© 2007 The Authors Entomologia Experimentalis et Applicata 123: 119–128, 2007

Journal compilation © 2007 The Netherlands Entomological Society 119

DOI: 10.1111/j.1570-7458.2007.00530.x

Blackwell Publishing Ltd

Estimation of foraging territories of Reticulitermes grassei through mark–release–recapture

T. Nobre1,2, L. Nunes2* & D.E. Bignell11School of Biological and Chemical Sciences, Queen Mary, University of London, Mile End Road, London E1 4NS, UK, 2Laboratório Nacional de Engenharia Civil, Núcleo de Estruturas de Madeira, Av. Brasil, 101, 1700-066 Lisboa, Portugal

Accepted: 14 December 2006

Key words: subterranean termites, Nile blue A, neutral red, bait, resource sharing, colony, Isoptera, Rhinotermitidae

Abstract Subterranean termites have highly cryptic life habits and their foraging activities are as a rule confinedto below-ground level gallery systems. Mark–release–recapture (MRR) using fat-soluble histologicaldyes is a candidate method for the study of foraging dynamics and territories, but has not hithertobeen standardized experimentally. A wide range of dye types and concentrations is reported in theliterature. In this study, six potential dyes were evaluated at different concentrations for markingworkers of Reticulitermes grassei (Clément) (Isoptera: Rhinotermitidae), under laboratory and simulatedfield conditions. Neutral red (1% wt/wt) was considered the most effective while showing acceptablylow toxicity. In a subsequent field trial using dye impregnated into a wooden bait, the MMR proce-dure was used to map the foraging territory perimeter of a single colony. Assumptions inherent in theinterpretation of MMR data are reviewed. To map the foraging territory perimeter by this method,two theoretical approaches are defined (a conservative and a non-conservative hypothesis). We showthat the approach adopted may affect the estimate obtained by as much as 100%. Results are discussedin the context of the ecology and behaviour of subterranean termite colonies.

Introduction

Studies on the foraging dynamics and territories of sub-terranean termite populations are inherently difficult becauseof their cryptic habit and spatially extensive colonies,but several authors have pointed out that mark–release–recapture (MRR) is a potentially suitable method, havingthe advantages of accuracy and economy of effort, in contrastto destructive sampling (e.g., Su & Scheffrahn, 1988; Marini& Ferrari, 1998; Tsunoda et al., 1999; Evans, 2001).

However, in practice, marking termites effectively isdifficult because of both their physical fragility and thevery large numbers typically found in individual colonies,such that there is no consensus on the optimum material(Evans, 2000). Hagler & Jackson (2001) defined an idealmarking material as durable, inexpensive, non-toxic, easilyapplied, and clearly identifiable; furthermore, the markershould not hinder the insect nor affect its normal behaviour,

growth, reproduction, or lifespan. The use of fat-solublehistological dyes to mark termites has been frequentlyexplored. The use of neutral red and Nile blue A are mostcommonly reported (e.g., Grace & Abdallay, 1989; Evanset al., 1998; Tsunoda et al., 1999; Stanley et al., 2001), butother dyes have also been evaluated, for instance Sudanblack, Sudan yellow, Sudan green, and Sudan red (Su &Scheffrahn, 1988; Grace, 1990; Salih & Logan, 1990; Evans,1997). A wide range of dye concentrations has beenemployed, again without agreement on an optimum. Forexample, for neutral red and Nile blue A, reported concen-trations range from 0.02% (wt/wt) up to 2%, a differenceof two orders of magnitude (e.g., Salih & Logan, 1990;Evans et al., 1998; Marini & Ferrari, 1998; Tsunoda et al.,1999; Evans, 2001; Stanley et al., 2001). Variations inreported concentrations have occurred not only betweenspecies under study, but also within the same species indifferent trials. The main disadvantages of dyes have beenreported to be non-homogeneous coloration, variablefade-out, and unintended transfer to other individuals bycannibalism and trophallaxis (Haagsma & Rust, 1993;Thorne et al., 1996; Curtis & Waller, 1997; Evans et al., 1998;

*Correspondence: Lina Nunes, Laboratório Nacional de Engenharia Civil, Núcleo de Estruturas de Madeira, Av. Brasil, 101, 1700-066 Lisboa, Portugal. E-mail: linanunes@lnec.pt

120 Nobre et al.

Suárez & Thorne, 2000). These concerns, and the fact that,in termites, only the foraging part of the population ismarked, raise questions of the veracity of the dye methodto estimate population sizes, as the basic assumptions ofthe MRR may be violated (Southwood, 1984). Recently,Crosland & Su (2006) stated that the use of MRR with termitesused to be primarily to estimate the foraging populationnumbers, but this was a misapplication of the technique,and its valid function should be the delineation of acolony’s geographical boundaries. Consensus is thereforegrowing that MRR is no longer acceptable for populationestimates but remains reliable for the less ambitious task ofdemonstrating foraging territories (e.g., Curtis & Waller,1997; Evans et al., 1998; Crosland & Su, 2006). Markedtermites can be used to confirm the interconnection ofmonitoring stations, thus enabling a partial delineation ofthe foraging territories of subterranean colonies. Field-based ecological and behavioural studies have also madeuse of colour-marking techniques (e.g., Grace, 1990; Ngee& Lee, 2002). Hagler & Jackson (2001) advised on the needto carry out preliminary studies before choosing a suitablemarker, as any given marking procedure is unlikely to beapplicable for all research purposes. This article reports theevaluation of several potential dyes and concentrationsfor marking Reticulitermes grassei (Clément) (Isoptera:Rhinotermitidae) workers, under laboratory and simulatedfield conditions. Subsequently, the foraging territory of acolony was estimated in the field through the establish-ment of a MRR programme, using an established baitinggrid. Foraging territory of a colony is defined as the areaencompassed by those bait stations containing markedtermites during the subsequent monitoring (as in, e.g., Su& Scheffrahn, 1988).

Materials and methods

Screening for the marker

Reticulitermes grassei were collected from rotting logs inbenchmark field sites in Portugal and maintained in a darkenedchamber (23 ± 1 °C; >75% r.h.) for a short period beforeinitiating the trials. The generalized method was as follows:filter papers (Whatman no. 1, Whatman InternationalLtd., Maidstone, UK; 40 mm) were dipped in aqueoussolutions of candidate dye and were allowed to dry atroom temperature. Papers were then placed in Petri dishes(45 mm diameter) and an extra filter paper, not treated,was glued to the lid with non-toxic and odourless UHU®glue stick (UHU GmbH & CO. KG, Baden, Germany), andwas moistened daily with distilled water (Nunes, 1997).This protocol was used throughout Experiments 1–3(described below). All experiments were carried out in adarkened chamber for 21 days, at 23 ± 1 °C and >75% r.h.

Experiment 1 – selection of dyes and preliminary assessmentof mortalities. Six candidate dyes were used: alizarin red S,bromocresol green, bromocresol purple, Nile blue A,neutral red, and Sudan black, each at initial concentrationsof 1, 0.75, 0.5, and 0.25% (wt/wt). Once it was establishedthat a dye could mark the termites successfully, givinga conspicuous coloration, additional workers were thenretested at 0.15, 0.10, 0.05, and 0.025%. The latter range ofconcentrations was chosen to correspond most closely tothose given in the existing literature (e.g., Su & Scheffrahn,1988; Grace, 1990; Salih & Logan, 1990; Grace et al., 1996;Evans et al., 1998; Marini & Ferrari, 1998; Tsunoda et al.,1999; Evans, 2001; Stanley et al., 2001). For each of thetests, 10 termite workers were introduced into each of 10replicate dishes, prepared at each dye concentration tested.Ten groups of workers were kept under the same conditions,feeding on undyed paper as controls. Mortality (as thedifference in number of live termites) and the number ofvisibly dyed termites were recorded daily for 21 days.Termites were recorded as dyed only if they were clearlydistinguishable from others in the same trial, as well asfrom the control group. Mortality was assessed by theabsence of movement and the loss of body turgor.

Experiment 2 – mortality due to the marking procedure.Two dyes were selected from the result of Experiment 1,and prepared at two concentrations each (neutral red at0.5% and 1%; Nile blue A at 0.25% and 0.5%). Termiteworkers (in groups of 150 in 90-mm Petri dishes) were fedwith dyed filter papers for an optimum marking period(also established in Experiment 1). After this incubation,they were transferred to 45-mm Petri dishes in groups of10, as described above. As controls, 10 groups of 10 termiteswere exposed to undyed filter papers under identicalconditions. Mortality and subjectively assessed fading ofthe dye were recorded daily for 21 days. Fading of themarker was defined as a decrease in the coloration thatwould no longer allow for a distinction between markedand non-marked termites.

Experiment 3 – marking after 24 h pre-starvation. A furtherset of dyes and concentrations were prepared as inExperiment 2, but offered to groups of termites submittedto 24 h starvation before transfer, in groups of 10, to Petridishes with dyed filter paper. The same regime of replicationwas employed, and mortality and subjectively assessedfading of the dye were recorded daily.

Experiment 4 – fading-out of the marker in soil. Dyes andconcentrations were again prepared as in Experiment 2.Groups of 150 termite workers were marked accordingto the procedure used in that experiment, with marking

Foraging territories of Reticulitermes grassei 121

periods in accordance with the optimum marking periodestablished in Experiment 1. After this, the number oftermites marked and non-marked was recorded andthen the marked termites were transferred to soil jars.The soil substrate consisted of an equal mixture of dryFontainebleau sand and a commercial soil (mixture forplant growth with 20% of organic matter and a waterholding capacity between 75% and 80%; brand nameBRICOBI®, AKI Bricolage España, Barcelona, Spain),sterilized at 100 °C for 30 min and moistened with distilledwater at 3:1 (wt/vol). Glass jars (50 mm diameter and65 mm height) were filled with this mixture to 70% of theircapacity. One block of maritime pine [Pinus pinaster Ait.(Pinaceae)], 10 × 10 × 30 mm, was placed at the surface ofthe soil as a food source. Six replicates were made per dyeand per concentration, together with an equivalent controlgroup without dye. The mortality and the number of dyedtermites were recorded after 30 days.

Statistical analysis. To test for significant differencesbetween treatments, and whenever the data allowed forthe performance of parametric analysis, comparisons weremade using one-way analysis of variance (ANOVA), followedby Tukey’s pairwise comparisons. Significant differenceswere always considered at P<0.05.

Mark–release–recapture

Field procedures. The MRR programme took place in afield site that comprised a 4.6-ha silvicultural plantation ofEucalyptus globulus Labill (Myrticeae), located in Peroguarda(Beja; Portugal) (38.11°N, 8.04°W, 160 m a.s.l.), wherea baiting grid system had already been established for4 years. This consisted of 10 rows of baits with seven baitsper row and a separation of 3 m between baits (total areabaited: 486 m2). Each bait consisted of a perforatedpolypropylene tube (9 cm diameter; 30 cm length), closedat the upper end with a plastic cap and sunk into theground. To accommodate the tube, the ground was firstbored to the size of the tube. The tube was then filled witha bait of several pieces of laminated, pesticide-free maritimepine (P. pinaster) and inserted into the ground, with thetop of the tube flush with the ground surface. Monthlysamplings of all baits were performed, with bait maintenanceand renewal as appropriate. For the initial marking event,one bait trap centrally located on the grid and fullyoccupied by subterranean termites was selected. The entirebait, still containing the termites, was removed and takento the laboratory, where termites were marked with neutralred 1% (wt/wt) according to the procedure selected asoptimum from the earlier experiments. They were thenreturned to the field and released via the same bait systemand in the same station used for collection. All baits were

subsequently checked for marked termites after 1 month.Termites collected from monitoring stations containingmarked individuals were considered nestmates of thefirst-marked termites, and were then stained by the samelaboratory procedure before release back to their respectivebaits. This procedure was repeated for a further 2 months,with a final sampling 3 months after marked termites werefirst put into the field.

At the end of the third cycle, an additional field test wascarried out. A filter paper (Whatman no. 1; 90 mm) dyedwith neutral red was left in one of the central bait trapswhere marked termites had been previously found. Theaim was to assess this procedure as an alternative markingmethod, applied directly in the field, without the stressprovoked by their dislodgment, transportation, and handlingin the laboratory.

Theoretical assumptions. To map the foraging territoryperimeter, two alternative approaches were considered:1. The grid system was schematized and points were

placed halfway between each bait occupied by markedtermites and the next bait position (in the same row andcolumn) in this case regardless of whether next bait wasoccupied or not. These points were then connected witha smoothened curve (a non-uniform rational B-spline –a mathematical model commonly used in computergraphics for generating and representing curves andsurfaces, usually shortened as NURBS). Non-occupiedbaits were thus not included in the foraging area. Thisapproach will hereafter be referred to as the conservativehypothesis.

2. The grid system was schematized but the points wereplaced halfway between each bait occupied by markedtermites and the next bait position (in the same row andcolumn) occupied by non-marked termites. If the nextbait was not occupied by termites, then the point wasplaced at this (latter) bait’s position. Therefore, baitsnot occupied were included in the foraging area. Allpoints were then connected with a smoothened curve(NURBS). This approach will hereafter be referred to asthe non-conservative hypothesis.

Results

Choice of the marker

Experiment 1 – selection of dyes and preliminary assessmentof mortalities. The dyes tested showed highly variableefficacy in marking the termites. For instance, alizarin redS only gave an occasional yellowish colour to the abdomenand in the majority of cases was not distinguishable fromcontrols. Bromocresol purple and Sudan black B wereable to darken the abdomen, but not differently from the

122 Nobre et al.

natural coloration that is acquired when wood is ingested.Bromocresol green, although able to mark the termiteswithout a differential mortality from the non-dyed controls(mortalitycontrol = 0.33 ± 0.49 and between all concentrationsof bromocresol green mortality = 0.47 ± 0.69; F4,5 = 0.48,P = 0.75), produced non-homogeneous coloration, rangingbetween brown (which can be mistaken for the naturalcoloration) and shades of blue. Neutral red marked thetermites with a homogeneous clear red colour, easilydistinguishable from controls and from any unmarkedtermites sampled in the field. For the eight concentrationstested, there were no significant differences in the mortalityobserved between the treatment groups and the controlbefore the 17th day of continuous exposure. At this timeand after, significantly higher mortality was observed at the0.75% dye concentration (mortalitycontrol = 0.20 ± 0.42 andmortality0.75% = 1.60 ± 1.71; F1,18 = 6.30, P = 0.02) and forthe 1% dye concentration (mortalitycontrol = 0.20 ± 0.42 andmortality1% = 1.00 ± 1.05; F1,18 = 4.97, P = 0.04). Figure 1Ashows mortality and the proportion of marked termites forall concentrations employed at the end of the trial (21 daysof exposure). The speed of marking (i.e., the number ofdays to achieve 90% marking), although lower than field

experience would suggest is optimal, was still consideredacceptable; it was 7 days at 1% dye concentration and 10 daysat 0.5% (Table 1). For all the other concentrations assessed,the speed of the dying procedure was considered too slow.

Nile blue A gave the termites a distinguishable bluishcolour. The higher concentration tested (1%) provokedsignificantly higher levels of mortality after 8 days of expo-sure (mortalitycontrol = 0.20 ± 0.42 and mortality1% = 1.00 ±0.94; F1,18 = 6.00, P = 0.03). For the other concentrations,no significantly different mortality was observed beforethe 13th day of exposure. In this case significantly highermortality was due to low survival in the trials at the 0.75%concentration (mortalitycontrol = 0.20 ± 0.42 and mortality0.75%

= 1.80 ± 1.93; F1,18 = 6.55, P = 0.02). After 16 days ofexposure, the 0.5, 0.25, and 0.15% dye concentrationsalso showed higher mortality than the non-dyed controls(mortality0.5% = 3.10 ± 3.31; F1,18 = 7.53, P = 0.01; mortality0.25%

= 1.10 ± 1.71; F1,18 = 4.63, P = 0.04; and mortality0.15% =1.70 ± 0.89; F1,18 = 6.01, P = 0.03). Figure 1B shows mor-tality and the proportion of marked termites for all theconcentrations at the end of the trial. The higher concen-trations tested allowed a visible marking of the termiteswithin 5 and 7 days of exposure (Table 1), and until termites

Figure 1 Marking (%) and mortality (%) observed in Reticulitermes grassei workers continuously exposed to (A) neutral red and (B) Nile blue A for 21 days – Experiment 1. Means of 10 replicate groups of 10 termites each.

Table 1 Number of days needed for 90% of Reticulitermes grassei workers to be marked, and the corresponding mortality at that time – Experiment 1. Ten replicate groups of 10 termites each

Concentration of the dye

Neutral red Nile blue A

Days to mark Marked (%) Mortality (%) Days to mark Marked (%) Mortality (%)

0.025 – – – 21 95 40.05 18 90 6 17 90 70.10 12 91 1 20 92 90.15 12 90 2 9 91 00.25 14 93 4 7 94 20.50 10 90 2 7 90 00.75 11 92 0 7 97 11.00 7 100 3 5 93 1

Foraging territories of Reticulitermes grassei 123

acquired the mark, there were no significant differences inmortality. Therefore, the choice of optimal concentrationwas made according to the level of mortality. As observedwith neutral red at the lowest concentrations tested, thespeed of marking was too slow; in many cases the end ofthe experiment was reached before a majority of termitesbecame marked. Based upon the results obtained, neutralred (at concentrations 0.5 and 1%) and Nile blue A (0.25and 0.5%) were selected for further evaluation.

Experiment 2 – mortality due to the marking procedure.Between the two concentrations of neutral red, significantlydifferent levels of mortality were only consistently registeredby the 11th day after exposure to the dye (Figure 2;mortality0.5% = 0.80 ± 0.79 and mortality1% = 0.20 ± 0.42;F2,27 = 3.68, P = 0.04), with neutral red at 0.5% showinghigher mortality than neutral red at 1% and the non-treatedcontrols. No significant differences from the controls wereobserved at the highest concentration of the dye. With Nileblue A, significantly different mortality was never observedduring the trial (Figure 2). Complete loss of the mark(fade-out) was not observed, although some termites markedwith Nile blue A, particularly at the lowest concentration,showed a weaker coloration at the end of the trial.

Experiment 3 – marking after 24 h pre-starvation. Ninetypercent of termites subjected to 24 h of starvation prior tomarking were dyed after 5 days of exposure with bothneutral red and Nile blue A. The same result was obtainedat both concentrations employed for each dye. For neutralred, the observed mortality was never significantly different

from that of non-marked controls (Figure 3). For Nile blueA, however, a significantly greater mortality was observedfrom the beginning of exposure to the dye at the higherconcentration (mortalitycontrol = 0.00 ± 0.00, mortality0.25% =0.00 ± 0.00, and mortality0.5% = 0.50 ± 0.53; F2,27 = 9.00,P = 0.00). No significant differences were found betweenthe lower concentration of Nile blue A tested and thecontrols.

Experiment 4 – fading-out of the marker in soil. Marking wascarried out as in Experiment 2, without subjecting theworkers to a previous period of starvation (efficacy shownin Table 2). Mortality and fading in soil at 30 days aftermarking with the different dyes and concentrations aresummarized in Table 3. Overall, more than 80% markingwas achieved (the one exception was 0.5% neutral red),with relatively low mortality. However, during the sub-sequent incubation in soil, Nile blue A showed either highmortality or high fade-out (Table 3).

Figure 2 Mortality (%) observed in Reticulitermes grassei workers after exposure to neutral red or Nile blue at different concentrations for 21 days – Experiment 2. Means of 10 replicate groups of 10 termites each.

Table 2 Mortality and efficiency (SD in parentheses) of the marking at the end of marking process for Reticulitermes grassei workers – Experiment 4. Means of six replicate groups of 150 termites each

Dye Days Mortality (%) Marked (%)

Nile blue A 0.25% 7 0.9 (± 0.9) 87.2 (± 8.8)0.5% 7 18.1 (± 11.7) 80.4 (± 11.1)

Neutral red 0.5% 10 6.3 (± 4.2) 76.3 (± 6.0)1% 7 1.7 (± 0.82) 86.6 (± 12.4)

124 Nobre et al.

Mark–release–recapture

Laboratory marking and field release. At the time of estab-lishment of the MRR programme (March 2004), 24% ofthe baits within the baiting grid were occupied. Markedtermites were then quickly found colonizing other baitsand at the end of the three campaigns seven baits had beenidentified that showed marked workers (Figure 4).

Reinforcement by field marking. Dyed paper (0.58 g at ambientconditions) was incorporated into the most central baitwithin the indicative territory of that group. The neutralred dyed filter paper was totally consumed within 1 month.Marked termites were always encountered on subsequentmonitoring of the site, up to 1 year after field marking. Thepattern of occupancy with marked termites was initiallyconstant. After two 6-month periods, an extension hadbeen observed (Figure 5), with three more baits beingoccupied by marked termites in comparison to the

previous, shorter MRR application. Likewise, the patternof baits occupied by non-marked termites also changed(with new baits being colonized), indirectly reflectingsome intensification of foraging during this specific period.The overall maximum linear dimension of the mappedarea where marked termites were found was 18.25 m. Thearea enclosed by the indicative territory corresponds to90.78 m2 (14% of the total area of the grid) under theconservative hypothesis and 184.67 m2 (29%) under thenon-conservative hypothesis.

Discussion

Of the six potential dyes, evaluated over a range ofconcentrations for their ability to mark R. grassei workers,only two were able to provide a conspicuous and consistentcoloration sufficient for field use: neutral red and Nile blueA. The exact amount of filter paper consumed was notmeasured, and thus different levels of consumption mayhave occurred between treatments and/or replicates, forinstance as a result of a general repulsion effect of the dyesor at particular concentrations. However, for the imple-mentation of an MRR campaign, the essential considerationwould be the ability of the dyed filter paper to mark theworkers effectively, independently of the amount of dyeconsumed.

The majority of the dyes tested were pH indicators. ThepH of the gut of Reticulitermes termites has been measuredby several authors (Bignell & Anderson, 1980; Brune et al.,1995; Nunes, 1997) and was found to differ little from neu-trality. Thus, loss of the original colour due to interactions

Figure 3 Mortality (%) observed in Reticulitermes grassei workers after 24 h of starvation and 5 days of exposure to neutral red or Nile blue at different concentrations for 21 days – Experiment 3. Means of 10 replicate groups of 10 termites each.

Table 3 Average mortality and fading (SD in parentheses) of the dye in Reticulitermes grassei after 30 days in soil – Experiment 4. Means of six replicate groups of 150 termites each

Dye and concentration Mortality (%) Fading (%)

Control 7.0 (± 1.9) –Nile blue A 0.25% 26.8 (± 12.1) 78.9 (± 8.4)

0.5% 46.7 (± 13.8) 0.2 (± 0.4)Neutral red 0.5% 25.6 (± 6.6) 16.4 (± 6.2)

1% 8.9 (± 2.8) 20.3 (± 9.8)

Foraging territories of Reticulitermes grassei 125

with intestinal secretions within the intestine is unlikely,and any fading out or failure of the colour to become estab-lished is presumably due to interactions with the gutcontents, or to physiological removal by excretion. In allmarking experiments, we found that dyes were only visiblewhen they reached the hindgut, which represents thegreater part of the intestinal volume and where ingestedmaterial is retained for the longest period (ca. 24 h). It isunclear that to what extent the dyes can spread to other

organs. Su et al. (1983) found that although the dye Sudanred 7B was largely kept in the gut particularly during thefirst few days, some proportion was transferred to othertissues, from where it disappeared much more slowly. Theperipheral zone of the hindgut lumen of termites is a rela-tively static area rich in micro-organisms attached to thegut wall (Bignell, 2000); in addition, lower termites such asReticulitermes accommodate a tightly packed populationof flagellates, which are also presumably capable of taking

Figure 4 Bait occupancy during the mark–release–recapture (MRR) programme of Reticulitermes grassei. A field grid is shown at different sampling dates, with the spacing scales in metres.

Figure 5 Bait occupancy in the end of the second period of Reticulitermes grassei marking and indicative colony foraging territory. A field grid is shown, with the spacing scales in metres.

126 Nobre et al.

up the dye, and any coloration achieved should be easilyvisible through the relatively thin cuticle in most individuals.

Sudan black was used in an MRR study with Coptotermesspp. (Rhinotermitidae) by Evans (1997), but was found tobe non-persistent in the field, with a reduction of 50% inmarked termites (Evans et al., 1998). In the present studythis dye failed to mark the termites with a conspicuouscolour, confirming the inadequacy of this compound as asuitable termite marker. Although Nile blue A marked thetermites successfully, higher mortality and fading made itless attractive for MRR. Mortality was noticeably greater atthe higher concentrations, although time for marking wasthe same as for neutral red. Concentrations of 0.5 and0.25% did not increase the mortality after marking.

Reticulitermes colonies are believed to comprise severalthousands individuals and the laboratory conditions ofthe experiments performed, and in particularly the lownumber of workers used for the first experiments, are notrepresentative of natural settings. However, the experimentsproved suitable to screen the possible use of dyes as termitemarkers, considering the practical constraints of time andbiological material. Furthermore, the response of the termitepopulations to the dyes seems to be strongly dependent onthe species (if not population) under study and thereforeit is advisable, prior to the implementation of an MRRtechnique, to perform some screening tests in the lab.Experiment 1 reported here constitutes a fast protocol forscreening dye and concentration range, together withExperiment 4, that assesses the fading-out of the markerwith time.

The inclusion of a starvation period before markingresulted in faster marking with Nile blue A, albeit withhigher mortality as a side effect at the higher concentration.The ‘fast’ marking procedure developed by Evans (2000)for Coptotermes acinaciformis and Coptotermes lacteus(where slightly desiccated termites are offered an aqueoussolution of fat-soluble stains) resulted in no increase inmortality levels and marking within 24 h. Long (2004)reported that, for Reticulitermes flavipes, a combinationof this method with the phagostimulatory effect of a 1%glucose solution resulted in a clear coloration with Nileblue A in 60 h. In the present study with R. grassei, we wereunable to reduce the duration of the marking processto less than 5 days, which perhaps resulted in a negativeimpact on termite survivorship and/or colour retention.The fading of the dye was negligible at 0.5%, but the mor-tality was near 50% in the marked population. On theother hand, at 0.25% Nile blue A, the fading observed wasvery high (almost 80%) but with mortality levels similar tothose obtained with neutral red. Thorne et al. (1996) alsoencountered high mortality rate and low dye retention fortermites marked with Nile blue A.

Although the use of Nile blue A is reported in MRRstudies with Reticulitermes (e.g., Su et al., 1993; Evans,2001), it proved a less suitable marker in the present study.The use of Nile blue A at 0.25% might be feasible in thefield if less than 30 days elapsed between marking andreleasing, and bait retrieval for recapture. In such cases, itis advisable that a prior trial in soil should be made (evenif only under laboratory conditions) to evaluate the lengthof the period during which the termites are likely to remainmarked. The use of a marker that fades rapidly affects theresults of an MRR programme, even if only used to delineateforaging areas. Likewise, a marker that causes differentiallyhigh mortality will also affect the final results.

Nile blue A has a record of success in several studies(Haagsma & Rust, 1993; Evans et al., 1998, 1999; Marini &Ferrari, 1998; Tsunoda et al., 1999; Evans, 2001; Stanleyet al., 2001), in concentrations ranging from 0.02% to 0.2%(wt/wt). In the present study, the lower concentrationsfailed to mark the termites within an acceptable time, andthe lowest concentration used for the fading trial (corre-sponding approximately to the higher concentrationreported in previous studies) resulted in a high level ofmarker loss.

Thorne et al. (1996) reported mark loss to be consider-ably different between colonies within species and justchanging the diet from filter paper to mixed wood resultedin significantly higher dye fading. The authors refer to thelimited exercise permitted to the termites by the necessaryconfinement of laboratory experiments, and therefore in afield situation where Reticulitermes are active in the soil,the mark losses are expected to be higher. The high levelsof dye fade-out, observed particularly for Nile blue(0.25%) in the experiment in soil, may partially reflect thecombined action of a wood diet and greater activity withinthe soil medium.

Neutral red therefore seems to be the best option for thePortuguese termites examined. Satisfactory coloration wasobtained with the higher concentrations (0.5% and 1%)and within a time feasible for practical work. The timeof continuous exposure to the dye had an influence onthe mortality, but a prior starvation treatment was able todecrease the effective marking time and no differentialmortality was observed subsequently. Fading of the neutralred marker was similar at both concentrations tested, andalthough around 20%, may be still considered acceptablefor an MRR programme aiming to delineate foragingareas. The estimation of foraging areas through baiting isalways an indirect approach, as the border of the territorycannot be defined with greater resolution than the distancebetween adjacent baits. Likewise, it can be argued that if abait is not occupied it does not follow that the bait repre-sents a territorial boundary. It might simply mean that the

Foraging territories of Reticulitermes grassei 127

bait in question, although still within the foraging range,was not encountered and used as a food source. This mightexplain situations where non-occupied baits are surroundedby baits that are being exploited.

The maximum linear foraging distance observed wasca. 18 m, corresponding to a territory of 91 m2 (by the con-servative hypothesis) and 185 m2 under the non-conservativeestimate. This illustrates that the assumptions made fordelineation of foraging ranges in mapping procedures mayaffect the estimate obtained by as much as 100%. Nonethe-less, the foraging territory area reported by Su et al. (1995)for R. flavipes (150 m2) falls reassuringly in between thepresent estimations. Although the assumptions made bySu et al. (1995) are not explicitly reported, the approachtaken seems to have been simply the linear connection ofthe outsider baits. Taking this approach in the presentstudy, the area estimation of the foraging territory wouldbe ca. 97 m2 (data not shown), which is close to the estimatemade under the conservative hypothesis. However, thisarea would include a bait occupied by non-marked termiteswhich would then have to be considered within the forag-ing territory of the marked group. Irrespective of whichapproach is chosen for the estimation of the geographicalboundary of a colony, it is important that the method isclearly stated, thus allowing comparisons between studies.The maximum linear distance found (18.25 m) was a littleless than the 24 m reported by Su et al. (1995) for R. flavipesand considerably smaller than the estimates of Grace(1990), which ranged between 41 m and 79 m.

The above estimates show a high variability in thereported foraging territories of Reticulitermes. AlthoughReticulitermes and Coptotermes species are quite differentin their colony organization, ecology, and behaviour, thesame variability can be found within the data available forCoptotermes. Su & Scheffrahn (1988) found that foraginggalleries of C. formosanus extended for more than 100 mand their foraging territory ranged from 162 to 3571 m2.In different studies (Su, 1994; Su et al., 1995), also forC. formosanus the maximum observed distances rangedfrom 30 to 185 m and foraging territories ranged from 143to 2189 m2. Part of this variability could be due to dif-ferences in habitat structure, although colony maturity isalso a factor to consider. Contrasting with these figures,Sornnuwat et al. (1996) encountered a maximum distanceof 5 m for Coptotermes gestroi in an urban area.

Acknowledgements

This work was partially supported by the grant given bythe Portuguese Foundation for Science and Technology(SFRH/BD/8761/2002). We thank Joao Pedro Cappas eSousa for making the field site available to us.

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