C. formosanus (Shiraki 1909) was based on some of C. gestroi collected in tropical Taiwan. In addition, t he syntypes of C. formosanus preserved in TARI were damaged to s ome degree and its collection information was incomplete. To prevent any further confusion in identification of C. formosanus it would be better to re describe C. formosanus based on both soldier and winged imago morphology, and included genetic sequence data for comparison. To assign alates and soldier castes collected from the same colony at northern Taiwan for a neotype and neoparatypes would be a practical method to prevent mismatching castes of C. formosanus with that of other Coptotermes spp.
24 Figu re 1 1. Tokuichi Shiraki (1882 1970) (A) ( Photo provided by the Entomology Dept of the National Taiwan University ) ; Masamitsu Oshima (1884 1965) (B) (Photo provided by the Entomological Society of Japan )
25 Figure 1 2. Coptotermes formosanus drawn by O shima in 1909 provided by the National Taiwan University Library Dealate (A); soldier (B); worker (C); front (D) and hind (E) wings
2 6 Figure 1 3. Coptotermes spp. collection sites in early 1900s (A) and in current study (B). White area, altitude > 50 0 m; light gray area, subtropical lowland; dark gray area, tropical lowlan d.
27 Figure 1 4 Soldier head capsules of Coptotermes sp p A solider collected in Taiwan by Oshima in 1909 resembled C. gestroi (A) (Photo provided by the National Taiwan Universit y Library) ; C. formosanus collected in Taipei or southern Japan by Oshima in 1912 ( Photo provided by the Entomology Dept. of the National Taiwan University ) (B); SEM pictures of C. gestroi (C) and C. formosanus (D) collected in southern and northern Taiwan respectively, in current study.
28 Figure 1 5 Syntypes of C. formosanus C. formosanus collect ion preserved in Taiwan Agriculture Research Institute (A); the autography of the label (B) anuscript (C); soldiers preserved in vials (D) with two setae (inset) on one side of fontanelle ; the most well preserved C. formosanus imago of the syntypes collected in 1908 (E). 1, label; 2, soldiers preserved in vials; 3, the best preserved imago; 4, im ago collected by Nitob e, only wings rema ining; 5, imago collected by Nit o b e, head and thorax remaining.
29 Figure 1 6 Winged i magoes of C. formosanus (A) and C. gestroi (B)
30 CHAPTER 2 PHYLOGEOGRAPHY OF COPTOTERMES FORMOSAN US AND COPTOTERMES GESTROI IN T AIWAN Introduction Coptotermes formosanus is a widely distributed pest species and found in several subtropical and temperate areas including southern China, Taiwan, Japan, Hawaii, the southern United States, and South Africa (Su 2003). Coptotermes gestro i is another important structural pest, but it is primarily found in tropical regions. The distribution of C. gestroi extends from s outheast Asia (Assam of India, Myanmar, Laos, Cambodia, Vietnam, Thailand, Peninsular Malaysia, Singapore, and the Indonesia n archipelago) through the Philippines, Taiwan, and Hawaii, to the New World including Florida, West Indies, Mexico, and Brazil (Wasmann 1896, Light 1929, Kirton and Brown 2003, Ferraz and Mndez Montiel 2004, Yeap et al. 2007). The three regions of distr ibutional overlap of these two species are Florida (Scheffrahn and Su 2005), Taiwan (Shiraki 1909, Tsai and Chen 2003), and Hawaii (Swe ze y 1914, Weesner 1965). Several studies of the origin and dispersal rout e s of C. formosanus and C. gestroi have been co nducted by using multiple methods. Studies on termitophilous beetles s uggest ed that C. formosanus is endemic to southern China (Kistner 1985) and some islands of southern Japan (Maruyama and Iwata 2002, Tsunoda 2006). Genetic studies by using mitochondrial COII gene sequences (Austin et al. 2006) indicated C. formosanus populations in the United State were introduced from China and Japan. Research on population genetic structure (Vargo et al 2003) by using microsatellite marker s indicated that C. formosanu s has been established in Japan for more than 300 years. With the same method, Vargo et al. (2006) concluded that C. formosanus invaded the United States mainland at least from two different sources. Jenkins et al.
31 (2007) u s ed the mitochondrial COII and 16 S gene sequences and revealed that C. gestroi collected in Ohio and Florida are closely related to populations from Singapore and Malaysia, respectively. These previous stud ies mainly focused on populations in the United States China, and Japan. Few sampl es were collected from the historically and geographically important locality of Taiwan. Taiwan is the type locality of C. formosanus (Shiraki 1909). The existence of C. gestroi in Taiwan had been suspected by Oshima (1911, 1912), but was only recently co nfirmed by Tsai and Ch en (2003). The objectives of this study were to examine the geographic distribution of the two species in the subtropical island of Taiwan and to investigate the phylogeographic relationship between Taiwanese populations and other dis tant populations. Material s and Methods Termite S amples a nd DNA Extraction During 2005 and 2007, Coptotermes spp. samples were collected from every county in Taiwan at urban areas and natural environments including several ecological reserves and national parks. Specimens were identified to species by using soldier or alate morphology. For phylogenetic study, 23 Coptotermes samples including 14 samples from Taiwan, five samples from Hainan Island, China, and four samples from Florida, United States, were id entified first by using morphological characters (Shiraki 1909, Oshima 1911, 1912; Light 1929, Tsai and Chen 2003). These samples were preserved in > 95% ethanol before DNA extraction (Table 2 1). The voucher specimens are deposited in the University of Fl orida Termite Collection, Fort Lauderdale Research and Education Center. Total genomic DNA from the same colony was extracted from three individual termites by using a DNeasy Tissue Kit (Qiagen Inc., Valencia, CA),
32 d stored at 20 C before polymerase chain reaction (PCR) attempts PCR and P urification Primers for mitochondrial COII, 12S, and 16S, and references are listed in Table 2 2. The 50 l PCR mixture (with final concentrations) contained 35 l water, Taq DNA polymerase incubation buffer (1X), dNTP mixture (0.2 mM), forward and reverse primers (0.2 M each) 2 l of termite DNA template, and 1.25 U of AmpliTaq Gold (Applied Biosystems, Foster City, CA). The thermal cycling program for all PCR was as follows: a precycle denaturation at 95 C for 5 min, followed by 35 cycles of denaturation at 95 C for 1 min, annealing at 55 C for 1 min, and extension at 72 C for 2 min, and a postcycle extension at 72 C for 10 min. The PCR products were cleaned by using Montag e PCR centrifugal filter devices (Millipore B illerica MA), following the Sequencing and P hylogenetic I nferences PCR products were sequenced in both directions at the University of Florida DNA Sequencing Core Laboratory using ABI Prism Big Dye Terminator cycle sequencing protocols developed by Applied Biosystems ( part number 4303153 Perkin Elmer, Foster City, CA) The sequences in this study were submitt ed to the GenBank database and accession numbers are shown in Table 2 1. Seque nces from other studies including Yeap et al. (2007), Fang et al. (2008), and Tsai (2003) were downloaded from GenBank and added for phylogeny analysis. T wo R flaviceps samples from Lanyu Island, Taiwan were included as the outgroup taxa (Li et al. 2008) (Table 2 1). Base compositional analyses were conducted using a computer program MEGA version 4.0.1 ( Tamura et al. 200 7 ). The pairwise analyses of base substitutions per site were conducted by using the
33 p distance model and the number of differences model in MEGA (Tamura et al. 2004, Tamura et al. 2007). DNA sequences were aligned by using Clustal W (Thompson et al. 1994). A c hi square test was performed in PAUP* 4.0b10 (Swofford 2002) to check for homogeneity in base frequencies. The model of base substitut ion in the COII, 12S rRNA, 16S rRNA genes sequences and combined data sets was evaluated using MODELTEST 3.7 (Posada and Crandall 1998). The Akaike supported model, the base frequencies, the proportion of invariable sites and the gamma distribution shape parameters and substitution rates were used in phylogenetic analyses. The t ree topology of each gene and their combin ed data set was performed by using Bayesian analysis ( MrBayes 3.1. 2, Huelsenbeck and Ronquist 2001) running the chain for 1 ,000,000 generat ions and setting the burnin at 1,000 after checking the saturation curve. The Markov Chain Monte Carlo method within a Bayesian framework was used to estimate the posterior probabilities of the phylogenetic trees (Larget and Simon 1999) using 50% majority rule. Sites with missing data or gaps were treated as missing characters for all analyses. The first second and third codon positions were partitioned for COII protein coding gene. Results Distribution and D ispersal F light S eason Thirty five C. formosanu s samples and seven C. gestroi samples were collected from 32 locations in Taiwan. Their distribution was mapped based on the combined data of current study, Tsai (2003), and Tu (1955) ( Figure 2 1A). Coptotermes formosanus is distributed throughout th e isl and of Taiwan, and C. gestroi was found only in the south west ern part of the island ( Figure 2 1A) Coptotermes formosanus was mostly found in man made structures, but C. gestroi was found in both man made structures
34 and natural wood sources such as tree st umps, dead branches, and dead tree trunks. Neither species was found in the mountainous area (> 500m) of Taiwan. A d ispersal flight of several C. gestroi alates (TW8) in Chiayi City w as observed on May 22, 2006 6 :00 PM, which is the first record of C. gestroi alates in Taiwan. During dusk the next day numerous C. formosanus alates swarmed at the same location. This observation s uggest s an overlap of the dispersal flight season s of the C. formosanus and C. gestr oi in Taiwan. Genetic A nalysis Partial fragments consisting of 660 663 bp of COII, 358 362 bp of 16S rRNA, and 396 401 bp of 12S rRNA were sequenced for the 23 samples obtained in the curre nt study In addition, 17 samples from Yeap et al. (2007), seven sa mples from Fang et al. (2008), and one sample from Tsai (2003) were obtained from Gen B ank and included for phylogenetic analysis (Table 2 1). All three mitochondrial genes of all ingroup taxa were A + T rich (62.9 1 % in COII, 65.93 % in 12S and 65.5% in 16S ) and had a base use with Among the three gene fragments, COII was the most variable (78.1% constant characters) and informative (19.3% informative sites) wh ereas 16S was the most conserved ( 89.9% constant characters ) and the least infor mative (7.9% informative sites) Based on Chi square tests for base frequency homogeneity among taxa, the base frequency distribution of the three gene fragments and their combined data set were homogenous ( P = 1.0). Sequence divergence of combined COII, 1 2S rRNA, and 16S rRNA genes varied from 5.2 to 5.8 % (73 82 bp) between C. gestroi and C. formosanus populations, and from 13.2 to 14.2 % (186 200 bp) between Coptotermes spp. and R. flaviceps (Table 2 3). Intraspecies variation of C. gestroi ranged from 0 to 1% (0 14 bp), and ranged from 0 to 0.3 % (0 4 bp) for C. formosanus
35 except for TW55 which was 0.7 0.8 % (10 11 bp) different from all of the other C. formosanus samples in this study. In Taiwan, t he seven C. gestroi s amples ha d identical COII, 1 2 S rRN A and 1 6 S rRNA sequences The COII gene sequences of Taiwanese C. gestroi in this study were also identical with the sample collected in Taiwan by Tsai (2003) (AY295078). Among the seven populations of C. formosanus distributed throughout Taiwan, the gene tic diversity ranged from 0 1.2% in COII, 0 0.7% in 16S, but was identical for 12S. T wo different DNA fragments were amplified (>70 bp, 10% difference) for each of eight C. gestroi samples from Taiwan and Florida by using two pairs of COII gene primers, A tLeu and B tLys (Jenkins et al. 2007), and C2F2 and B tLys (Yeap et al. 2007) (Table 2 2) using the same PCR conditions. The sequence amplified by using A tLeu and B tLys primers has a single nucleotide deletion relative to the sequence obtained using C2F 2 and B tLys primers. After comparing the two sequences with other COII sequences of C. gestroi deposited in Gen B ank, it was considered that the sequence with a deletion nucleotide was a pseudogene and not used in tree construction. However DNA fragments of the 15 C. formosanus samples in this study amplified by using the two pairs of COII primers were not different. Phylogenetic A nalysis and P opulation S tructure Bayesian trees showed the phylogenetic relationships inferred from COII ( Figure 2 2) and combi nation of COII, 12S rRNA, and 16S rRNA ( Figure 2 3). The 12S and 16S trees can be viewed at http://flrec.ifas.ufl.edu/su/hou feng li.shtml Using R. flaviceps as the outgroups, C. gestroi and C formosanus were well separated in two distinct clades. Among C. gestroi populations, t he COII and 12S rRNA trees placed Indonesian into a
36 distinct clade with support of 100% (COII) and 85% (12S). The 16S rRNA gene tree offered few clues to population str ucture of these C. gestroi populations. The tree generated from three mitochondrial gene sequences combined ( Figure 2 3) revealed that the 21 samples of C. gestroi could be divided into three geographical groups : g roup I: Taiwan, the Philippines, and Hawai i populations (support of 88%); g roup II: Thailand, Malaysia, and Singapore population (support of 65%); g roup III: Indonesian populations (support of 98%). The Florida sample was inferred to be close to g roup II. Among C. formosanus populations, there are more samples available in COII, making it more informative for investigating population structure with this gene than 12S rRNA and 16S rRNA trees. The COII tree supported that Taiwanese, Japanese, and some Chinese samples (haplotype B and G) were grouped into a clade (support of 96%). Samples from Florida, Hawaii, and China (haplotype D) were separated into another clade (support of 83%). The Chinese haplotype A, C, E, and F and samples from Hainan Island were not resolved. There was only 0 3 bp difference s (< 0.5%) between the 26 COII sequences ( Figure 2 2) except for TW55 collected from southeastern Taiwan (6 8 bp, 1% difference). Although TW55 was separated into another clade in the 16S rRNA tree, it was not separated from the others in COII and 12S rRNA trees, and no morphological differences were found between TW55 and other samples. Discussion Distribution Among East Asian archipelagos, C. formosanus has been reported from the southern part of Japan including southern Honshu ( 35 N), Shikoku, and Kyush u,
37 through Ryukyu islands (Mori 1987) to the southern tip of Taiwan ( 22 N) (Tu 1955, Tsai 2003). Coptotermes gestroi has been reported from the middle of Taiwan (Taichung City) ( 24 N) (Tsai and Chen 2003) through the Philippines to the Java islands ( 8 S ) (Yeap et al. 2007). The zone of overlap of the two species lies between 22 and 24 N in Taiwan ( Figure 2 1A). A similar distribution pattern of the two species in America has also been reported. Coptotermes formosanus has been reported from North Carolina ( 35 N) (Su 2003) to Florida City, Florida ( 25.5 N) (Scheffrahn and Su 2005). Coptotermes gestroi has been reported from Riviera Beach, Florida ( 27 N) (Scheffrahn and Su 2007) to the Caribbean islands such as Little Cayman Island, Turks and Caicos Islan ds (Scheffrahn and Su 1990, Su et al. 2000). The overlapping area of the two species lies between 25.5 and 27 N in Florida ( Figure 2 1B). Low temperature is believed to be a limiting factor for termite activity (Sponsler and Appel 1991, Fei and Henderson 2 002). Even though Florida is further north than Taiwan, the average January temperature of the two overlapping zones are similar ( Figure 2 1) (Henry et al. 1994, Lee et al. 199 7 ) because of the warming effects of the Gulf Stream that passes costal southeas tern Florida. The average temperature of the overlapping zone ranged from 14 to 20 C in January. Neither C. formosanus nor C. gestroi have so far been found in the central mountainous area of Taiwan and the middle wetland area of south Florida, which sugge sts the distribution of the two species is also limited by geography. Phylogeography Before early 1900s, the reported distribution of C. formosanus was restricted to China, Taiwan, and Japan (Su 2003). Most Taiwanese are immigrants from south
38 China, and fr equent shipping between China and Taiwan has been recorded since the Ming Dynasty (1600s) (Su 1986). Japan has imported many agricultural products such as sugar cane, rice, tea, and logs from Taiwan since the late 1800s (Su 1986). The frequent transportati on of infested materials between these three areas may have increased the gene flow among C. formosanus populations. Genetic data in this study support that most Taiwanese populations of C. formosanus are closely related to Japanese and some Chinese popula tions which is consistent with human mediated movement of these termites. In contrast to C. formosanus which showed a high level of divergence within Taiwan, the seven C. gestroi colonies distributed over 170 km in southwest Taiwan had identical sequences of COII, 12S rRNA, and 16S rRNA. Results suggest that the C. gestroi invaded Taiwan recently from a limited or point source. I hypothesize that the Taiwanese population of C. gestroi was introduced from the Philippines because it was closest to the Philip pine populations both geographically and genetically. The frequent shipping between Taiwan and the Philippines could be traced back to the 17th century when Spanish colonized in both areas and Dutch colonized in southern Taiwan (Andrade 2008). The tendency of C. gestroi to infest boats and ships may have contribute d to its dispersi on (Scheffrahn and Su 2005). Jenkins et al. (2007) did not include Philippine and Hawaiian samples for determining the source of Taiwanese C. gestroi partially because that the P hilippine and Hawaiian populations were thought to be another species, C. vastator Light, which was proved to be a junior synonymy to C. gestroi recently (Yeap et al. 2007).
39 When Light (1929) described C. vastator (= C. gestroi ) in the Philippines, he repo rted C. gestroi was already a major pest to manmade structures there. However, C. gestroi was not recorded in Hawaii until 1963 (Weesner 1965). In this study, the Hawaiian populations of C. gestroi were also closely related to the Philippine populations. B ased on the historical records, geographic distance, and genetic data, I speculate the C. gestroi in Hawaii originated from the Philippines. Guam is located at midway among Taiw an, the Philippines, and Hawaii ; hence, although the genetic data was not avail able for the present study, the C. gestroi (= C. havilandi ) populations in Guam (Su and Scheffrahn 1998 a ) may be also close to those from these three areas. Origin of C. formosanus and C. gestroi Southern China is considered the origin of C. formosanus bec ause of high species diversity of Coptotermes (24 species) (Li 2000) and the association of termitophil ous beetles in C. formosanus nests (Kistner 1985). Populations of endemic species generally have higher genetic diversity in the center of origin than in troduced areas, and this principle is applicable to termites (Tsutsui et al. 2000, Austin et al. 2006). However, Fang et al. (2008) analyzed COII gene sequences of 35 C. formosanus colonies from six provinces in China and reported a low level of genetic va riation (0 0.5%) compared with other termite species in the same area. Fang et al. (2008) considered that the low level of genetic variation was caused by frequent human activity and also pointed out that higher genetic diversity was found in areas with lo w transportation development in China such as Guangxi province. Coptotermes formosanus is highly adapted to the urban environment, and has been dispersed by railway and ships (Austin et al. 2008, Jenkins et al. 2002, Scheffrahn and Su 2005) through 10 sout heastern U.S. states in the past 50 years (Su 2003). The rapid expansion suggests a possibility that some of
40 the 12 provinces in China may have been infested relatively recently through human activity. Taiwan and mainland China were connected during the Pl eistocene (Ota 1998, Voris 2000), and high genetic variation of Taiwanese populations found in this study suggests that C. formosanus could be endemic to Taiwan. In the curr ent study, the COII gene variation among Taiwanese populations (0 1.2%) was higher than that among Chinese populations (0 0.5%) due to sample TW55, which was collected in southeast Taiwan from an isolated area surrounded by mountains ( Figure 2 1A). The 16S rRNA gene sequences of TW55 also showed difference from other samples to a certain degree (0.7 1.0%). More C. formosanus samples collected from east Taiwan for genetic analysis could further confirm the high genetic variation among Taiwanese populations. Coptotermes gestroi populations possesses higher genetic variation among several ge ographic areas than C. formosanus (Table 2 3), which supports the hypothesis that southeast Asian countries including the Philippines, Malaysia, Singapore, and Indonesia are the center of origin for C. gestroi Obtaining genetic sequences from samples coll ected in other southeast Asian countries, such as Vietnam, Laos, Cambodia, and the type locality, Myanmar, will be helpful to infer the specific origin. In conclusion, C. formosanus was found to be distributed throughout Taiwan whereas C. gestroi was limi ted to the south. The zone of overlap for the two species was between 22 and 24 N with average temperatures of 14 20 C in January. The genetic data support that Taiwan is one of the endemic areas for C. formosanus but C. gestroi is an introduced species f rom the Philippines.
41 Table 2 1. Termite species, localities, and GenBank accession numbers Species Code Location GenBank Accession No. COII a 12S 16S Samples from this study C. gestroi TW8 Taiwan Chiayi C ity, West District EU805750 EU805704 EU805727 C. gestroi TW11 Taiwan Chiay i C ity, East District EU805751 EU805705 EU805728 C. gestroi TW19 Taiwan, Pingtung County Pingtung C ity EU805752 EU805706 EU805729 C. gestroi TW21 Taiwan, Pingtung County Hengchun Township EU805753 EU805707 EU80573 0 C. gestroi TW24 Taiwan Tainan C ity, East District EU805754 EU805708 EU805731 C. gestroi TW29 Taiwan Tainan C ity, North District EU805755 EU805709 EU805732 C. gestroi TW30 Taiwan Tainan C ity, North District EU805756 EU805710 EU805733 C. gestroi FLC G U.S.A., Florida, Key West EU805757 EU805711 EU805734 C. formosanus TW49 Taiwan, Taoyuan County, Taoyuan City EU805758 EU805712 EU805735 C. formosanus TW50 Taiwan, Taichung City, Situn District EU805759 EU805713 EU805736 C. formosanus TW51 Taiwan, Tain an City, South District EU805760 EU805714 EU805737 C. formosanus TW52 Taiwan, Yilan County, Nan ao Township EU805761 EU805715 EU805738 C. formosanus TW53 Taiwan, Yilan County, Nan ao Township EU805762 EU805716 EU805739 C. formosanus TW54 Taiwan, Hualien County, Hualien City EU805763 EU805717 EU805740 C. formosanus TW55 Taiwan, Taitung County, Taitung City EU805764 EU805718 EU805741 C. formosanus H1 China, Hainan Province, Sanya City EU805765 EU805719 EU805742 C. formosanus H2 China, Hainan Province, S anya City EU805766 EU805720 EU805743 C. formosanus H3 China, Hainan Province, Sanya City EU805767 EU805721 EU805744 C. formosanus H4 China, Hainan Province, Qionghai City EU805768 EU805722 EU805745 C. formosanus H5 China, Hainan Province, Qionghai City EU805769 EU805723 EU805746 C. formosanus WBBR U.S.A., Hallandale Beach, Broward County, Florida EU805770 EU805724 EU805747 C. formosanus LNDN U.S.A., Hallandale Beach, Broward County, Florida EU805771 EU805725 EU805748 C. formosanus 437GI U.S.A., Hallan dale Beach, Broward County, Florida EU805772 EU805726 EU805749 Yeap et al. 2007 C. gestroi CG1MY Malaysia, Penang EF379945 EF379982 EF379963 C. gestroi CG4MY Malaysia, Kuala Lumpur EF379951 EF379987 EF379969 C. gestroi CG5MY Malaysia, Mua r EF379952 EF379988 EF379970 C. gestroi CG1SG Singapore, Serenity Terr. EF379946 EF379983 EF379964 C. gestroi CG2SG Singapore, Serangoon EF379949 EF379985 EF379967 C. gestroi CG1TH Thailand, Bangkok EF379947 EF379977 EF379965 C. gestroi CG2TH Tha iland, Bangkok EF379950 EF379986 EF379968
42 a COII gene sequences from Gen B ank used for analysis include seven haplotypes (A G) of C. formosanus fr om mainland China, EF056702, EF056705, EF056706, EF056709, EF056714, EF056729, and EF056738 (Fang et al. 2008), and one C. gestroi sample, AY295078, from Taiwan (Tsai 2003). C. gestroi CG1IN Indonesia Cibinong EF379944 EF379981 EF379962 C. gestroi CG2IN Indonesia Bogor EF379948 EF379984 EF379966 C. gestroi (= C. vastator ) CV1HW U S A Hawaii, Oahu EF379953 EF379990 EF379971 C. gestroi (= C. vastator ) CV1PH Philippines Los Banos, Lag una EF379954 EF379989 EF379972 C. gestroi (= C. vastator ) CV2PH Philippines Los Banos, Laguna EF379955 EF379991 EF379973 C. gestroi (= C. vastator ) CV3PH Philippines Los Banos, Laguna EF379956 EF37 9992 EF379974 C. formosanus CF1JP Japan, Wakayama EF379941 EF379978 EF379959 C. formosanus CF2JP Japan, Wakayama EF379942 EF379979 EF379960 C. formosanus CF3JP Japan, Okayama EF379943 EF379980 EF379961 Li et al. 2008 R. flaviceps TW223 Taiwan, Taitung County, Lanyu Township EU627782 EU627778 EU627780 R. flaviceps TW224 Taiwan, Taitung County, Lanyu Township EU627783 EU627779 EU627781
43 Table 2 2 PCR and sequencing primers used in the present study Name Gene Ori entation Sequence Reference a A tLeu b COII Forward 1, 2, 3 4 C2F2 c COII Forward 1, 5, 6 B tLys COII Reverse 1, 2 4, 5, 6 16Sar 16S Forward 1, 7 16Sbr 16S Reverse 1, 7 12S F 12S Forward 1 5, 8 12 SR 12S Reverse 1, 5, 8 a References: (1) Simon et al. 1994; (2) Miura et al. 1998; (3) Liu and Bechenbach 1992; (4 ) Jenkins et al. 2007; (5) Yeap et al. 2007; (6) Hayashi et al. 2003; (7) Marini and Mantovani 2002; (8) Kambhampati 1995. b Forward primer was used for amplifying COII gene of C. formosanus c Forward primer was used for amplifying COII gene of C. gestroi
44 Table 2 3. Distance matrix (percentage, above diagonal) and number of difference of nucleotides (below diagonal) of combined mitochondrial COII, 12S rRNA, and 16S rRNA gene sequences. All positions containing gaps and missing data were eliminated fro m the dataset ( c omplete deletion option). There were a total of 1406 positions in the final dataset. The rectangle with solid lines and dotted lines indicate the intraspecific difference of C. gestroi and C. formosanus respectively. No. species, country, code 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 1 C. gestroi Taiwan a 0.4 0.4 0.4 0.3 0.6 0.6 0.7 0.6 0.6 0.9 0.8 5.7 5.8 5.4 5.7 5.8 5.7 5.7 5.8 14.2 14.1 2 C. gestroi USA Hawaii CV1HW 5 0.0 0.1 0.5 0.7 0.7 0.8 0.7 0.7 0.9 0.9 5.5 5.6 5.3 5.5 5.6 5.5 5.5 5.6 13.9 13.8 3 C. gestroi Philippines CV1PH 5 0 0.1 0.5 0.7 0.7 0.8 0.7 0.7 0.9 0.9 5.5 5.6 5.3 5.5 5.6 5.5 5.5 5.6 13.9 13.8 4 C. gestroi Philippines CV2PH 6 1 1 0.6 0.8 0.8 0.9 0.8 0.8 1.0 0.9 5.5 5.5 5.2 5.5 5.5 5.5 5.5 5.5 13.9 13 .8 5 C. gestroi Philippines CV3PH 4 7 7 8 0.8 0.8 0.9 0.8 0.8 1.0 0.9 5.7 5.8 5.4 5.7 5.8 5.7 5.7 5.8 14.1 13.9 6 C. gestroi USA Florida FLCG 9 10 10 11 11 0.1 0.2 0.1 0.1 0.5 0.6 5.5 5.6 5.3 5.5 5.6 5.5 5.5 5.6 13.9 13.8 7 C. gestroi Malaysia b 9 10 1 0 11 11 2 0.1 0.0 0.0 0.5 0.6 5.5 5.6 5.3 5.5 5.6 5.5 5.5 5.6 13.8 13.7 8 C. gestroi Singapore CG1SG 10 11 11 12 12 3 1 0.1 0.1 0.6 0.6 5.6 5.7 5.3 5.6 5.7 5.6 5.6 5.7 13.9 13.7 9 C. gestroi Singapore CG2SG 9 10 10 11 11 2 0 1 0.0 0.5 0.6 5.5 5.6 5.3 5.5 5.6 5.5 5.5 5.6 13.8 13.7 10 C. gestroi Thailand c 9 10 10 11 11 2 0 1 0 0.5 0.6 5.5 5.6 5.3 5.5 5.6 5.5 5.5 5.6 13.8 13.7 11 C. gestroi Indonesia CG1IN 12 13 13 14 14 7 7 8 7 7 0.1 5.7 5.8 5.4 5.7 5.8 5.7 5.7 5.8 13.9 13.7 12 C. gestroi Indonesia CG2IN 11 12 12 13 13 8 8 9 8 8 1 5.8 5.8 5.5 5.8 5.8 5.8 5.8 5.8 13.8 13.7 13 C. formosanus Taiwan d 80 78 78 77 80 78 78 79 78 78 80 81 0.1 0.7 0.0 0.1 0.1 0.1 0.2 13.7 13.4 14 C. formosanus Taiwan TW50 81 79 79 78 81 79 79 80 79 79 81 82 1 0.8 0.1 0. 1 0.2 0.2 0.3 13.7 13.4 15 C. formosanus Taiwan TW55 76 74 74 73 76 74 74 75 74 74 76 77 10 11 0.7 0.8 0.7 0.7 0.8 13.9 13.7 16 C. formosanus Japan e 80 78 78 77 80 78 78 79 78 78 80 81 0 1 10 0.1 0.1 0.1 0.2 13.7 13.4 17 C. formosanus Japan CF3JP 81 7 9 79 78 81 79 79 80 79 79 81 82 1 2 11 1 0.2 0.2 0.3 13.7 13.4 18 C. formosanus USA Florida f 80 78 78 77 80 78 78 79 78 78 80 81 2 3 10 2 3 0.1 0.1 13.7 13.4 19 C. formosanus China, Hainan g 80 78 78 77 80 78 78 79 78 78 80 81 2 3 10 2 3 2 0.2 13.5 13. 2 20 C. formosanus Hawaii CF1HW 81 79 79 78 81 79 79 80 79 79 81 82 3 4 11 3 4 1 3 13.7 13.4 21 R. flaviceps TW223 200 196 196 196 198 196 194 195 194 194 195 194 192 192 196 192 193 192 190 193 1. 1 22 R. flaviceps TW224 198 194 194 194 196 194 192 19 3 192 192 193 192 188 188 192 188 189 188 186 189 15 a Samples include TW8, TW11, TW19, TW21, TW24, TW29, and TW30. b Samples include CG1MY, CG4MY, and CG5MY. c Samples include CG1TH and CG2TH. d Samples include TW49, TW51, TW52, TW53, and TW54. e Sa mples include CF1JP and CF2JP. f Samples include WBBR, LNDN, and 437GI. g Samples include H1, H2, H3, H4, and H5
45 Figure 2 1. The distribution of C. formosanus and C. gestroi in Taiwan (A) and Florida (B). The termite collection sites in Taiwan incl uded 32 sites in this study and data from Tsai (2003) and Tu (1955). The Florida map after Scheffrahn and Su (2005) included 4 collection sites from this study. The collection sites of samples used for molecular analysis in this study were noted with sampl e codes. The gray scale indicates the January average temperature in Taiwan (Lee et al. 1997) and Florida (Henry et al. 1994)
46 Figure 2 2 The 10001st Bayesian tree inferred from COII gene sequences. Posterior probability values exceeding 50 are given on appropriate clades. > 75 changes.
47 Fig ure 2 3. The 10001st Bayesian tree inferred from combined COII, 12S rRNA, and 16S rRNA gene sequences. Posterior probability values exceeding 50 are gi ven on appropriate clades. *, > 65 changes, **, > 170 change s.
48 CHAPTER 3 INTERSPECIFIC COMPET ITION AND TERRITORY DEFENSE MECHANISMS OF COPTOTERMES FORMOSAN US AND COPTOTERMES GESTROI Introduction Coptotermes formosanus and C. gestroi originated in Asia and have been dispersed to North America, South America, and Af rica by human transportation in the past century (Su 2003, Scheffrahn and Su 2005, Austin et al. 2006, Jenkins et al. 2007). To date C. formosanus is primarily found in the subtropics and warm temperate zones, and C. gestroi is reported mostly from the tro pics (Su 2003) Their distribution overlaps in some subtropical areas such as south Florida (Scheffrahn and Su 2005), Hawaii (Weesner 1965), and Taiwan (Li et al. 2009 a ). Coptotermes fo rmosanus colonized these three areas earlier than C. gestroi did. Coptotermes formosanus is endemic to Taiwan (Li et al. 2009 a ) while C. gestroi is an invasive species first recorded in 1911 by Oshima (Oshima 1911). In Hawaii, C. formosanus and C. gestroi ( = C. vastator ) were first found in 1913 (Swezey 1914) and in 1963 (Bess 1970), respectively. In south Florida C. formosanus and C. gestroi (= C. havilandi ) were first reported in 1980 (Koehler 1980) and in 1996 (Su et al. 1997), respectively. Both species are subterranean termites and structural pests. Their ecological niches probably overlap in the sympatric areas. Termites are territorial, and intra and inter specific competition has been consider ed to be an important regulatory factor to maintain coloni al territories (Jones and Trosset 1991, Levings and Adams 1984, Jmhasly and Leuthold 1999). The outcome of interspecific competition between C. formosanus and C. gestroi may affect their distribution and overlapped zone. Field studies on subterranean termi tes typically use above or in ground monitoring stations in combination with mark recapture methods or molecular tools to
49 delineate colony boundary and abundance (Haverty et al. 1975, Su and Scheffrahn 1988 b Jones 1990, Su et al. 1993, Vargo 2003, Messenger et al. 2005) These field investigations revealed that more than one species of subterranean termite colony could maintain their neighboring territories for a long period of time (years) and keep their territori es in distance from each other. However, monitoring stations are inadequate to serve as a window in the field for observing interactions among subterranean termite species in real time. Only a few field observations based on serendipitous discovery were re ported (Jones and Trosset 1991, Jmhasly and Leuthold 1999), and the mechanism forming territory spatial distribution remains unknown. To date, most experiments related to interspecific competition of termites were conducted in Petri dish bioassays (no choi ce bioassay) (Thorne and Haverty 1991, Polizzi and Forschler 1998, resulting from encounters of two subterranean termite species. These laboratory experiments usually resulted in high mortality within hours, which contradicts field observations in which several neighboring intra or interspecific colonies might survive for years. The objective of this study was to examine the territory defense behavior between C. formosanus and C. gestroi in a foraging arena bioassay which allowed us to observe the tunnel progress and encounters of these two species in real time. T he results generated from a foraging arena bioassay were further compared with that of a Petri dish bioassay.
50 Materi al s and Methods Termite S pecies Individuals of three field colonies of both C. formosanus and C. gestroi were used in this study. Coptotermes formosanus were collected in New Orleans, Louisiana (colonies F1 and F2) and Hallandale, Florida (F3). Coptotermes gestroi were collected in Broward County, Florida (G1), Miami Dade County, Florida (G2), and Monroe County, Florida (G3). Before testing, termites were kept at 28C in one liter cylindrical plastic jars with pieces of moist wood. Voucher specimens were pr eserved in absolute ethanol and deposited in the University of Florida Termite Collection, Fort Lauderdale Research and Education Center. Three colonies each of the two species were used to create a 3 x 3 combination of interspecifically paired interaction To differentiate species in each pair, two species of termites were fed on non dyed or dyed filter paper containing 0.1% Nile blue (wt/wt) for 5 days before each test (Su et al. 1991) resulting in 18 (3 x 3 x 2) possible pairs Each pair was tested three times in the Petri dish bioassay and once in the foraging arena bioassay resulting in 54 and 18 tests, respectively During the experiments, the room C Petri dish Bioassay One group of nine workers and one soldier each o f C. formosanus and C. gestroi were placed in a Petri dish (60 x 15 mm) lined with moist filter paper. The initial behavioral response was observed and recorded through a camcorder ( Sony DCR TRV9, Tokyo, Japan ) for 1 min and the number of surviving indivi duals of each species was counted at 24 h
51 individuals of both species survived, and the rest of results were in the dominant as the winner. A chi square test was used to examine the dye and species effects on interspecies competition. Foraging Arena Bioassay The exp erimental arena (Figure 3 1A) was constructed of two sheets of transparent Plexiglas (60 x 60 x 0.6 cm in thickness) separated from each other by Plexiglas laminates (5 cm in width and 0.15 cm in thickness on each side) placed between the outer margins to form a 0.15 cm gap of 50 x 50 cm and held together with screws ( Figure 3 1A). In the arena, f ive pieces of wood (3.7 x 3.7 x 0.15 cm) were fasten ed at each of two corners by injecting glue into a 0.6 cm diameter hole in its center which encompassing a sq uare space (8.4 x 8.4 x 0.15 cm) at each of the two corners. Sand was used to fill the rest of space b etween the two Plexiglas sheets. The top Plexiglas sheet had four 1 cm diameter access holes in the center for injecting water to moisten sand. A 5 cm dia meter disc placed in center of the arena served as a rotatable switch of the central access holes and also maintained the arena gap. Two hundred termites (180 workers and 20 soldiers) were introduced into the square space encompassed by wood pieces through a 0.6 cm diameter access hole on top Plexiglas at each of the two corners. Termites accessed the foraging sand through the four 0.5 cm width gateways between wood pieces. A ll access holes on top Plexiglas were closed and plugged immediately after the sand was moistened and termites were introduced. After introducing termites into the arena, back lit digital images of the entire arena were taken every five minutes for five days yielding 1,440 images per test. The images
52 of each test were combined into a vid eo for quick review by using Windows Movie Maker version 6.0 (Microsoft Corp. Redmond, WA) and were converted into a filmstrip for detail analysis by using Acrobat 6.0 Professional (Adobe Systems Inc. San Jose, CA). Tunnel interceptions of the two species, and the subsequent agonistic behavior and tunnel blockage patterns were reviewed ( Figure 3 1). The distance between the tunnel junction and resultant tunnel blockages, and time length from tunnel interception to the formation of tunnel blockages, were mea sured. Live termites of each species were counted on the fifth day from the image on the computer monitor. Results Petri dish Bioassay Immediately after releasing termites of the two species into Petri dishes, termites expressed agonistic behavior toward each other in most cases Soldiers of both species moved rapidly and chased the individuals of the other species. Soldier mandibles flared and slashed the head and abdomen of the other species. Workers bit legs, antennae and abdomens of soldier and workers of the other species. It was frequently observed that several workers simultaneously attacked a single soldier or worker of the other species. Fierce combat was generally observed and several individuals died in the first minute. After 24 h, based on the number of surviving individuals, only four of 54 test species effect on interspecies competit ion ( 2 =0, p =1). Coptotermes gestroi won most of the agonistic encounters over C. formosanus ( 2 =32, p <0.0001). The overall survival rates
53 were 44.44 4.30% (Mean SE) and 8.15 3.37% for C. gestroi and C. formosanus respectively. Tunnel Interception and B lockage In the foraging arena bioassay, both C. gestroi and C. formosanus excavated several tunnels through the gateways simultaneously and deposited sand into the two respective square spaces. The first encounter of tunnels of the two species occurred wit hin 1 2 d. First interception is when each primary tunnel extended from the gateways and encountered a tunnel of the other species for the first time ( Figure 3 1 A ), and 44 such first interceptions were observed in the 18 replications (Table 3 1) during the entire 5 d experiment al period After a tunnel interception, termites from both sides rapidly encounter s. The worker caste is the major caste involved in fights. Some individuals were woun ded and died in tunnels and body fluids stained the surrounding sand. Both species buried the connected tunnel at several locations shortly after tunnel interception and agonistic encounters One species might excavate another tunnel and bypass the blocked portion or reopened the blockage, and then encounter ed tu nnel s of the other species again, which is defined as additional interception (Table 3 1 and Figure 3 1 C ). In total, there were 80 tunnel interceptions, 59 tunnels of C. gestroi encountered the tunn els of C. formosanus 17 tunnels of C. formosanus encountered the tunnels of C. gestroi and only in four cases, leading tunnels of both species encountered each other. Coptotermes gestroi was more active than C. formosanus in tunnel excavation in the fora ging arena bioassay, but in most cases (17 of 18 replications) both species survived
54 to the end of experimental period. The survival rates were 38.89 3.06% and 51.56 5.19% for C. gestroi and C. formosanus respectively. Numbers of tunnel blockage induc ed by tunnel interception are shown in Table 3 2. In most cases (72 of the 80 observations), the first tunnel blockage was made within an hour after tunnel interception, which completely separated both species, and 77.5% of tunnel interceptions induced mor e than one tunne l blockage On average, each tunnel interception resulted in 2.71 0.16 tunnel blockages, and > 60% of them were made in the first hour (Table 3 2). The average distance between tunnel interception and blockages was 6.42 0.53 cm, and ove r 78% of all blockages were within 10 cm of the interception. In the total 217 of tunnel blockages, 185 of them (85.3%) were induced in the presence of termite cadavers, and 32 of them (14.7%) were initiated without termite cadavers. Surviving termites cov ered cadavers with sand and then further filled a section of the tunnel. During the 5 d experimental period, only 5.4% (10 of 185) tunnel blockages containing cadavers were reopened, but 40.6% (13 of 32) blockages without cadavers were reopened, which indi cate s that termites avoided contact with cadavers. Discussion Interspecific Competition Both the Petri dish bioassay and the foraging arena bioassay showed C. gestroi was more aggressive than C. formosanus In the Petri dish bioassay, C. gestroi killed mos t of the C. formosanus within 24 h. In the foraging arena bioassay, most first tunnel interceptions (36 of 54) and most additional tunnel interceptions (23 of 26) resulted from leading tunnels of C. gestroi intercepting the tunnel of C. formosanus (Table 3 1). These results may partially explain how C. gestroi successfully infested the areas such as
55 south Florida and Hawaii where C. formosanus had been previously established for several decades. Low temperature is a limiting factor for termite activity (Spo nsler and Appel 1991, Fei and Henderson 2002) that affects their distributions (Li et al. 2009 a ). If climate changes, the distribution of C. gestroi and C. formosanus may change accord ingly. The average temperature of their overlapping region in the United States ranged from 18 to 20 C in January which may shift toward north, and the distribution of C. gestroi may expand. Based on the current study, the presence of C. formosanus is not likely to be a limiting factor for further expansion of C. gestroi Terr itory Defense Mechanism In the foraging arena bioassay, both C. formosanus and C. gestroi quickly responded to tunnel interceptions and blocked tunnels within an hour resulting in territory separation and avoidance, which differed from the high mortality r esults of the Petri dish bioassay (no choice bioassay). The death of some individuals might have been caused by the fight and suicide cramming (Messenger and Su 2005a) in the foraging arena bioassay, and termite cadavers were usually the starting point of tunnel blockages. I speculated that chemicals released from termite cadavers may trigger a series of behaviors with multiple adaptive functions. Freshly killed termites triggered the burying behaviors by the surviving termites leading to avoidance of patho genic organisms such as bacteria and fungi produced from such cadavers (Su 1982, Jones et al. 1996, Chouvenc 2003). Surviving termites further filled a section of tunnel with deposited sand which prevented invasion of the other species. During decompositio n of the buried cadavers, the continued release of chemicals served as a deterrent which
56 prevented both termite species from reopening the blockages and encountering each other again through the same tunnel. Territory Dynamic Equilibrium In light of the te rritory defense mechanism, I further speculate that neighboring territories of subterranean termite colonies in the field equilibrate in a dynamic state. During the active season in summer, subterranean termites actively excavated tunnels and expanded t hei r territories (Waller and La Fage 1987, Messenger and Su 2005b). The tunnels of neighboring colonies may encounter each other and cause some agonistic responses which results in tunnel blockage. Based on the current study, the multiple tunnel blockages indu ced by a single tunnel interception were only 6 cm from the tunnel intercepting point on average. Tunnel blockages separated the neighboring colonies from each other by relatively short distance s Once one species bypasses the tunnel blockage and keeps exc avating, an additional interception may happen. Hence, borders of neighboring territories are delineated by minor fights induced by tunnel interception. The borders may shift over time according to each tunnel intercepting point but it is not likely that o ne species or one colony could easily overwhelm the others. Tunnel interceptions of two neighboring colonies also provide information on the colony status to each other. Once one colony is no longer able to maintain their tunnels, the dynamic equilibrium m ay shift. The stronger colony could expand their territory quickly through the tunnel systems left by the weaker colony. This observation was recently reported. After one colony was eliminated by feeding them with a chitin synthesis inhibitor, the vacated territories were occupied by neighboring colonies with in days (Messenger et al. 2005, Lee et al. 2007).
57 In conclusion, C. gestroi was more aggressive than C. formosanus in both bioassays. Coptotermes formosanus is not likely to be a limiting factor for fur ther expansion of C. gestroi The results of the current study support the long held assumption that interspecific competition is an important regulatory factor to maintain termite colonial territories. The territory defense mechanism of C. formosanus and C. gestroi is a series of tunnel blockages resulting from tunnel interceptions. Severe fights with heavy loss between neighboring colonies, as results from the no choice Petri dish bioassay are unlikely as long as the colo nies are healthy in the field. I s peculate that neighboring territories equilibrate in a dynamic state.
58 Table 3 1. The tunnel interception sequences between C. gestroi and C. formosanus F, a tunnel of C. formosanus intercepted the tunnel of C. gestroi G, a tunnel of C. gestroi intercep ted the tunnel of C. formosanus B, lea ding tunnels of both species encountered each other. Primary interception Additional interceptions 1 st 2 nd 3 rd 4 th 5 th n F 10 F F 1 F F G 1 F G 1 F G G G G 1 G 23 G F 1 G G 10 G G G G 2 B 3 B G 1 Table 3 2 Tunnel block a g es induced by tunnel interception of C. gestroi and C. formosanus Time after tunnel intersecting Number of b lockages Number (%) of blockages made in each time period a 1 st 2 nd 3 rd th < 1 h 72 38 13 8 131 (60.4%) 1 2 h 6 14 15 3 38 (17.5%) 2 3 h 0 3 5 11 19 (8.8%) > 3 h 1 7 8 13 29 (13.4%) Number (%) of each sequential blockages induced by tunnel interceptions b 79 (98.8%) 62 (77.5%) 41 (51.3%) 35 (43.8%) a The total number of blockages observed in all replicates was 217 b The total number of tunnel interceptions observed in all replicates was 80.
59 Figure 3 1. Time courses study of tunnel interceptions and consequent tunnel blockages in the foraging arena bioassay. (A) Two hundred individuals of C. gestroi and C. formosanus were released in the square space at the two corners. Termites excavated several primary tunnels from the space. After 22h, one lead ing tunnel of C. gestroi was going to intercept the tunnel of C. for mosanus The encounter point was denoted as 1* and classified as a primary interception. The square with dotted line s represents the correlated position in the arena of the following three figures. (B) After the first tunnel interception, termites of both species blocked the tunnel at four locations in 65 min which were denoted as 1a, 1b, 1c, and 1d. (C) Coptotermes gestroi continually excavated from a branch of the same primary tunnel and then intercepted the tunnel of C. formosanus again. The second encou nter point was denoted as 2* herein and classified as additional interception. (D) The tunnel blockage resulting from the second tunnel interception was built within 20 min and was denoted as 2a.
60 CHAPTER 4 TERMITE PESTS AND TH EIR CONTROL IN TAIWA N Introd uc t ion Termites are serious pests in Taiwan which have caused severe damage to manmade structures for over 200 years (Su 2003) So far, 17 named termite species of four families and 12 genera have been recorded, including Termitidae: Na kinoshitae (Hozaw a), Na. parvonasutus Na. takasagoensis O formosanus P nitobei S mushae ; Rhinotermitidae: C. formosanus C. gestroi Prorhinotermes japonicus (Holmgren), R flaviceps R. chinensis Snyder; Kalotermitidae: Cr yptotermes domesticus (Haviland), G fuscus G. satsumensis (Matsumura), I inamurae N koshunensis and Termopsidae: Hodotermopsis sjoestedti (Holmgren) (Chung and Chen 1994, Tsai and Chen 2003, Li et al. 2009 b ). Among the Taiwanese termites, Coptotermes spp. were considered most destructive to wo oden structures, books, clothes, and furniture (Oshima 1909, Yi 1954, Tsai and Lai 2004). Reticulitermes flaviceps and Cr. domesticus were also known to damage wooden structures but their economical importance i s uncertain. O dontotermes formosanus was most common species in Taiwan and caused damages to vegetables tea, fruit trees sugar cane, rice, and wooden structures at a point of wood soil interface (Tu 1954 Yi 1954 ). Nasutitermes parvonasutus and S. mushae were recorded as agricultural pests of sugar cane (Yi 1954) and wild rice (Tu 1954), respectively, and H. sjoestedti caused damaged in silviculture (Huang 2000) In addition, it has been reported several times that C. formosanus and O. formosanus chewed through the lead sheathing of electrical and t elephone cables and caused short circuits ( Yi 1954, Tsai et al. 2004 )
61 Coal tar creosote was the first chemical used for wood treatment in Taiwan in the early 1900s (Oshima 1909, 1911, 1912). In 19 40s and 19 50s, arsenic compounds were widely employed for s oil and wood treatment (Yi 1954). The local manufacture of chlorinated hydrocarbons, such as DDT, aldrin, lindane, and chlordane began in the mid 19 50s (Yi 1954), and they became popular between 19 60s and mid 19 80s. After most of chlorinated hydrocarbons w ere banned in late 19 80s, organoph os phates, particularly chlorpyrifos, became the major termiticide in Taiwan. Due to the public concern over its potenti al contamination of groundwater in 2005, the overall use of chlorpyrifos was cancelled in the United St ate s and it is also being phased out from the market in Taiwan. Tsai and Lai (2004) considered that chemical treatment was still the major tool for termite control in Taiwan then, but no quantitative data on termiticide use was reported in their study. Th e objective of the current study includes a verification of the economic status of termites as pests, the quantification of current termiticide use, and an estimation of the annual revenue of termite control business in Taiwan Material s and Methods A ques tionnaire ( A ppendix A and its translated version, Appendix B ) was designed for licensed pest control operators (PCOs) to quantify termite damage and the control measures used in Taiwan in 2008. Six questions were included: 1. How many cases of termite cont rol were done by your company in each county of Taiwan in 2008? 2. Among these infested buildings, what were the percentages of residential premises, industrial buildings, commercial constructions, historical constructions, and others? 3. What kinds of ter miticide were used and their ratio? 4. What was the proportion of each termite species? 6. If termites were identified, what species were they and their ratio ?
62 Th e qu estionnaire s w ere delivered to PCOs through the Taiwan Environmental Pest Management Association and then phone interviews were conducted to obtain further information and answers of missed question s. All reported cases from the valid questionnaires were l umped and used for c alculating the p ropotions of control cases from each county, propotions of infested buildings by each termite species propotions of infested buildings of each type, and percentage of each termiticide used by PCOs The average cost of e ach termiticide was based on the data offered by PCOs who used such termiticide s Posted information on the website of the Environmental Protect Administration (EPA) of Taiwan regarding permitted pesticides (Anonymous 2009a) and licensed pest control compa nies (Anonymous 2009b) w ere used for related analysis. The correlation between t he number of termite control cases in each couty reported from questionnaires and the number of registered PCOs database were tested by correlation ana lysis ( SAS Institute 1985 ). Results and Discussion Questionnaire and Registered Pest Control Companies The administration of the EPA of Taiwan requires PCOs to take a one week training and a certified exam to obtain a license. There were 732 registered p est control companies in Taiwan (Anonymous 2009b) and they were primarily located in three densely populated counties Taipei, Taichung and Kaohsiung (Table 1). A total of 17 completed questionnaires was returned for this study. The number of termite cont rol cases in each county reported from the questionnaires was strongly correlated to the number of registered pest control companies in each county ( r = 0.95, P < 0.0001; proc corr, SAS Institute 1985) (Table 1) which indicated that these questionnaires w ere representative.
63 Termite Pests and Infested Buildings Twelve of the 17 PCOs ( ) identified termite species when they offered termite control service. In the total 1, 638 of termite control cases, > 87% was caused by C. formosanus or C. gestroi Cr. domestics O. formosanus and less than 1% by R flaviceps Coptotermes formosanus and C. gestroi were usually found in lowland area (< 500 m) especially in urban environments (Li et al. 2009 a ) The drywood termite, Cr. domestics was found to b e a major pest on wooden constructions, such as historical buildings and temples. Cryptotermes domestics distributed through out the island while most were found in southern Taiwan. The fungus growing termite, O. formosanus was the most common termite spec ies found in both urban and natural environments under the altitude of 1,000 m throughout Taiwan (Huang 2004) Odontotermes formosanus may occasionally enter houses from the yards but seldom caused severe damage. Reticulitermes flaviceps is a minor pest an d found in either northern Taiwan or in mountainous areas of southern Taiwan Of the total 1,934 of termite control cases reported from the queationnaires of them industrial constructions, and < 1% on trees. The modern buildings were usually constructed of steel and concrete in order to survive frequent earthquakes and typhoons on the island. No specific construct ion code or pre treatment is required for preventing termite damage in Taiwan ; hence PCOs primarily off ered remedial control Termites did not cause damage to frame structures of most Taiwanese buildings, but furniture, wooden floor, ceiling, and other wo oden decorations were seriously threatened.
64 Termiticide Currently, 80 pesticide products were labeled for termite control use and seven of them were specialized for this purpose (Table 2). The active ingredients of the seven termiticide products are boric acid, hexaflumuron, fipronil, and cypermethrin. Organoph o sphates and pyrethroids are the primary active ingredients of the non termite specific pesticides. Among the 80 pesticides, 56 (70%) we re Restricted Use Pesticides (RUPs), which are only purchased an d used by licensed PCOs. Based on the questionnaire survey, the liquid pesticide with fipronil as the active ingredient was used i cases. Chlorpyrifos and pyrethroid s only had 4% market share. Even though 90% of the 80 termiticides we re made in Taiwan 95% market share belonged to two imported products, Termido r, a liquid termiticide with fipronil, and Sentricon Termite Colony Elimination System with hexaflumuron baits. Termite bait systems were introduced into Taiwan around 1996, but its use was limited due to its high cost. The cost of bait systems accounted for total cost but that of liquid pesticide (Table 2). Recently, due to the concern of potential impact of liquid pesticides on the environment, baiting has been better accepted in subterranean termite control, especially at cultura lly important sites (Su and Hsu 2003). Most urban constructions are multi story buildings without yards, requiring above of cases instead of in ground baiting stations, which we re the most used type in the United S tates. Since termite bait systems are not effective against drywood termites and higher termites, such as Cr. domesticus and O. formosanus PCOs applied a combination of both bait system and chemical the current survey.
65 Business Revenue of Termite Control Industry In this study, I attempted to estimate the total business revenue of termite control industry based on all termiticide sale of Taiwan in 2008. However, there was a concern that Termidor and other liquid termiticides might be partially used on non termite pest control. Hence, I chose Sentricon, which is only used to control termites, to estimate the total business revenue in 2008 based on the equation the total business revenue = [(total sa les of Sentricon )/ ( Sentricon cost /service charge )]/ (market share of Sentricon ), which w There is a need to further estimate the cost of termite control conducted by non licensed PCOs and repair cost s in order to obtain the total monetary expenditure associated w ith termite control and damage. In conclusion, C. formosanus and C. gestroi are the most serious termite pests in Taiwan and responsible for over 87% termite control cases in 2008. Most infested buildings were re sidential premises (75%). The liquid termiticide with fipronil and the termite bait with hexaflumuron were the two primary products used by PCOs, and they had 75% and 20% market shares, respectively. The annual business revenue of termite control industry
66 Table 4 1. Distribution of licensed PCOs and termite control cases in Taiwan Area County No. of licensed PCOs (%) a No. of cases b Northern Taiwan Taipei c 315 (43.0%) 787 (40.7%) Taoyuan 55 (7.5%) 164 (8.5%) Hsinchu d 22 (3.0%) 133 (6.9%) Miaoli 1 (0.1%) 17 (0.9%) Central Taiwan Taichung e 94 (12.8%) 192 (9.9%) Changhua 25 (3.4%) 51 (2.6%) Yunlin 7 (1.0%) 5 (0.3%) Nantou 9 (1.2%) 12 (0.6%) Southern Taiwan Chiayi f 16 (2.2%) 25 (1.3%) Tainan g 60 (8.2%) 360 (18.6% ) Kaohsiung h 92 (12.6%) 153 (7.9%) Pingtung 11 (1.5%) 21 (1.1%) Eastern Taiwan Yilan 2 (0.3%) 1 (0.1%) Hualien 10 (1.4%) 4 (0.2%) Taitung 8 (1.1%) 6 (0.3%) Islands Penghu 2 (0.3%) 2 (0.1%) Kinmen 2 (0.3%) 1 (0.1%) Lienchiang 1 (0.1%) 0 (0.0%) total 732 (100.0%) 1934 (100.0%) a Information was obtained from the website of the Environment al Protection Administration, RO C (Taiwan). b Total cases of termite control of each county in 2008 reported in 17 valid questionnaires. c Taipei City and K eelung City were included. d Hsinchu City was included. e Taichung City was included. f Chiayi City was included. g Tainan City was included. h Kaohsiung City was included.
67 Table 4 2. Type, cost, and use of termiticide in Taiwan Registered termiticide s a Termiticide use b Active ingredient category No. of termiticides (specialized for termite) No. of RUPs (%) No. of products made in Taiwan No. of cases (%) Termiticide cost/ service charge SE Pyrethroids c 46 (3) 31 (67%) 44 (96 % ) 8 (0.39%) 20.00% Pyrethroids + o rganoph o sphates 7 (0) 6 (86%) 7 (100%) Pyrethroids + Imiprothrin 3 (0) 0 1 (33%) Organoph o sphates d 18 (0) 18 (100%) 16 (89%) 66 (3.39%) 11.25 1.25% Carbamate e 2 (0) 0 2 (100%) Boric acid 2 (2) 0 2 (100%) Hexaflumuron 1 (1) 0 0 385 (19.89%) 25.98 2.62% Fipronil 1 (1) 1 (100%) 0 1446 (74.76%) 18.59 2.20% total 80 (7) 56 (70%) 72 (90%) 1905 (98.44%) f a Information obtained from the website of the Environmental Protection Administration of Taiwan, RO C. (Anonymous 2009a) b Data was generated from questionnaires. c Pyrethroids includes permethrin, bifenthrin, cypermethrin, alphacypermethrin, etofenprox, deltamethrin, phenothrin, tetrameth rin, pyrethrins, and esbiothrin, d tetramethrin. d Organopho sphates includes ch lorpyrifos, pirimiphos methyl, and fenitrothion. e Carbamate only includes propoxur. f No termiticide was used in 29 cases.
68 APPENDIX A QUESTIONNAIRE FOR QU ANTIFYING TERMITE DA MAGE AND THE CONTROL MEASURES USED IN TAI WAN ( ) ____________________________ ______________________ ________________________ ________________________ ________________________ 2008 1. (2008.1~2008.12) ______ ______ ______ ______ ______ ______ ______ ______ ______ ______ ______ ______ ______ ______ ______ ______ ______ ______
69 2. a. ____% ____ b. ____% ____ c. ____% ____ d. ____% ____ e. ____% ____ f. __________ ____% ____ g. __________ ____% ____ h. __________ ____% ____ i. __________ ____% ____ 3. A 2008 100 60 20 10 10 A a. 60 % b. 20 % a. + 10 % a. 10 % a. __________ ____% b. __________ ____% c. __________ ____% d. __________ ____% e __________ ____% f __________ ____ %
70 a. ____________ + ____________ ________% b. ____________ + ____________ ________% a. __________________ ____ _____________ ________% b __ _____________________ ____________ ______ __% 4. B 20,000 2,000 20,000 4 ,000 B __________% x a. 10 % 2,000/20,000 b. 20 % 4 ,000/20,000 __________% a. ____________________ ____ _% b. ____________________ ____ _% c ____________________ ____ _% d ____________________ ____ _% e ____________________ ____ _%
71 5. ( ) ( ) 6. a. ( Coptotermes spp. ) ______________% b. ( Cryptotermes spp ) ______________% c. ( Odontotermes spp. ) ______________% d. ( Reticulitermes spp. ) ______________% e. _______________ ______________% f. _______________ ______________% g. _______________ ______________% h. _______________ ______________%
72 APPENDIX B TRANSLATTED QUESTIONNAIRE 1. How many cases of termite control were done by your company in each county of Taiwan in 2008? Taipei__ ; Taoyuan__ ; Hsinchu__ ; Miaoli_ ; Taichung__ ; Changhua__ ; Yunlin__ ; Nantou__ ; Chiayi__ ; Tainan__ ; Kaohsiung__ ; Pingtung__ ; Yilan ___ ; Hualien__ ; Taitung__ ; Penghu__ ; Kinmen__ ; Lienchiang__ 2. Among these infested buildings, what were the percentages of the following locat ions? Residential premises__ % ; industrial buildings __ % ; commercial constructions __ % ; historical constructions __ __ % ; others ( )__ __ % 3. What kinds of termiticide were used and their ratio? Only use one termiticide: a.__________________ _______(termiticide name)__ % b ._________________________(termiticide name)__ % Only use two termiticides: a.____________ + ____________ (termiticide names)__ % b .____________ + ____________ (termiticide names)__ % Only use multiple termiticides: a.___ ________________________________(all termiticide names)__ % b .___________________________________(all termiticide names)__ %
73 charge? a.__________________ (termiticide na me)__ % b.__________________ (termiticide name)__ % c.__________________ (termiticide name)__ % 5. Was an effort made to identify termite species? Yes: __ No: __ 6. If termites were identified, what species were they and their ratio ? a. Coptoter mes spp.: __ %. b. Cryptotermes domesticus : __ %. c. Odontotermes formosanus : __ %. d. Reticulitermes spp.: __ %. e. Other termite _____________ ( termite name ): __ %.
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82 BIOGRA PHICAL SKETCH Hou Feng Li was born in Taiwan 1979, and he was fascinated with insects during his childhood and wished to be an entomologist since he was ten years old. In high school, he founded an insect lecture in the student Biology Club, and studied e ntomology under Dr How Jing Lee in the Department of Plant Pathology and Entomology, National Taiwan University. In 1997, he graduated from high school and was invo lved in physiologica l research on insect biological clock s In 2001 2003, he did compulsory military service as a second lieutenant in a tank company. In 2003 summer, he returned to his previous research team as a research assistant for a year. Since the fall of 2004 Hou Fen g studied and did research on termite biology under Dr Nan Yao Su in the Department of Entomology and Nem atology, Fort Lauderdale Research and Education Center, University of Florida He explored several research areas including taxonomy, ethology, phylog eography, and practical control of the destructive termite pests of Florida and Taiwa n Hou Feng was selected as the recipient of the 2008 William L. and Ruth D. Nutting Award for outstanding achievements in basic termite biology under the North American S ection of the International Union for the Study of Social Insects. He recei ved his M.S and Ph.D. degrees in May 2006 and in December 2009, respectively. Hou Feng married Rou Ling Yang another Ph.D. trained entomologist in May 2007 He and his wife enjoy their lives and research together.