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surviving termites recorded. Group effect on fungil growth. Five worker temites were placed in a 30 ml glas petri dish (ten repliates) containing a moistened 4.25 ...

The d e of social behaviour of R e t i c u l i t n m e s ~ v i p e s(Kollu) (Isoptera: Rhinotermitidae) in defence against the fungal pathogen Metarhizium ansiopliae (Metschnikoff) Sorokin (Deuteromycotina:Hyphomycetes)

A thesis submitted in conformity with the requirements for the degree of Master of Science in Forestry

Graduate Department of Forestry University of Toronto

O Copyright by Barry H.Strack 1998


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The role of social behaviour of Reticul i termesflavipes (Kollar) &optera: Rhinotennitidae) in defence against the fun@ pathogen Metarhiziurn anisopl iae (Metsdinikoff) Sorokin (DeuteromycotM: Hyphomycetes) Master of Science in Forestry 1998

Barry H.Stradc Graduate Department of Forestry

University of Toronto The feasibility of ushg the entomopathogenic fungus, Me ta rhizium a nisopl iae (Metçchnikoff), for the biological control of Re ticuli termesflavipes

(Kollar) was investigated. Individual termites were removed h m their colonies and dusted with the conidia of M. anisopliae, then immediately rehimed among their nestmates. Termite interactions resulted in the dissemination of conidia through populations maintained in petri dishes, resulting in termite mortality.

These interactions, however, were mostly antagonistic. It was observed that individu& carrying the conidia elicited aggressive behaviour in their nestmates.

Additional studies reveaied that termites are able to recognize individu& dusted with the conidia of M. anisopliae and prevent them from integrating into the

colony. In a soi1 environment, dusted termites were frequently buried alive by

their nestmates or groomed untii no conidia remained on the5 cutide. Further investigations showed that termite agonism is elicited by the presence of M. anisopliae on termite cutide, but not by the symptoms associateci with fungal infection.

I would first like to thank my supervisor Dr. Timothy G. Myles. Without his

guidance, encouragement and encyclopedic knowledge of termites, this research project would not have been possible. Collectively, the members of my cornmittee,

Dr. Dave Malloch, Dr. h d y Kenney, and Dr. Çandy Smith, were a weU-spring of knowledge and experience, thereby ensuring that my researdi remained focused and of the hghest caliire. Individually, each was a source of inspiration and insight and 1 extend to them my çincere gratitude. I would also like to thank the biinistry of the

Environment for Ontario and the Rosamind Gilles Fellowship from the University of Toronto for providing financial support during the initial stages of this project. Desenring of special adaiowledgement and thanks are Ralph Toninger,

Dr. Devaiah Munivanda, and JohnSisson, three friends at the University of Toronto who frequently provided invaluable assistance during innumerable moments of crisis. 1 must also thank JohnIMcCarronand Ian Kennedy for extending to me their expertise, with very litüe grumbling, whenever needed.

My wife, Susan Strack, is to be thanked most of dl. Her love, support and kindness is magical and it is entirely because of her that 1 was able to undertake and complete this most interesting investigation.




Introduction to the biologicd control of the eastem subterranean termite with the h g a l pathogen Metarhizium anisopliae (Metschnikoff) Sorokin

Termite biology and distribution Conventional termite control Biological control Mection of insect hosts by entomopathogenic fun@ h e c t responseç to huigal infection Subterranean termite problem in Ontario The research project


The transmission of the fungal pathogen Meta r h i z i u m a n isopl iae (Deuteromycontina:Hyphomycetes) Sorokin within laboratory colonies of the eastem subterranean termite (Isoptera: Rhinotermitidae)


Subterranean termite colonies Termite control nie huigal biocontrol agent Metarhiziu m anisopliae (MetschnikofO Termite social behaviour

Termite collection

I.. Il

iii vii

Termite populations in bare pehi dishes Termite populations in petri dishes with agar Termite populations in soil cups Behâviourd defences in the soi1 environment Data analysis

RESULTS Termite populations in bare petri dishes Termite populations in petri dishes with agar Termite populations in soil cups Behavioural defences in the soil environment



CHAPTER THREE Termite recognition of nestmates infected with the fungai pathogen, M e tarhizium anisopl iae (Metschnikoff) Sorokin

ABSTRACT INTRODUCTION Termite agonism Interspeafic agonsim Agonistic behaviour in termites of the same colony Recognition factors Research objectives

Termite collection Coating of termites in conidia pathogenic and non-pathogenic conidia Infection of termites by M. anisopliae

The effects of conidia on nestmate recognition The effects of in€edionon nestmate recognition


CHAPTER FOUR The influence of density and group behaviour in the dissemination of pathogenic conidia in laboratory colonies of the eastern subterranean termite (Içoptera:Rhinotermitidae)


INTRODUCTION Disease in termite populations Research objective

Density experiment Group effect on fungal growth RESULTS Density experiment Group effed on fungal growth DISCUSSION



Thesis stllzunary and conclusions


TABLE 21 Types of behavioural responses displayed by populations of ReficulitermesfIaoipes following the introduction of a nestmate dusted with the conidia of Meta rhiziurn a nisopl iae. TABLE 2.2

Frequency of behaviour in petri dishes (n=5 dishes) containing ReticulitermesJlaoipes following the introduction of two termites dusted with increasing quantities of Metarhizium anisopliae conidia.

TABLE 2.3 Mean mortality among groups of Re ticrilitermesflav ipes after 14 days in bare petri dishes following the introduction of two termites dusted with increasing quantities of M e f a r h i z i u m m i s o p l i a e conidia. TABLE 2.4

Frequency of behaviour observed in petri diçhes of sterile agar (n=5 dishes) containing Reticuliterrnesflavipes following the introduction of two termites dusted with increasing quantities ofMetarhizium anisopliae conidia.


;Mean rnortality among groups of Re t icu litermes flaa ipes after

14 days in petri dishes of sterile agar following the introduction of two termites dusted with increasing quantities of Me ta rhiz i u m anisopliae conidia.


Mean mortality of groups of Ret iculitermesflaoipes after 14 days in soi1 cups foliowirtg the introduction of 12 termites dusted with different doses of Meta rhizium anisopl iae conidia.


Mean number of different behavioural responses displayed by populations of Reticulitermesflnaipes following the introduction of termites dusted with either the conidia of iMetnrhizium anisopliae orPenicillium breoicompactum, or with talcum powder.



Frequenqr of Metent types of behaviour disphyed in observational chambers (n=5 Chambers) containing ReticulitermesfIauipes following the introduction of two termites dusted with eitherhfetnrhiziurn anisopliae or Penicilliurn breoicompactum, or with talcum powder (conttol).



Types of behavioural responses displayed by populations ofReticulitermesflaaipes following the introduction of a nestmate either coated with the conidia of Meta rhiz iu m an isop line, suffering from mycosis, or dead h.om fun@ infection.


Frequency of d m behaviour observed in petri dishes (nt5 dishes) of Ret iculi t e r m e s f l a ~ i p e sfoIlot\.ing the introduction of a single termite dusted with either the conidia of Metarhizium anisopliae, Penicillium brevicompactum, or with talcum powder.


The mean number of Merent behavioural responses displayed by populations of Ret iculitermesfIavipes in petri dishes (n=5 dishes) following the introduction of a single termite either coated in the conidia of Metarhiziurn an isop 1 iae, moribund from fungal infection, or dead.


Frequency of behaviour displayed by Reticuli terrnesJlav ipes in petri dishes (n=5)foUowing the introduction of a single treated nestmate.


Mean mortality among groups of Reticul itermesflaaipes under different density regimes for 14 days following the introduction of a single termite dusted with Metarhizium anisopliae.



TABLE 4.2 The percentage of termite cadavers in pehi dishes (n=10dishes) that were either buried under termite feces, supporting abundant fungal growth or showing signs of deliquescence after king iniected with the h g a l pathogen Meta rhiziu m anisopliae.


Introduction to the biologicd control of the eastern subtemnean termite with the fun& pathogen Metarhirium ansiopliae (Metsdurikom Sorokin

Termites play an important role in the decomposition of wood and the cyding of nutrients through their consumption of lignoce11ulosic materiai (Martius

1994). While they are an integral component of terrestriai ecosystems, their dietary

habits frequently place them in connict with human interesb. Approximately 300 speaes of termites are considered pests of economic importance, due to the damage they inflict upon tree crops, lumber, utility p l e s and residential properties (Gay 1969; Logan et al. 1990).

Termite biology and distribution

There are over 2300 species of termites, in seven fiunilies, comprising the order Isoptera (Shellman-Reeve 1997). They are approximately distributeci between

4S0N and 4 5 5 and below elevations of 3000 m (Wood 1988). Largely considered tropical insects (Krishna 1970), termites are most diverse in the Ethiopian (Bouillon 1970), Oriental (Roonwal1970)and Australian regions (Gay and Calaby 1970). Relatively few speaes have become established in the cooler Nearctic and Palearctic regions (Weesner 1970; Harris 1970). Only two genera, Reticuliterrnesand

Zoo ter mopsis, have naturd distributions which extend into Canada, and both are reshided to the pa&c coast in southem British Columbia (Weesner 1970; Myles 1995). Accidental introduction of termites occur on a large scaie through shipments

of wood products, and it is likely in this way that the eastern subterranean termite, Reticuliterrnesflaoipes (Kollar), was introduced to Ontario during the 193û's (Gay



Termite colonies are often established through date pairs. Mate termites

disperse in large tlights fmm mature colonies at certain times of the year and following a brief flight, select a mate and enter into wood or soil to copulate, lay eggs and rear brood (Nutting 1969). Alternatively, some subterranean termites are able to

establish colonies through a process referred to as budding whereby a cohort of neotenic and worker termites separate from the primary colony and create their own nest (Myles 1988). This type of colony formation cm result in a number of intercomected nests, or caü, (Noirot 1970) extending over a large area and supporting a population that can exceed a million individuals ( F o d e r and Townsend 1996). Controlling infestations of subterranean termites is notoriously difficult due to the possibility that if disturbed, a single large colony might divide into severai smder colonies, each capable of sustaining itself and requiring separate treatment (Pawson and Gold 1996).

Conventional termite control

The method used in controllhg termite infestations depends on the moishue requirements of the pest species (Spear 1970). Dry-wood termites are able to establish colonies within pieces of wood that have very little mois&,

such as

packkg mates, furnihue and the foundations of homes. The entire colony is selfcontained, without requiring outside sources of moisture. These infestations are generally treated with chernical insediades that can either be pumped into the

galleries created by termites within infesteci wood or deployed in vapour form as funiigants that penetrate the entire home (Spear 1970; Scheffrahn et al. 1997). Conversely, subterranean termites have high moisture requirements and

must dways remain in contact with the soi1 so that they have access to water. As they begin excavating their galleries within the foundation of a home, they line the

galleries with soil, which is cemented together with saliva and feces. There iç a constant movement of termites between the surrounding soii and the infested structure as they replenish the moisture within their galleries. This system allows

them to maintain high humidity while within a dry environment. Consequently, only a portion of the adud colony may be within the structure, the remainder may

be in the surrounding soil and in adjacent buildings. Because their colonies encompass large areas, treatrnent for subterranean termites is largely preventative

in nature. Chernical barriers are usually establiçhed in the soil around vulnerable structures, which prevents the immigration of termites from nearby sites of

infestation (French 1991; Su et al. 1997). Research has also been conducted on the feasibility of non-chernical barriers,

using materials which subterraneat termites cannot burrow through. Finely crushed cord, of a diameter ranging between 1.l8 mm to 2.8 mm, has been succesçful in laboratory trials at exduding Coptotermes formosanus Shiraki, (Su et

ai. 1991). Stainless steel alloy meshes are available in Australia and are reportedly very effective (Lenz and Runko 1994), but their cost can be prohibitive. Uiatomaceous earth, which is us& as an abrasive desiccant in pest control, has also been investigated, but it does not appear to prevent penetration by termites (Grace

and Yamamoto 1993). Once subterranean termites become established in a home, it rnay become

necessary to replace all parts of the structure that are infested or have sustained major damage. The sumundmg soi1 is then treated with chernical insecticides to

block the entry of termites (Spear 1970). However, public resistance to the use of toxic pestiades in the urban environment has resulted in research into c o n t r o h g

termite infestations with naturally-occurring pathogens.

Biological control Numerous organisms have been investigated for controlling populations of insect pests. Unlike chemical insectiades, pathogenic organisms have the ability to be self-replicating and can be moved throughout the environment by the behaviour

of their host (Villani and Wright 1990). Entomopathogenic nematodes (Berry et al. 1997), insect parasitoids (Sweezey and Vasquez 1991)and fun@ (Gottwald and Tedders 19û4) have been used with varying degrees of success to control pests in agricultural systems. Interest in the biological control of subterranean termites haç led researchers to assess the kasibility of deploying pathogenic nematodes (Trudeau

1989; Logan et al. 1990), ants and ant-derived compounds (Comafius and Grace 1995; 1996; 1997)to suppress termite populations. Entomopathogenic fun@ have been widely investigated for controlling subterranean termites (Kramm et al. 1982; Hanel

and Watson 1983; Delate et al. 1995; WeUs et al. 1995; Zoberi 1995; Jones et al. 1996)

and show considerable promise because the social behaviour of termites may fadtate h g a l dissemination through termite colonies.

The social nahm of termites demands that colony members participate in activities that require frequent body-to-body contact. For instance, the coordination

of foraging, bail-following and colony defense relies upon the exchange of semiochemicals between nestmates (Grace 1991)and the transfer of these chernicals is likely accomplished through mutual grooming and trophollaxis (M&Iahan 1969;

Wilson 1971). Such interactions have been effective at transferring fungd

pathogens between nestmates in laboratory experiments (Myles 1992; Rosengaus and Traniello 1997). Fungi are also excellent candidates for termite control because the soi1 environment inhabited by subterranean termites is ideal for the propagation of

certain pathogenic fungi (Zimmerman 1981; Inglis et al. 1997).

Infection of insect hosts by entomopathogenic fungi

Fungal infections, or mycoses, often follow a typical pattern of development.

The h g a l spore, or conidium, is the infective agent and adheres to the insecYs cutide through a combination of electrostatic forces and mucous production

(Bidodika and Khachatourians 1994). Before conidial germination can occur, the fungus must recognize the insect as an appropriate host. This requiTes that the host cuticie meet the physiological (Tanada and Kaya 1994; Hajek and S t teger 1994) and

nutritional requirements (St. Leger et al. 1994; hiilner et al. 1996) of the conidia. If the h g u s were to corne into contact with an incompahile host species, the fungus

may fail to develop (St. Leger 1993). Penetration of the host's cutide is accomplished through a combination of

mechanical pressure and cuticle-degrading proteases secreted by the fungus (Goettel et al. 1989). Once the body cavity, or hemocoel, of the insect has been readied, the fungus invades the intemal organs and the host evenhially dies, usually from damage to vital organs or by the various toxins that the fungus produces (St. Leger

1993). The Me cycle of the h g u s is complete when it emerges through the host's cutide and b e g h sponilating (Hanel 1982). At this stage, the insect cadaver is a

source of infective propagules and is capable of spreading the disease. Lacking a suitable host, the conidia of some entomopathogenic fungi are able to persiçt in the soil for up to six years, all the while retaining their infective capabilities (Keller and

Zimmerrnan 1989; Weseloh and Andreadis 1997).

Insect responses to fungal infection

The insect immune system consists of several layers of mechanical and

cellular defenses designed to prevent infection by pathogenic fungi. The first b d e r encountered by a huigus is the array of numerous hairs, or setae, whidi cover the inçects exoskeleton. These structures can suspend an infectious conidium

above the cuticle and its supply of nuhients, where it will eventually die (St. Leger 1991). If the surface of the host is reached, germination and hyphal growth c m be

inhibited by the presence of certain lipids found within the the layer of wax on the cutide (Lecuona et al. 1997). In addition, hyphal invasion of the cutide can initiate

melanization directly below the developing fungus, preventing penetration (St.

Leger 1991; Tanada and Kaya 1993). Furthemore, the extrusion of hemolymph into the wound created by the hyphae can seal the route of penetration and initiate

cellular and humoral immunoresponses (Ratdiffe 1993). If the hingus succeeds in breaching the integument and reacheç the hemocoel, it is likely to be recognized by free-floating hemocytes, which rnay destroy it through phagocytosis or encapsdation (Gillespie et al. 1997). The recent discovery of antifmgal proteins in

insect hemolymph rnay provide additional defence against invasion (Gillespie et al. 1997).

The behaviour of insects rnay change dramatically when they become infected by parasites or pathogens, possibly affecting the dessemination of disease (Horton

and Moore 1993). Inçects that are n o m d y nochunal rnay become active during the

day, or insects that are predominantly subterranean rnay move to the soil surface. These authors are of the opinion that such behaviour rnay benefit the overd fitness of the host, as it results in the removal of the infected individual from the viQnity of its kui, reducing the lïkelihood k t they will become infected.

Social insects rnay take an active role in maintainhg the health of their colonies by removing nestmates that are near death. Wilson (1971) and Holdobbler and \ ' i l i n (1990) des&

how ants are able to recognize dead or dying nestmates

and wiu remove them from the colony. Similar observations of necrophoric

behaviour in dry-wood termites by Pearce (1984) reveal that healthy termites bury si& and dead nestmates within chambers in their nests, usually after covering them in feces or resinow secretions. The apparent ability of termites to recognize

and isolate morbid and dead nestmates may h t r a t e attempts at controlling termites with infectious disease as it may M t the exposure that healthy termites

have to diseased members of the colony.

Subterruiean termite problern in Ontario The eastem s u b t e m e a n termite,

K.jla v ipes (Isoptera: Rhuiotermitidae), is

found in 32 muniQpalities in southern Ontario (Myles and Grace 1991). By 1988, almost 5000 homes in Toronto had been treated for termite infestations. It has been

estimated that termite colonies in Toronto encompass entire city blocks and may contain over a million individu& (Grace 1990; Myles 1996). Traditional treatments

in Toronto entail the use of soi1 termiticides, which a d as diemical barriers to foraging termites. However, these barriers do not suppress termite populations and are susceptible to termite penetration as the chernicals leach into the soi1 or degrade over time. The use of entomopathogenic fun@ for suppressing termite populations

may be an alternative to cherrtid treatments, though it is necessary to determine whether termites carrying the conidia of pathoge~cfimgi are able to mix freely with the colony.

The research projed This thesis assesses the feasibilïty of using the h g a l entomopathogen Me fa r h i z i u m a n isop l iae (Metsdmikoff) as a biological control agent against the eastern subterranean termite, Reficulitermesflaoipes (KoUar). The research is


divideci into three areas. First, investigations were carrieci out to determine if termite social behaviour is effectiveai tansferring conidia between nestmates. Also investigated was the question of whether termites are able to recognize individuals

that are infected by pathogenic h g i and prevent their mixing with the rest of the

colony. The second area of researdi determined how termites are able to recognize nestmates that are carrying the conidia of M.a nisopliae. The final topic investigated the influence of termite density and group behaviour on the epizootiology of

h g a l diçeases in termite coIonies.

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The transmission of the fungal pathogen Metarhizium anisupliae (Metschnikoff) Sorokin (Deuteromycotina:Hyphomycetes) within laboratory colonies of the eastem subterranean termite asoptera: Rhinotermitidae)


The transmission of the h g a l pathogen Me ta rhizium a nisopl iae (Metschnikoff) through laboratory colonies of the eastem subtmanean termite, Ref ictîlit ermesflaoipes (KoLiar) was hvestigated. Termites dusted with different quantities of pathogenic conidia were introduced into groups of termites

maintained in bare petn dishes, petri dishes filled with sterile agar, and cups of soil. The behaviour of the resident termites towards those that were dusted with conidia was observed and the mortality recorded. In the bare and agar-filled petri dishes,

resident termites displayed darm behaviour and either groomed or attad