Zoology and Ecology

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Studies on the development of captive colonies

A. K. Harit, S. Gajalakshmi & S. A. Abbasi

To cite this article: A. K. Harit, S. Gajalakshmi & S. A. Abbasi (2016): Studies on the development of captive termite colonies, Zoology and Ecology, DOI: 10.1080/21658005.2016.1228731

To link to this article: http://dx.doi.org/10.1080/21658005.2016.1228731

Published online: 16 Sep 2016.

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Download by: [171.51.18.27] Date: 16 September 2016, At: 09:29 Zoology and Ecology, 2016 http://dx.doi.org/10.1080/21658005.2016.1228731

Studies on the development of captive termite colonies

A. K. Harit, S. Gajalakshmi and S. A. Abbasi

Centre for Pollution Control & Environmental Engineering, Pondicherry University, Kalapet, India

ABSTRACT ARTICLE HISTORY Different authors have tried in the past to develop captive colonies of in their Received 15 June 2016 laboratories, starting with capturing of alate pairs and confining them in Petri dishes or Accepted 23 August 2016 small boxes to facilitate mating, then transferring incipient colonies into progressively larger KEYWORDS containers. Twenty-three termite species of the genera Coptotermes, Cortaritermes, , Swarming; termite; alates; , , Mastotermes, Microtermes, Odontotermes, Pseudacanthotermes, Macrotermes; colony Reticulitermes, , and Zootermopsis have been explored following this method in 33 initiation reported studies. However, in most of the attempts, incipient colonies did not grow beyond the population of a few hundred and tended to die off in a few months. In this paper, we report the efforts made to develop colonies of Hypotermes obscuriceps, Macrotermes convulsionarius, Microcerotermes cameroni, Odontotermes brunneus, Pericapritermes sp., and Trinervitermes biformis found in the study area. Several strategies were attempted to develop termite colonies to maturity. However, none of these efforts resulted in any greater success than the previous attempts of other authors had been, albeit targeted at the development of different species. Although some useful information on termite biology was obtained in the course of the process, the studies performed indicate that termitaria are possibly based on too fine a tuning of food, architecture, humidity, and temperature to be amenable to simulation in laboratory conditions.

Introduction dwelling, and shifted the colony to a bigger container as it grew. Eventually they transferred the colony to the Beginning with the work of Ausat et al. (1960), 33 reports below-ground soil. In the process, even though a giant have appeared on attempts to develop captive termite 6 m tall mound came up and was enveloped by the colonies within the confines of a laboratory (Table 1). But, authors in a glass case (to make it a show-piece), the as may be seen from the summary provided in Table 1, in colony was not really a captive one. more than half of the reported studies, incipient colonies An estimated 3000 species of termites belonging perished within less than 400 days. In most of the colonies, to 281 genera exist in the world. Of these, six species which survived longer, termite populations persisted of higher termites dwell in the area where authors of for a few hundred or fewer days. Recently Connétable, this study work (Kaur 2014) and, arguably none of these Robert, and Bordereau (2012) have reported that they species has been explored earlier for ab initio colony managed to maintain colonies of Pseudacanthotermes development. Moreover, whereas the previous attempts spiniger and P. militaris – both higher termites that make at termite colony development were largely aimed to their nests in wood – for about 20 years. But they needed find ways to control those species of termites, the aim to feed the termites with the fungi Termitomyces eurhyzus of these authors was to use the colonies as ‘bioengines’ and T. auriantacus obtained from fungus combs har- for the processing of specific biowaste. vested from nests existing in nature. Moreover, even at their largest size, the colonies were small enough to be housed in 175 l smaller containers. Use of termites in controlled fashion to process The only instance of human-mediated development biowaste of a full-fledged colony of a mound-building termite species has been described by Leuthold, Triet, and It may sound surprising, but is very true that there is only Schildger (2004). They initiated a colony of M. jeanneli one solid waste treatment technology (indeed only one in confinement, supplied it with small pieces of the fun- pollution control technology) in existence today which is gus comb when the first workers appeared, made elab- centered around a multi-cellular : vermicomposting. orate arrangements to maintain humidity in the termite All other bioprocesses in environmental engineering are

CONTACT S. A. Abbasi [email protected] © 2016 Nature Research Centre 2 A. K. Harit et al. ee ( 2011 ) Reference oka ( 2004 ) ashiro ( 2007 ) ashiro G ( 2007 ) Y ( 2008 ) ( 2010 ) Bordereau ( 2012 ) Bordereau ( 2013 ) dam and Mitchell ( 2009 ) onnétable, Robert,onnétable, and ong, Thorne, and Breisch and Breisch Thorne, ong, Matsuura, Fujimoto, and Fujimoto, Matsuura, L Matsuura, Kobayashi, and Kobayashi, Matsuura, Menzel and Elena D iehl Menzel A Tian et al. ( 2009 ) et al. Tian Shimada and Maekawa N eoh and L houvenc et al. ( 2012 ) et al. C houvenc C Kawatsu and Matsuura ( 2013 ) and Matsuura Kawatsu Howard et al. ( 2013 ) et al. Howard i et al. ( 2013 ) L i et al. Janowiecki, Jones, and BryantJanowiecki, Jones, houvenc and Su ( 2014 ) C houvenc - - Objective of the culture omparison of colony foundation success in terms of survival and the in terms success rates foundation omparison of colony number of progeny between single-female colony foundation (F), female-female foundation single-female between colony number of progeny (FM) (FF), and female-male foundation foundation in parthenogenesis, of alates, chromosomes of the ploidy restoration Examination and parthenogen reproductives supplemental embryos of parthenogenesis determination markers: gens using microsatellite lifespan of founding reproductives, the response of colonies to the loss of founding the loss of founding to of colonies the response reproductives, of founding lifespan ratios factorscaste of intrinsic in determining and the contribution kings and queens, R. flavipes colonies of the laboratory reared and individual body sizes facultatively parthenogenetic facultatively termite tive strategies, and if so, whether different combinations of individuals lead to the of individuals lead combinations whether different and if so, strategies, tive success same reproductive from pairing; assessment of the possible strategies developed for energy conserva energy - for developed of the possible strategies pairing; assessment from and mortality development the establishment, of colonies investigating tion by vation of nesting and mating behavior, oviposition and egg hatching, molting, and molting, and egg hatching, oviposition behavior, of nesting and mating vation differentiation caste the development of reproductive characteristics (vitellogenic ovariole numbers and ovariole characteristics (vitellogenic of reproductive the development of queens and kings diameter) testis their analysis in terms of colony composition and sex ratio and sex composition of colony in terms their analysis stimuli that elicit the buryingstimuli that response P. militaris P. species by comparing incipient colony success and success colony incipient comparing species by Reticulitermes tion in six different pair-units different between behavior founding , which were allowed to interact, merge and merge interact, to allowed , which were of Z. nevadensis and helpers in colonies frequency in colonies’ of mixed-family Measurement in the laboratory. develop settings natural entry time, physical damage to antennae, and fresh body weights of male dealates; of male dealates; body weights and fresh antennae, damage to entry physical time, in partner selection involved which factors are elucidate to in order through the parameters of numbers, castes and/or developmental stages, and stages, and/or developmental castes of numbers, the parameters through biomass and laboratory colonies, and mature field colonies of C . formosanus field and mature and laboratory colonies, etails of biometric descriptors of both immature and sexual castes of C. dudleyi and castes and sexual of both immature etails of biometric descriptors O. hainanensis . O bser and O. formosanus of O. colonies incipient ulturing methods for and observations of incipient, mature and observations mature in C . formosanus of incipient, pathway development aste

(1) c (2) (3) in queens of F and FF pairs their partheno of heterozygosity c haracterization To obtain demographic information regarding colony growth rate and longevity, and longevity, rate growth colony regarding information obtain demographic To Size, hatching rate, and hatching period of sexually and asexually produced eggs in the produced and asexually period of sexually and hatching rate, hatching Size, shows faculta - shows C. fulviceps males, ascertain of reproductive whether in the absence To over a period of 25 weeks a period of 25 weeks trinervoides over of T. colonies dynamics within incipient Energy C hanges in wood digestion abilities (endogenous cellulase gene expression levels) and levels) gene expression cellulase digestion abilities (endogenous C hanges in wood D Burial behavior of young reproductives of incipient colonies and examination of the and examination colonies of incipient reproductives of young Burial behavior P. spiniger and P. termites of fungus-growing development colony and D ispersal flight bility of parthenogenesis and occurrence of homosexual female-female colony initia - A bility female-female of parthenogenesis of homosexual colony and occurrence Use of genetic markers to determine the family of origin of reproductives, soldiers, soldiers, of reproductives, the family of origin determine to of genetic markers Use , including the colony origin, origin, in R. chinensis , including the colony choice four factorsEffects on mate of the during the first year of R. flavipes during the first monogamous colonies dynamics of inbred G rowth C finement attained finement and 712 ml Duration of study and Duration - of con maximum size 400 days: not reported 400 days: 11 years; 5.1 l 11 years; 235.8 ml 150 days; 10.92 l 150 days; 25 weeks; 84.8 ml 25 weeks; 90 days and 45 days; 600 l and 45 days; 90 days 400 days; 20 ml 400 days; N ot reported 50 days; not reported 50 days; round 20 years; 175 l 20 years; A round 100 days; 127 ml 100 days; 2 years; 35.6 ml 2 years; N ot reported 1 year; 87 ml 1 year; 7 months; 39.3 ml 7 months; 76 mm) culture × Container and substrate used for used for and substrate Container wet and sterilized filter paper, and paper, filter and sterilized wet with a thin soil layer covered soil stacked together with a 6-mm space with a 6-mm space together stacked filled with the was them that between medium culture yellow-clay 8 g of mixed sawdust 8 g of mixed with wet clayish soil clayish with wet paper birch paper pad and a combination of moist paper pad and a combination mulch softwood and hardwood moistened organic soil organic moistened Petri dish containing moist filter paper moist filter dish containing A Petri A plastic container glass cell (52 A glass cell Petri plates were used, autoclaved, with autoclaved, used, were plates Petri Flat-bottom glass pill vials with moist Flat-bottom 3 120 W mm) (150 L 3 120 W mm) glass plates Two 20 ml glass vial with approximately A 20 ml glass vial with approximately N ot reported Petri dish with moistened sand dish with moistened A Petri Small rectangular plastic boxes filled up Small rectangular plastic boxes Petri dish with sawdust bait blocks dish with sawdust A Petri Petri dish with field-collected decayed dish with field-collected decayed A Petri Petri dish with moist filter paper dish with moist filter A Petri plastic nest container with moist filter with moist filter A plastic nest container A plastic cylindrical vial with 6 g of - graphic location graphic frica Termite species and geo Termite A O . hainanensis , C hina France , France P. militaris ttempts made to develop incipient colonies in laboratories. colonies incipient develop made to ttempts , US A speratus Reticulitermes Reticulitermes flavipes , US A Reticulitermes , Japan speratus Reticulitermes , Brasil Cortaritermes fulviceps Trinervitermes trinervoides , South Trinervitermes and formosanus Odontotermes , Japan speratus Reticulitermes Cryptotermes dudleyi , Malaysia Pseudacanthotermes spiniger , Pseudacanthotermes Pseudacanthotermes spiniger and Pseudacanthotermes sp., Japan sp., Reticulitermes , US A nevadensis Zootermopsis C hina chinensis, Reticulitermes Reticulitermes flavipes , US A Reticulitermes , US A formosanus Coptotermes 15. 14. 13. 12. 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. 1. Table 1. A Table Sample no. Zoology and Ecology 3 ishida ( 2001 ) euthold ( 1981 ) ancello ( 2004 ) ancello rab, and A rab, eonardo, asarin ( 2004 ) ( 2004 ) ( 1993 ) ( 2004 ) C usat et al. ( 1960 ) et al. usat osta- L euthold, Triet, and Schildger Triet, euthold, A Watson ( 1974 ) Watson Watson et al. ( 1978 ) et al. Watson Sieber and L Johnson ( 1981 ) Kirchner and MinkleyKirchner ( 2003 ) Howard et al. ( 1981 ) et al. Howard Raina et al. ( 2003 ) Raina et al. Sieber ( 1983 ) Rojas and Morales-Ramos Rosengaus and Traniello Rosengaus and Traniello Ferraz and C Ferraz Brent and Traniello ( 2001 ) Traniello and Brent L Matsuura and N Matsuura C Wiltz et al. ( 2001 ) et al. Wiltz Fei and Henderson ( 2003 ) Fei - with basidiospores with basidiospores . montanus To study the beginning of colony foundation among single–female, female–fe foundation of colony study the beginning To and male–femalemale, units mortal female indices: - three by as measured success unit foundation compare To per developmental and proportion of progeny per female, number of progeny ity, stage laboratory tion of development and origin of soldiers in colonies of different age of different of soldiers in colonies and origin tion of development and females of different species, and pairs of conspecific females conspecific and pairs of species, of different and females of the first foraging workers of M. michaelseni and O workers foraging of the first sp. Termitomyees of the corresponding mushrooms collected from primary reproductives by using incipient colonies grown on survival grown colonies using incipient primary by of C. formosanus reproductives artificiala nutritionally balanced diet growth of incipient colonies in the damp wood termite in the damp wood colonies of incipient growth choice by reproductive pairs, colony survival rate, pre-oviposition survival number period, colony rate, pairs, reproductive by choice mortality instar period, of worker worker rate period, incubation and viability of eggs, as some important as well behavioral of soldiers, molting and percentage instars, substrates in different responses tion in laboratory after orphaning colonies of the formosan subterranean termite subterranean of the formosan incipient colonies incipient C. formosanus escription of various methods used for the maintenance of small colonies in the of small colonies the maintenance methods used for escription of various aste ratios in a long-established, laboratory colony neotenic-headed ratios aste sp. in Microtermes establishment and fungus comb development olony

D Morphological differences between soldiers and their mature counterparts; examina - soldiers and their mature between differences Morphological C To observe and describe behavioral elements of different castes in incipient colonies in incipient castes of different elements observe and describe behavioral To C est mate recognition in the ‘lower’ termite H. mossambicus termite ‘lower’ in the recognition N est mate To study incipient colony formation with males and females of the same species, males of the same species, with males and females formation colony study incipient To Reproductive biology of the Formosan subterranean termite subterranean biology of the Formosan Reproductive To investigate whether the fungus comb establishment could be induced by feeding feeding by be induced could establishment whether the fungus comb investigate To To study the effect of three selected chitin synthesis inhibitors on fecundity and on selected inhibitors chitin synthesis study the effect of three To Effect of the sibship of primary reproductives on mate mortality, survivorship mortality, Effect and of the sibship primary on mate reproductives colonies, viz. substrate viz. substrate colonies, C. gestroi of incipient behavior study the post-swarming To To determine whether sex-specific cues regulate reproductive maturation in females in maturation reproductive regulate cues whether sex-specific determine To To describe the method of incipient colony breeding in laboratory conditions breeding colony describe the method of incipient To (1) (2) To elucidate some aspects- induc elucidate neotenic of the secondary in C. gestroi, reproduction To Effect of a nutritional metabolism disrupter on the development of incipient colonies of incipient on the development Effect of a nutritional metabolism disrupter Effect of sibship (sibling and non-sibling) and colony origin on growth and mortalitygrowth on origin of colony Effect of sibship (sibling and non-sibling) round 3 years; 722.27 ml 3 years; A round 2 years; not reported 2 years; 18 years; not reported 18 years; 6 months; 190 ml 6 months; 13 weeks; 255 ml 13 weeks; 3.042 l 200 days; 56.07 ml 200 days; 2 years; 80 ml 2 years; 105 and 146 days; 617.4 ml 105 and 146 days; 180 days; 17.7 ml 180 days; 4 years; 117.75 ml 4 years; 2 years; 95.4 ml 2 years; 60 days; 67 ml 60 days; 20 years; 6 m 20 years; 150 days; not reported 150 days; 3 years; not reported 3 years; 100 days; 198.5 ml 100 days; 100 days; 143 ml 100 days; - -

3 agar from a termite mound a termite from towel and approximately a 11 cm and approximately towel sterile soil sterile paper towel towel and wood towel (saw dust, sand, vermiculite, and filter and filter vermiculite, sand, dust, (saw paper) vivarium house with sieved moist house with sieved vivarium soil mineral ven cloth made of 100% pulp, and a cloth made of 100% pulp, ven bait sawdust block of mixed plastic containers filled with decayed filled with decayed plastic containers sp. and Eucalyptus sp. of Pinus sawdust with distilled water moistened ed bait or bait treated with the nutri - ed bait or treated which is tional metabolism disruptor, made of oxypurinol and xanthine acetone-rinsed block floral absorbent ontainers (not reported)ontainers Petri dish with moist soil, saw dust, dust, saw dish with moist soil, A Petri Petri dish and large containers dish and large A Petri C Petri dish with sieved moistened soil moistened dish with sieved A Petri Petri dish containing a moist paper dish containing A Petri lass plates and aluminum foil with and aluminum foil G lass plates Plastic containers with soil containers Plastic Petri dish and moistened filter paper filter dish and moistened A Petri Petri dish with agar-sawdust mixture dish with agar-sawdust A Petri Petri dish with moist soil A Petri 11.3 liter storage boxes lined with a wet lined with a wet boxes storage 11.3 liter Petri dish lined with a moist paper A Petri Petri dishes with different substrates substrates dishes with different Petri Plastic cups containing birch sawdust birch cups containing Plastic Plastic boxes, a plastic bucket, and a a plastic bucket, boxes, Plastic Petri dish lined with moistened unwo dish lined with moistened A Petri Petri dish containing sawdust and sawdust dish containing A Petri Plastic dishes containing either untreat dishes containing Plastic igeria ermany ustralia ustralia A A Switzerland G , G ulfport virginicus ulitermes O rleans , Kenya montanu Odontotermes O rleans Boston O rleans Odontotermes Mastotermes darwiniensisMastotermes , Mastotermes darwiniensisMastotermes , Macrotermes michaelseni , Macrotermes , US A formosanus Coptotermes sp., N sp., Microtermes Hodotermes mossambicus , Hodotermes Reticulitermes flavipes and Retic - Reticulitermes , N ew formosanus Coptotermes Macrotermes michaelseni and Macrotermes , N ew formosanus Coptotermes , angusticollis Zootermopsis , Brazil gestroi Coptotermes , U. S. A , U. angusticollis Zootermopsis Macrotermes jeanneli, Bern Macrotermes , Japan speratus Reticulitermes , Brazil gestroi Coptotermes , N ew formosanus Coptotermes 33. 32. 31. 30. 22. 29. 21. 28. 20. 27. 19. 26. 18. 25. 17. 24. 16. 23. 4 A. K. Harit et al. based on the direct use of enzymes, microorganisms or earthworms can fluently reproduce within reactors, botanical species. and thus new worms are generated even if the old ones We have been studying vermicomposting from the die. But the same is not possible with termites, because bioprocess engineering perspective in order to under- worker termites do not ordinarily breed, or even survive stand the mechanism of the process, to model it, and to if separated from their colony. This makes it necessary develop criteria for the design, operation, and control of that captive termite colonies should be developed within vermireactors in such a way that the maximum output the confines of a box or a room so that they could be is achieved at the minimum cost, and in a sustainable then made to forage upon the waste we wish to pro- fashion. While conducting experiments to assess the cess. It is like ‘domesticating’ termites for use in degrad- preference of different earthworm species for different ing ligninous and other ‘hard’ solid waste, which resists substrates, we have repeatedly encountered situations composting/vermicomposting. when ligninous and other ‘hard’ biowaste was either not The present paper reports attempts to develop colo- consumed by earthworms or, if forced to feed upon it, the nies of Hypotermes obscuriceps, Macrotermes convulsion- lost weight and died long before their normal arius, Microcerotermes cameroni, Odontotermes brunneus, life span. This type of waste was also not amenable to Pericapritermes sp. and Trinervitermes biformis, which are composting (Ganesh et al. 2007a, 2007b). the species found in the region where the authors of But most of the biodegradable solid waste such as the current paper work. None of these species appear MSW (municipal solid waste) and weeds like ipomoea to have been explored for this purpose before. contain appreciable portions of ligninous and other The initiation of all termite colonies occurs in nature ‘hard’ substances, which defy composting and vermi- by winged reproductive individuals (alates), which composting. Very few animals in nature – mostly a few emerge from pre-existing colonies at certain times of species of insects – are capable of digesting lignin. Of the year. Alates then swarm towards different sources of these, termites are most numerous and play a decisive light. This enables a large number of individuals to come role as scavengers (Premalatha et al. 2013). We are try- together and facilitate mating. As a prelude to the pres- ing to harness this ability of termites to make controlled ent work, studies on the swarming behavior of different use of them in treating lignocellulosic biowaste. But, species in terms of period/season of emergence, dura- unlike earthworms, termites cannot be easily cultured tion and frequency of swarming etc. were first carried out as explained below. (Harit, Gajalakshmi, and Abbasi 2014). Simultaneously,

traps were designed and the designs were optimized for non-destructive capturing of alates (Harit, Gajalakshmi, Special challenges in developing termite colonies and Abbasi 2016). The present work is the next step in in captivity the sequence. Termites are highly eusocial insects, and the social class that is utilizable to feed upon waste (i.e. worker termites) Materials and methods does not breed (except in the case of a few species of the ‘lower’ termites, and in special circumstances). Also Alates of different species were collected using light they are animals with a rigid hierarchy and sharp division traps as detailed elsewhere (Harit, Gajalakshmi, and of labor. Except under special circumstances, and only Abbasi 2016). Information on the number of times differ- in certain species, auxiliary colonies can be started by ent species swarmed, and the number of alates of each worker/soldier termites when they get separated from species trapped by us to initiate colonies is summarized the parent colony. Otherwise workers do not reproduce, in Tables 2 and 3. and no termireactor can be operated at any significant After each swarming, the trapped alates were brought length of time with worker termites picked from the to the laboratory, placed in containers and were allowed field. In vermicomposting, the sustainability of reac- to lose their wings. The sex of the alates was determined tor operation is made very easily achievable because on the basis of sternum and styli arrangement (McMahan

Table 2. The number of times alates of different species swarmed in 2009 and the number of colonies of each species that were initiated in different types of containers. Number of dealates in each Number of colonies started in experiment Petri-dish + Plas- Petri-dish Plastic container tic container Petri- Termite species Number of swarmings Numbers % Numbers % Numbers % dish Plastic container Hypotermes obscuriceps 2 55 15.5 25 16.4 80 15.8 4 6 Macrotermes convulsionarius 4 203 57.5 83 54.6 286 56.6 4 6 Odontotermes brunneus 3 55 15.5 27 17.7 82 16.2 4 6 Trinervitermes biformis 2 40 11.3 17 11.1 57 11.2 4 6 Total 11 353 152 505 Zoology and Ecology 5

Table 3. The number of times alates of different species swarmed in 2010 and the number of colonies of each species that were initiated in different types of containers. Number of dealates in each Number of colonies started in experiment Petri-dish + Plas- Petri-dish Plastic container tic container Number of Termite species swarmings Numbers % Numbers % Numbers % Petri-dish Plastic container Hypotermes obscuriceps 3 15 2.6 25 17.7 40 5.7 4 6 Macrotermes convulsionarius 1 200 35.8 20 14.1 220 31.4 4 6 Microcerotermes cameroni 3 38 6.8 13 9.2 51 7.2 4 6 Odontotermes brunneus 5 170 30.4 45 31.9 215 30.7 4 6 Pericapritermes sp. 1 72 12.9 30 21.2 102 14.5 4 6 Trinervitermes biformis 2 63 11.2 8 5.6 71 10.1 4 6 Total 15 558 141 699

1960; Pearce 1997; Hu and Forschler 2012). Those alates Rosengaus, and Traniello (2007) for Zootermopsis angus- that did not shed their wings were dealated by breaking tics but the dimensions of the plastic boxes used by the off the wings by bending them forward over the head. two groups of authors were different from each other. After dealation, all the insects were transferred into Adam and Mitchell (2009) have attempted to culture the plastic boxes or Petri dishes species-wise. Based on the colony in flat-bottle glass pill vials with Trinervitermes number of individuals trapped in each swarming, a num- trinervoides. We have used containers of different sizes ber of experiments (including replicates) were started and shapes to see if any particular type leads to better accordingly as detailed in Tables 2 and 3. Into each Petri results. dish and plastic box, four and six alates were introduced Five substrates were used as media for termite cul- respectively. After the introduction, it was observed that ture: normal soil (red-ferralitic), mound soil, sawdust, a the males pursued the females, with their head close to mixture of sawdust and mound soil, and filter paper. All or in contact with the tip of the female abdomen, and plastic boxes and Petri dishes were filled with respec- keeping their antennae in contact with the sides of her tive substrates to about half of the container height. All abdomen. It is tandem pairing similar to the one reported the substrates were previously sieved and oven-dried by several other authors (Raina et al. 2003; Park, Bland, (except filter paper) to prevent contamination with fungi and Raina 2004; Husseneder and Simms 2008; Hanus et and other microorganisms as reported by Ferraz and al. 2009; Kawatsu and Matsuura 2013). Cancello (2004). Matsuura, Fujimoto, and Goka (2004), Costa-Leonardo, Arab, and Casarin (2004), Matsuura and Nishida (2001), Calleri, Rosengaus, and Traniello (2007), Culture containers and substrates used for culture Ferraz and Cancello (2004), Adam and Mitchell (2009), For rearing of incipient colonies, three types of con- Howard et al. (1981), and Ausat et al. (1960) have also tainers were used – polystyrene Petri dishes (10 cm used sawdust, filter paper, and soil. However, Thorne, diameter), rectangular plastic boxes of different sizes Breisch, and Haverty (2002), Park, Bland, and Raina (20 × 12.5 × 6.5 cm, 23 × 15 × 8.5 cm, 30 × 20 × 10 cm, (2004), Ferraz and Cancello (2004), and Tian et al. (2009) 38 × 24 × 12 cm), and cylindrical plastic boxes have used yellow clay, mixture of sawdust and agar, ver- (18.5 × 21.8 cm, 10.4 × 12.5 cm, 6.3 × 7.3 cm). Matsuura miculite and decayed white birch for termite rearing. In (2005, 2006), Ferraz and Cancello (2004), Matsuura, this study, several substrates were used to determine Fujimoto, and Goka (2004), and Matsuura and Nishida which substrates facilitate colony development and (2001) have used Petri dishes of 9 cm diameter to rear therefore are more suitable. termites in laboratory conditions, whereas Rosengaus Two or three pairs of randomly selected dealates and Traniello 1993 have used Petri dishes of the same (two in each Petri dish and three in each plastic box) dimensions as reported in this study. Costa-Leonardo, were placed in each container. They were placed on Arab, and Casarin (2004), Costa-Leonardo, Arab, and the top of substrate. To maintain moisture, a few drops Hertel (2005), Thorne, Breisch, and Haverty (2002), of water (10–12 ml for Petri dishes, 100–200 ml for and Watanabe and Noda (1991) have employed Petri plastic containers, and 0.5–1.0 ml for filter paper) were dishes of 6 cm diameter to rear the colonies. However, added to moisten and soften the substrate. This was the Petri dishes used for termite rearing by Tian et al. to make it easy for termite dealates to construct their (2009), Park, Bland, and Raina (2004), Raina et al. (2003), nuptial chambers. Since the chance of mortality of Fei and Henderson (2003), Sieber (1983), and Sieber and the collected alates was high, more than one pair of Leuthold (1981) were of different diameters: 18, 5, 4.5, alates was introduced into each container to enhance 6.5, and 11 cm respectively. Plastic boxes were used by the possibility of colony initiation. The reproductives Connétable, Robert, and Bordereau (2012) for culturing were seen to groom each other, confine themselves in Pseudacantotermes spiniger and P. militaris, and Calleri, a corner of the container, and begin constructing the 6 A. K. Harit et al.

Figure 1. A typical nuptial chamber (A) and eggs (B) of Hypotermes obscuriceps. nuptial chamber. In a few instances, when more than Colonies of other species never grew large enough to one pair survived, one chamber for each surviving pair necessitate their transfer to large boxes. All the containers was seen. All the containers were kept closed. Within were maintained at 25–30 °C and 80–90% relative humidity. a day, in most of the containers, tandem behavior was They were covered with a black plastic sheet to prevent observed and the termites were seeking a suitable desiccation of soil and termites. When the soils appeared place to establish the colony and construct the nuptial dry on touch they were moistened with distilled water. chamber. Antennae mutilation also occurred within a All colonies were observed daily during the first day. A total of 1204 colonies were started: 911 in Petri 10 days of their inception. If any individual was found dishes and 293 in plastic boxes. trapped in condensed moisture, it was freed. Dead Of all the termite species studied in this work, dealates were replaced with live ones. After the first 10 Macrotermes convulsionarius was the most successful in days, dead dealates were removed but not replaced. terms of the survival rate of its juveniles and adults, and Subsequent inspections were made thrice a week for the construction of its tunnels and mounds. This has been following month, then twice a week for the next month, detailed in the next section. As the number of individ- once a week thereafter until the experiment was termi- uals increased in the Petri dishes containing M. convul- nated or dealates were dead. sionarius, the contents of 30 Petri dishes, containing reproductives, eggs, nymphs, workers, and soldiers were Results and discussion transferred into larger plastic boxes. This was done by cutting the edge along the circumference of each Petri Mating and colony initiation dish so that transfer of the contents could be made In general, in all species mating was observed within 24 h without causing major disturbance. The Petri dishes after the start of the experiment. In all cases a male and were placed inside the box with lids closed. Fifteen of a female engaged in extensive grooming of each other the plastic boxes were filled with normal soil and another before positioning themselves for the tail-to-tail mating. 15 with mound soil. In each set of 15 with both kinds of They then found a favorable place, and built a nuptial soil, five types of feed (wood pieces, leaf litter, wooden chamber (Figure 1). Courtship was concluded with cop- chip, grass, and filter paper) were supplied in triplicate. ulation, which was periodically repeated throughout the After one week, the colonies existing in six of the Petri life of the pair. In most of the cases, only one nuptial dishes shifted to the plastic container. In the remaining chamber was seen. In a few rare instances, when there sets, the termites continued to stay in the Petri dishes. In was more than one pair of dealates in a nuptial chamber, six experiments, mortality was recorded within 2 weeks. only one of the pairs took an active part in copulation, When the termites did not shift to the plastic box or while the other one became passive and eventually died. did not find forage in the plastic box, the lids of the corre- In two previous reports on Odontotermes obesus (Ausat sponding Petri dishes were removed in order to provide 1960) and Odontotermes hainanesis (Tian et al. 2009), greater opening for the termites to move to larger con- nuptial chambers were found to be constructed in 30 tainers. It was noticed that in a week, adults and young min. In our study, it was also found that nuptial chambers ones shifted to plastic boxes from almost all Petri dishes. were constructed within 30 min by all species. However, from four Petri dishes, termites did not move; instead, their workers brought wooden chips and small pieces of grass from the boxes to their Petri dishes. Survival of dealates, egg production and Once the existing colony shifted from a Petri dish to incubation a larger box, and moved below the soil surface, it was The time from the first copulation to the laying of eggs very difficult to record whether its members were alive varied with species, ranging between 4 and 30 days in or dead until the termite-processed soil of distinct visual most cases (Tables 4 and 5). The highest percentage of appearance was seen on the surface. eggs laid within 5 days from the start of the experiment Zoology and Ecology 7

Table 4. Oviposition, incubation and maximum survival period of the incipient colonies started in 2009. Time taken for the Time taken for the appearance of the first appearance of the first Species Experiment started on batch of eggs (days) larvae from eggs (days) Maximum survival period Hypotermes obscuriceps 2 September 2009 6 28 3 months 28 days 11 35 5 November 2009 12 30 3 months 15 days Macrotermes convulsionarius 16 August 2009 11 32 7 months 29 days 15 21 3 September 2009 7 33 9 months 8 33 13 34 5 November 2009 4 32 5 months 26 days 44 10 November 2009 4 15 7 months 13 days 7 34 Odontotermes brunneus 26 August 2009 – – 8 days 2 September 2009 8 30 7 months 23 days 11 35 5 November 2009 7 34 4 months 25 days Trinervitermes biformis 31 October 2009 30 17 7 months 3 days 1 November 2009 130 34

Table 5. Oviposition, incubation and maximum survival period of the incipient colonies started in 2010. Time taken for the Time taken for the appearance of the first appearance of the first Species Experiment started on batch of eggs (days) larvae from eggs (days) Maximum survival period Hypotermes obscuriceps 29 October 2010 8 27 4 months 5 days 30 October 2010 6 28 3 months 25 days 8 27 11 November 2010 – – 5 days Macrotermes convulsionarius 24 August 2010 6 33 8 months 24 days 7 33 9 34 11 33 Microcerotermes cameroni 1 May 2010 18 18 2 months 26 days

20 19 5 June 2010 17 19 3 months 21 days 20 20 23 June 2010 18 31 2 months 18 days Odontotermes brunneus 23 August 2010 4 27 6 months 29 days 5 29 24 August 2010 6 27 7 months 5 days 8 28 25 August 2010 5 26 5 months 22 days 6 26 4 October 2010 6 33 3 months 3 days 28 October 2010 5 30 4 months 16 days Pericapritermes sp. 18 May 2010 6 25 4 months 15 days 7 25 Trinervitermes biformis 25 August 2010 5 29 5 months 7 days 9 November 2010 7 28 6 months 11 days

Table 6. Appearance of the first batch of eggs in the incipient position of the eggs in the nuptial chamber, but moved colony. them in groups when they did so. As the time passed, Number of days for the appearance of the first batch of eggs the eggs increased in size, became opaque and lost their Days Percentage smoothness, indicating the development of nymphs 0–5 17.5 in them. In a few instances, when the eggs were with- 6–10 47.5 out adult animals, they got diseased and died. Sands 11–15 17.5 16–20 12.5 (1965) observed the oviposition in Trinervitermes germi- More than 21 5.0 nates within 5 days, whereas in Odontotermes obesus, it occurred 3–4 days after pairing (Ausat et al. 1960). It took 8–11 and 2–5 days for O. formosanus and O. hainanensis was 18%. The maximum percentage (48%) of egg pro- respectively (Tian et al. 2009) to produce eggs. The time duction was recorded between 6 and 10 days and the required for the appearance of the first instar larvae after minimum (5%) after 21 days (Table 6). the hatching of eggs also varied with species ranging Reproductive termites produced eggs in batches between 12 and 44 days. Of these, 83% of H. obscuriceps (Figure 2). The freshly laid eggs appeared smooth and and 73% of O. brunneus eggs hatched within 28 ± 2 days. uniform in size. Parent termites frequently changed the The corresponding periods were 19 ± 1 days for 80% of 8 A. K. Harit et al.

Figure 2. Reproductive termites with eggs and nymphs: Odontotermes brunneus (A) and Trinervitermes biformis (B).

Figure 3. The adult and the nymphs of Macrotermes convulsionarius exhibiting trophallaxis (A) and the pillar-like structure constructed by M. convulsionarius inside the nuptial chamber (B).

M. cameroni and 33 ± 1 days for 77% of M. convulsionarius with M. convulsionarius. The pillar-like structure was eggs. Leuthold, Triet, and Schildger (2004) have reported constructed in Petri dishes with filter paper and sawdust that it takes Macrotermes jeanneli instar larvae 40 days as culture media 95 days after the start of the experi- to emerge, and Sands (1965) has found the correspond- ments (Figure 4). In experiments conducted in plastic ing period in Trinervitermes sp. to range between 26 and containers (with mound soil and normal soil as culture 42 days. media), small mound-like structures were visible after The first instar larvae were milky white in color and 10 days of the start of the experiments, indicating that immediately after hatching were motionless. They started the individuals had matured and had started to forage. to move a little in the following 48 h. Only after 48–72 h, It is reported that the fungus-growing termites of the they started to move around briskly. Similar observation family eat wood and litter, which after has been made by Ferraz and Cancello (2004). The first passing through their gut is deposited as pellets in a larvae were subjected to mouth-to-mouth feeding by comb-like structure in the nest (Wood and Thomas 1989). the parents (stomodeal trophallaxis) (Figure 3(A)). When In the present study, the pillar-like structure of M. con- workers matured, the authors provided them with small vulsionarius composed of small aggregates of soil was pieces of feed (such as grass, leaf litter, small wooden constructed inside the chamber, indicating that the col- piece, ice cream scoop) inside the closed containers. Of ony had developed to the stage at which workers start to these, the most preferable feed was leaf litter followed forage. Similarly, short pillar-like structures were reported by pieces of wood, and grass. by Johnson (1981) in the copularium of Microtermes sp. Workers and soldiers (about 15–20 workers and 1–2 comprising several balls of soil (fecal pellets), which were soldiers per colony) were noticed 30–32 days after the attached to the floor of the copularia. Sieber (1983) also appearance of the first larvae (Figure3 ). The number of reported the presence of gray pillars in a chamber of days it took all the species used in the present study to Macrotermes michaelseni and Odontotermes montanus, produce eggs, to become nymphs and then turn into whereas Sieber and Leuthold (1981) reported the depo- workers/soldiers is given in Tables 4 and 5. sition of fecal pellets on the fungal comb of the incipient colony of M. michaelseni. The number of individuals in the first brood of work- Foraging and construction of pillars and mound- ers, the timing of their appearance, and the beginning like structures of food-collection by them are important indicators for Out of the six species studied, small pillar, bridge, or the likely success of an emerging colony. Lepage and mound-like structures were constructed in containers Darlington (2000) have reported that in , the Zoology and Ecology 9 development of steriles is faster than in lower termites. and Pyemotes scolyti have been reported to parasitize It is reported to be 30–32 days for Macrotermes bellicosus laboratory cultures (Becker 1969). They attach them- (Collins 1977), 22 days for M. subhyalinus (Lepage 1974), selves onto the head capsule or other parts of the termite 24 days for M. michaelseni (Okot-Kober 1980; Lepage body. Their development in a weak termite colony can 1990), and 23–28 days for Allodontermes giffardii (Bodot lead to its extinction. Early removal of infected animals, 1967). Cheo (1948) recorded the appearance of workers if possible, is the only effective way of controlling and in O. formosanus culture after 110 days, whereas Ausat reducing mites. et al. (1960) reported the foraging of O. obesus workers When an individual seemed to have died due to an after 65 days. infection, the other members of the colony either sealed the dead body off in a small chamber or completely cov- ered it with soil/sawdust to prevent the spread of the Mortality pathogen. A dead individual was never left as it is in any The highest mortality rate of primary reproductives was existing colony, and if not isolated/buried as reported recorded during the first week, after which it decreased. above, was eaten up. The body parts not utilized were Mortality was also observed after the incubation, which kept aside or transported away from the nuptial chamber then reduced once the first workers appeared. Pearce by other colony members. Sieber and Leuthold (1981) (1997) has also reported similar observations concerning have reported that no dead soldier or reproductive indi- soil dwelling termites. Mortality was due to fungal and/ vidual was ever left unattended in a nest but was always or bacterial infections as witnessed by the presence of eaten up. The unpalatable parts of carcasses, such as fungal growth on bodies of dead individuals, and the head capsules and legs were transported out of the nest. appearance of exoskeleton. In some cases (approxi- Sieber (1983) has reported that 58 and 82 days after mately 10% of the cases), mite attacks were observed the emergence of M. michaelseni and O. montanus (in (mostly after one month of the start of the experiment). the laboratory colony) workers respectively, they started Some mites are parasitic to termites (Becker 1969; dying. They were soon followed by soldiers and larvae Pearce 1997). In particular Tyrolichus casei (Gallo 1955) and finally the two reproductives. After 105 and 146 days

Figure 4. Mound-like structures made by Macrotermes convulsionarius.

Figure 5. Dead bodies of termites attacked by (A) fungi and (B) mites. 10 A. K. Harit et al. all colony members were dead. However Cheo (1948) experience of other authors in colony development of reported survival of the oldest pairs of O. formosanus for 23 other termite species has been similar, it can be con- 200 days, while Tian et al. (2009) reported a 50% survival cluded that the external control of termite diet and envi- rate after 180 days of the establishment of the incipient ronment right up to the maturing of their colony may colony and survival of O. obesus individuals for 947 days be impossible given the very fine tuning that termites (Ausat et al. 1960). In the present study, the last colony of are capable of achieving (and the template of which is M. convulsionarius survived for up to 9 months and that not known). of O. brunneus for 7 months and 23 days. Other authors have advanced various explanations Authors’ contributions for the reasons why despite a successful start of incip- ient colonies no full-fledged termite colony has been All the three authors have made equally substantial con- developed in laboratory conditions. Two of the reasons tributions to the conception, and design, and interpre- suggested are as follows: competition for the available tation of the experiments as well as to the manuscript resources (Guerreiro et al. 2007), and the limited amount drafting. of energy available to incipient termite colonies (Adam and Mitchell 2009). Rosengaus and Traniello (1993) have Acknowledgement reported that in Zootermopsis angusticollis non-sibling pairs suffered significantly higher mortality than sibling The authors are thankful to the Department of Biotechnology, pairs. Some authors have reported infections caused by Government of India, for financial support in the form of a major project BT/PR11488/AGR/21/289/2008. Harit thanks mites, fungi and/or bacteria (Becker 1969; Pearce 1997) University Grants Commission (UGC) for the Rajiv Gandhi as the reason for the discontinuation of colony develop- National fellowship, and SAA thanks the UGC for Emeritus ment. In the present study, more than one of the causes, Professorship and associated grants. including a fungal infection and a mite attack (Figure 5), seem to have terminated incipient termite colonies. Funding This work was financially supported by the Department of Biotechnology, Government of India (R and D project) [Project Summary and conclusion number BT/PR11488/AGR/21/289/2008].

Attempts to develop colonies of six termite species (Hypotermes obscuriceps, Macrotermes convulsionarius, References Microcerotermes cameroni, Odontotermes brunneus, Pericapritermes sp., and Trinervitermes biformis) have Adam, R. A., and J. D. Mitchell. 2009. “Energetics and Development of Incipient Colonies of the Harvester been described. None of these species seems to have Termite, Trinervitermes trinervoides (Sjöstedt) (Termitidae, been studied for this purpose earlier. In each case, alates ).” Insectes Sociaux 56: 21–27. of the concerned species were herded together and were Ausat, A., P. S. Cheema, T. Koshi, S. L. Perti, and S. K. Ranganathan. found to mate within 24 h after the start of the exper- 1960. “Laboratory Culturing of Termites.” In Termites in the iment. Construction of nuptial chambers commenced Humid Tropics, Proceedings of the New Delhi Symposium, 121–125, UNESCO, Paris. within 30 min of mating. The time between the first cop- Becker, G. 1969. “Rearing of Termites and Testing Methods Used ulation and the laying of eggs varied from species to spe- in the Laboratory.” In Biology of Termites. Vol. 1, edited by K. cies ranging between 4 and 30 days in most cases. The Krishna and F. M. Weesner, 351–385. New York: Academic time taken for the appearance of the first instar larvae Press. after the hatching of eggs also varied from species to Bodot, P. 1967. “Observations sur l’essaimage et les premières species ranging between 12 and 44 days. The first lar- étapes du développement de la colonie d’Allodontermes giffardii Silv. [Observations on the swarming and early vae were subjected to mouth-to-mouth feeding by the development of the colony Allodontermes giffardii Silv] parents (stomodeal trophallaxis). (Isoptera, Termitidæ).” Insectes Sociaux 14: 351–358. The period of survival of the incipient colonies var- Brent, C. S., and J. F. Traniello. 2001. “Influence of Sex-specific ied both from species to species and within species. The Stimuli on Ovarian Maturation in Primary and Secondary maximum survival period (9 months) was recorded for Reproductives of the Dampwood Termite Zootermopsis angusticollis.” Physiological Entomology 26: 239–247. M. convulsionarius, and the minimum (2 months 18 days) Calleri, D. V., R. B. Rosengaus, and J. F. Traniello. 2007. for M. cameroni. Generally, mortality was triggered by a “Immunity and Reproduction during Colony Foundation fungal infection or a mite attack. in the Dampwood Termite, Zootermopsis angusticollis.” As described in this paper, the authors initially began Physiological Entomology 32: 136–142. by starting incipient colonies in Petri dishes and plas- Cheo, C. C. 1948. “Notes on Fungus-growing Termites in Yunnan, tic boxes with the hope that the colonies will be grown China.” Lloydia 2: 139–147. Chouvenc, T., A. Robert, E. Sémon, and C. Bordereau. 2012. “Burial further by controlling their feed, ambient temperature, Behaviour by Dealates of the Termite Pseudacanthotermes humidity, etc. Reproduction did begin and small incip- spiniger (Termitidae, Macrotermitinae) Induced by Chemical ient colonies did develop but then they died off. As the Signals from Termite Corpses.” Insectes Sociaux 59: 119–125. Zoology and Ecology 11

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