Journal of Insect Physiology 58 (2012) 1568–1574

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Journal of Insect Physiology

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Patterns of venom production and temporal polyethism in workers of Jerdon’sjumping ant, Harpegnathos saltator

Kevin L. Haight ⇑Social Insect Research Group, School of Life Sciences, Arizona State University, P.O. Box 874501, Tempe, AZ 85287-4501, USA

a r t i c l e i n f o

Article history:Received 5 September 2012Received in revised form 24 September 2012Accepted 25 September 2012Available online 4 October 2012

Keywords:AntsVenom productionPolyethismLongevityHarpegnathos

0022-1910/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.jinsphys.2012.09.011

⇑ Tel.: 1 480 727 0954; fax: 1 480 965 6899.E-mail address: kevin.haight@asu.edu

a b s t r a c t

Ants are chemical factories, and among their more noticeable products are their venoms. Though manystudies have addressed the properties and activities of ant venoms, basic venom-related physiologicalquestions, such as how venom production and replacement may vary with age, are rarely addressed.The answers to these questions are fundamental to understanding the physiological capabilities of theseorganisms, as well as the parameters within which potential optimization of their investment in venomproduction must take place. The only previous investigation into venom production in ants found it to belimited to early life in workers of the fire ant, Solenopsis invicta (Haight and Tschinkel, 2003). Because sim-ilar studies have not been conducted for comparison, it is unclear whether or not this is a common phys-iological pattern in ants. As a parsimonious way to address this question, and, more generally, to increasethe currently scant information available regarding the venom-producing capabilities of ants, the longev-ity, temporal polyethism, age-related venom production, and age-related venom replacement capabilitiesof workers of Jerdon’s jumping ant, Harpegnathos saltator were investigated. Longevity varied from10 days to nearly 2 years, with a median lifespan of 206 days. Workers remained in the nest when young,transitioned to outside work (foraging) after 50 days of age, and reached a plateau in their tendency to beoutside the nest at 74 days of age. They eclosed with empty venom sacs, filled them by about 57 days ofage, and were able to replace venom at all three ages tested (though at a higher rate when aged 100 daysthan 30 and 206). So, venom-production ability is not limited to early life in H. saltator workers, andaspects of venom physiology and exploratory behavior appear to coincide in a manner likely to resultin foraging efficiency benefits; venom sacs reach fullness around the age workers begin their foragingcareers, and venom replacement rate is highest around the age workers become the most dedicatedforagers.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Ants have been called ‘‘chemical factories’’ due to the myriadchemicals they are capable of producing (Grasso et al., 2004;Morgan, 2008; Martin and Drijfhout, 2009). Among the morenoticeable of these chemicals are their venoms, which they usefor defense, food acquisition, and communication (Schmidt,1982; Hölldobler and Wilson, 1990). Studies of ant venoms haveshown them to be both chemically and pharmacologically diverse(Schmidt, 1986; Blum, 1988; Piek et al., 1991; Orivel and Dejean,2001; Orivel et al., 2001; Zelezetsky et al., 2005; Hoffman, 2010),as well as potentially medically useful (Altman et al., 1984; Pieket al., 1991; Zelezetsky et al., 2005; Dkhil et al., 2010; Badr et al.,2012). Though the chemical properties and activities of ant venomshave been addressed in many studies, basic venom-related physi-ological questions have received less attention. For example, how

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venom-production and replacement ability may vary over workerlifetime is largely unknown (but see Haight and Tschinkel, 2003).This has left a gap in our knowledge regarding the physiologicalcapabilities of these organisms, as well as the parameters withinwhich potential optimization of their investment in the costly pro-cess of venom production must take place (Billen, 1990; Nisaniet al., 2007).

This study was conducted in order to increase the currentlyscant information available regarding venom production in ants.In the only previous investigation on this topic, Haight andTschinkel (2003) found that workers of the imported fire ant, Solen-opsis invicta, are apparently unable to replace spent venom afterthey have reached 30 days of age (S. invicta workers can live70–250 days depending on conditions; Calabi and Porter, 1989).Because there have been no similar studies for comparison,whether or not the limitation of venom-production capability toearly life is a common physiological pattern in ants is unknown.However, given that S. invicta (an opportunistic predator/scaven-ger; Tschinkel, 2006), does not rely on venom for food acquisition,

K.L. Haight / Journal of Insect Physiology 58 (2012) 1568–1574 1569

contrasting its venom-producing capabilities with those of astrictly predacious ant species represents a parsimonious way toaddress this question.

In ants, and other eusocial Hymenoptera, the tasks workers per-form change in a predictable way with age, a phenomenon knownas temporal (or age) polyethism (Hölldobler and Wilson, 1990,2009; Bourke and Franks, 1995). In-nest chores, such as queenand brood care, tend to be performed by younger workers, whileolder workers tend to perform outside tasks such as nest defenseand finally foraging for food. Since it is the oldest workers whichforage, the ability to produce venom late in life (if it occurs in ants)should most likely be found in a strictly predacious ant specieswhich relies on venom for food acquisition. The ability of olderworkers (foragers) to replace spent venom should be selected forin such a species, assuming it follows the typical temporal polyeth-ism pattern observed in other ants. Thus, if venom-production abil-ity were limited to early life even in a strictly predacious antspecies, it would suggest such limitation may be a common phys-iological pattern in ants.

Harpegnathos saltator Jerdon (Hymenoptra: Formicidae) is astrictly predacious ant species found in Southern India, Sri Lanka,and Southeast Asia (Do Nascimento et al., 1993). It hunts solitarilyusing its large eyes, long mandibles, and notable jumping abilitiesto locate and capture live arthropod prey which it subdues bystinging and returns to the nest (Maschwitz et al., 1979;Shivashankar et al., 1989; Musthak Ali et al., 1992; Peeters et al.,1994). Though its venom has not yet been characterized chemi-cally, it is at least partly proteinaceous (denatures in ethanol; pers.obs.) and is known to cause irreversible paralysis in insects(Maschwitz et al., 1979). Though Jerdon (1851) remarks H. saltator‘‘stings very severely,’’ in this author’s experience human reactionsgenerally consist of initially acute localized pain which attenuatesand disappears within 30 min, and a slight redness and/or low weltaround the sting site which usually disappears within an hour orso. This is consistent with their venom being targeted primarilytowards insect prey and only secondarily towards potential verte-brate predators (J.O. Schmidt, pers. comm.). In some cases a slightlyitchy bump, similar to an old mosquito bite, may be noticed at thesting site one to several days later. Though it can often be difficultto identify the exact location of an H. saltator sting several hoursafter having received it, more severe reactions, consisting oflimited, localized swelling, have been experienced on the rareoccasions when multiple stings have been delivered to the samearea. As H. saltator stings are painful but do not seem to causetissue damage, it appears unlikely that their venom contains highlevels of active phospholipase. However, deeper investigation intothe chemical and pharmacological properties of this venom isneeded before its various components and activities can be as-sessed fully.

In broad terms, the present study was conducted in order tobetter understand the physiological capabilities (and putative lim-itations) of venom production in ants (which could be of practicaluse in cases where ant venoms prove medically useful but the syn-thesis or purchase of the pharmacologically active compounds maynot be feasible). More narrowly, it was conducted to increase thebasic biological and physiological information available for H. sal-tator, a species poised for increased interest since the publicationof its genome and its proposal as an experimental model for thestudy of epigenetics in aging and behavior (Bonasio et al., 2010;Schneider et al., 2011). Indeed, such information has been calledfor as essential to the correct interpretation of genomic data andas a guide to asking the right questions using such data (Gadauand Helmkampf, 2012). Specifically, the present study reportsquantitative findings on the longevity, temporal polyethism, age-related venom production, and age-related venom replacementcapabilities of H. saltator workers.

2. Materials and methods

2.1. Study organism

All H. saltator colonies used in this work were descendents ofthose collected by Liebig et al. (1998) from the Western Ghats insouthern India. They were maintained in the laboratory at approx-imately 25 �C with a 12 h/12 h light/dark cycle (0700–1900 h) dur-ing these studies. All colonies were fed live crickets (Achetadomesticus) twice a week, and had the plaster floors of their nestsmoistened as needed to maintain humidity within their nest boxes.Nest box dimensions and features varied by experiment, and arelisted in further detail below.

2.2. Wire marking

Unique wire marks (Fig. 1) were used to positively identify cer-tain individual workers within colonies. Wire marks consisted of asingle loop of 34 gauge colored wire (Artistic Wire Ltd., Elmhurst,IL, USA; www.artisticwire.com) tied around the petiole either ante-rior or posterior to the petiolar node (this allows a single color tobe used to positively identify two different workers within a col-ony). Marking workers with wire eliminates mortality due to paintsolvents, which cause desiccation through the soft cuticle of callow(recently eclosed) workers, and provides a more permanent markthan paints which may be lost during grooming. Wire markingwas done under magnification (Leica MZ95 dissecting scope; LeicaMicrosystems Ltd., Heerbrugg, Switzerland) with the aid of animprovised knot tyer (Fig. 1) and fine scissors to trim away the ex-cess wire from outside the knots.

2.3. Quantification of venom sac volume

The volume of venom contained in individual workers wasquantified by measuring scale-included digital images of their ve-nom sacs (Fig. 2). Venom sacs were removed whole by dissection in95% ethanol under magnification using a Leica MZ125 dissectingscope. Digital images were taken using a microscope-mounted dig-ital camera (Spot Insight camera model #4.2, Diagnostic Instru-ments, Inc., Sterling Heights, MI, USA). The long and short axes ofeach venom sac were measured (mm) using ImageJ computer soft-ware (http://rsb.info.nih.gov/ij/; National Institutes of Health,Bethesda, MD) and volume was subsequently approximated usingthe formula for the volume of a prolate spheroid: V = 4/3pab2,where a = 0.5 long axis, b = 0.5 short axis. The resulting volumesin cubic millimeters were then converted to nanoliters using theequivalency 1 cm3 = 1 ml. As H. saltator worker size has been foundto vary directly with the cube of worker headwidth (K.L. Haightunpublished data: Dry weight, mg = 0.997(Cube of Headwidth,mm3) + 0.085, R2 = 0.77, n = 72, slope = 1 t-test p > 0.50, inter-cept = 0 t-test p > 0.50), worker heads were also imaged and mea-sured (Fig. 2) to account for potential effects of worker size.

2.4. Worker longevity and temporal polyethism

The colonies used in this study, consisting of at least one gamer-gate (mated, reproductive workers which supersede a senescent/dead queen; Peeters and Hölldobler, 1995) and approximately200–500 adult workers and their brood, were housed in plasticnest boxes with lids (Pioneer Plastics, Inc., North Dixon, KY, USA;box style 395c: 318 mm long � 256 mm wide � 97 mm high). Eachnest featured a plaster floor (Modern Materials Labstone Blue)approximately 25 mm thick with a rectangular cavity 235 mmlong � 200 mm wide � 9 mm deep covered by clear plate glass253 mm long � 228 mm wide � 5 mm thick. An uncovered

Fig. 1. Knot tyer used for wire marking ants. Tyer constructed from a bent length of 1/16 inch (1.6 mm) diameter steel wire with ends that had been pounded flat and drilled.A pre-looped marking wire (examples shown) is passed through the holes and held in place with wooden pegs (toothpick tips). The loop is then placed as desired around theant and the knot is tightened by gently squeezing the tyer as indicated by the arrows. Fine scissors are then used to trim excess wire from outside the knots. All elements shownto scale, except magnified window illustrating wire mark tied anterior to petiolar node. Replacing the hole-and-peg feature with alligator clips improves ease of use.

Fig. 2. Examples of worker venom sac and head. Left image: L = lancet, DG = Du-four’s gland, VS = venom sac. Right image: dorsal view of head with white arrowindicating location of headwidth measurements.

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25 mm � 25 mm extension of the nest cavity served as a nestentrance. Red acetate film (Grafix Colored Clear Lay Acetate film)covered the plate glass to simulate darkness in the nest interior.Fluon (Northern Products, Inc., Woonsocket, RI, USA) was appliedto the insides of the nest box walls to inhibit escapes when the lidswere removed.

In order to assess the lifespan of H. saltator workers, twelveworkers from each of three colonies were wire marked on theday of their eclosion (0 days old) and returned to their respectivenests to be subsequently followed throughout their lives. Colonieswere checked daily for natural mortality of marked workers. Thedate of death and the headwidth of each marked worker were re-corded. To facilitate locating the wire-marked workers, paintmarks (Testors Pactra enamel, Rockford, IL, USA) were applied totheir thoraces after they had aged enough to have fully sclerotizedand darkened cuticles which are capable of resisting the desiccat-ing effects of the paint solvent.

In order to assess how worker age corresponds to relative loca-tion within the nest (and thus contributes to polyethism) everyMonday, Wednesday, and Friday each marked worker was visuallylocated and given a location score of 1 if they were observed insidethe nest (under the glass), 2 if they were observed at the nest en-trance, or 3 if they were observed outside the nest (above theglass). These observations were generally made between 0900and 1100 h (H. saltator is a crepuscular forager; Shivashankar

et al., 1989) and before colonies were fed (if it was a feedingday). As not all workers eclosed on the same day, workers of asmany as seven different ages were present during any observationday.

2.5. Venom production and worker age

The colonies used in this study (consisting of at least onegamergate and approximately 200–400 adult workers and theirbrood) were housed in plastic nest boxes with lids (PioneerPlastics, Inc., box style 195c: 270 mm long � 194 mmwide � 95 mm high). Each nest featured a plaster floor approxi-mately 25 mm thick with a rectangular cavity 155 mm long �130 mm wide � 9 mm deep. The cavities were not covered by glassin this experiment to facilitate access to workers for marking andsampling purposes. Fluon was applied to the insides of the nestbox walls to inhibit escapes when the lids were removed.

In order to assess how venom production changes with workerage, the venom sac volumes of workers from nine different agegroups were quantified as described in Section 2.3. At least twoworkers from each age group (0, 1, 15, 29, 43, 57, 71, 85, and99 days old) were sampled from each of 10 colonies. On the daythey eclosed, workers to be sampled were randomly assigned anage group and were wire marked (zero day old workers were notmarked as they were sampled immediately). Before marking be-gan, all callow (i.e., recently-eclosed) workers were removed fromthe colonies to ensure the eclosion dates of subsequently emergingworkers were known. After being marked, workers were replacedin their nests until they reached their assigned ages and were sam-pled. Sampled workers were killed individually by placing them in12 ml plastic vials and freezing them over night at �30 �C. The nextmorning water and a cotton plug (to keep the ants submerged)were added so workers were stored in ice to prevent desiccation.Pre-stung crickets only were fed to these colonies over the courseof this study to help ensure marked workers did not expend theirvenom.

2.6. Venom replacement and worker age

The colonies used in this study (also consisting of at least onegamergate and approximately 200–400 adult workers and theirbrood) were housed (and fed) as described in Section 2.5.

Fig. 3. Frequency histogram of H. saltator worker lifespan (days) with mean,median, and sample size listed for complete data and data excluding oldestextremes. Numbers inside bars indicate observed worker lifespans. Two oldestworkers, 700 and 701 days old, were sacrificed for another study and thus did notreach their natural lifespans. Complete data n = 35 instead of 36 due to theexclusion of one worker accidentally killed during the study.

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In order to assess how the ability of workers to replace spentvenom changes with worker age, three different age groups wereexamined; young workers (30 days old), mid-aged workers(100 days old), and old workers (206 days old). These ages werechosen based on the data collected as described in Sections 2.4and 2.5 (see Section 3.3 for rationale). Six workers from each agegroup were sampled from each of 15 colonies. Workers to be sam-pled were wire marked on the day they eclosed and were randomlyassigned to an age group/subgroup combination. Each subgroup(either Undepleted, Depleted, or Synthesis) consisted of two work-ers per colony. Before marking began, all callow workers were re-moved to ensure the eclosion dates of emerging workers wereknown. After being marked, workers were replaced in their nestsuntil they reached their assigned ages. Upon reaching theirassigned ages Undepleted (U) workers were sampled/killed as de-scribed in Section 2.5, while Depleted (D) and Synthesis (S) work-ers were removed from their nests and placed individually intoplaster-floored boxes with lids and fluon-coated inner walls(Pioneer Plastics, Inc., box style 29c: 95 mm long � 95 mmwide � 78 mm high; plaster floors �20 mm thick). In these boxesthe D and S workers were allowed to sting and subdue two livecrickets (A. domesticus, �10–13 mm long), provided one at a time.In cases where workers ignored their cricket prey (usually youngerworkers), they were harried until they stung defensively. The Dworkers were then sampled/killed as described in Section 2.5,while the S workers were replaced in their respective nests andwere sampled/killed 14 days later.

The venom sac volumes of all workers sampled in this experi-ment were quantified as described in Section 2.3. The ability ofworkers to replace spent venom was assessed within each agegroup by comparing the venom sac volumes of their D and S sub-groups. Comparisons between each age group’s U and D subgroupswere also made to assess the success of the depletion treatment.The rates of post-depletion venom production (i.e., venom replace-ment) were also compared between age groups; within each agegroup venom replacement rates were calculated for each colonyas the mean venom sac volume of S subgroup workers minus thatof D subgroup workers, divided by 14 days.

2.7. Statistical analyses

Statistical analyses were performed using either MINITAB sta-tistical software (Minitab, 1999) or as per Zar (1999). Whereverdata did not meet parametric assumptions, the appropriate non-parametric analyses were applied.

Fig. 4. Average location score across worker age. Average location scores below 2(shaded area) indicate a tendency to be found inside nest; average location scoresabove 2 indicate a tendency to be found outside nest. Sample sizes, n, shown forlabeled ages starting at 1, ending at 344. Data from ages 345 to 701 are excludedfrom the figure since they include sample sizes of 0 in many cases. Lines are leastsquare mean lines. Ninety-five days of age was identified as the break pointbetween the sloping and horizontal portions of the data by sequentially fitting leastsquare-mean lines starting with data for the 1–80 day old workers and increasingthe ages included in one day increments (up to 114 days of age). A peak in theresultant R2 values was observed at 95 days of age, indicating that the proportion ofthe variation in average location score that is associated with variation in workerage, as described by the straight-line model, declines as data for ages above 95 daysare included. Average location score for ages 1–95 days = 0.018 (age) + 1.093,R2 = 0.795. Average location score for ages 96–344 days = 0.0003 (age) + 2.3973,R2 = 0.004. These least-square-mean lines intersect at �74 days of age.

3. Results

3.1. Worker longevity and temporal polyethism

The lifespans of H. saltator workers in this study varied betweenjust over a week to nearly 2 years, with indications that longer life-spans are possible (Fig. 3). Lifespan was positively associated withworker size as measured by the cube of worker headwidth: Spear-man Rank Correlation, rs(c) = 0.563, n = 33, p < 0.001 (two deadworkers accidentally discarded without measuring headwidths).This association remains even when the three extremely long-livedworkers are excluded: Spearman Rank Correlation: rs(c) = 0.512,n = 30, p < 0.005. The relationship can be described mathematicallyas lifespan, d = 118.4 (Cube of Headwidth, mm) – 841.9, R2 = 0.17,n = 33, or, with the three oldest workers excluded, as lifespan,d = 71.4 (Cube of Headwidth, mm) – 448.9, R2 = 0.20, n = 30.

H. saltator workers were found to follow the typical pattern ofage polyethism observed in other ants (Hölldobler and Wilson,1990, 2009; Bourke and Franks, 1995) and social insects in general;

workers tend to remain within the nest when young and move to-wards the nest periphery and ultimately outside the nest as theyage (Fig. 4). These data indicate a transition age, the age afterwhich workers tend to be found outside the nest rather than inside,to be about 50 days of age (Fig. 4). After 50 days of age, the ten-dency to be found outside the nest continued to increase until aplateau starting around 74 days of age (Fig. 4).

Fig. 6. Venom sac volume (means ± 95% C.I.s) of Undepleted, Depleted, andSynthesis sub groups for three different age groups of H. saltator workers.⁄⁄⁄ Indicates p < 0.001, ⁄⁄ p < 0.005 (Mann�Whitney U tests, one-tailed, withBonferroni corrections: Age 30 Undepleted vs.Depleted, W = 1316.0, n1 = n2 = 30,p < 0.001; Age 30 Depleted vs. Synthesis, W = 589.0, n1 = n2 = 30, p < 0.001; Age 100Undepleted vs. Depleted, W = 1323.0, n1 = n2 = 30, p < 0.001; Age 100 Depleted vs.Synthesis, W = 546.0, n1 = n2 = 30, p < 0.001; Age 206 Undepleted vs. Depleted,W = 1129.0, n1 = n2 = 30, p < 0.001; Age 206 Depleted vs. Synthesis, W = 736.0,n1 = n2 = 30, p < 0.005). Worker size did not vary by worker age (ANOVA,F2,267 = 1.22, p = 0.30), nor by treatment group within age groups (ANOVA 30 dayolds, F2,87 = 0.05, p = 0.95; ANOVA 100 day olds, F2,87 = 0.33, p = 0.72; ANOVA 206day olds, F2,87 = 2.29, p = 0.11).

1572 K.L. Haight / Journal of Insect Physiology 58 (2012) 1568–1574

3.2. Venom production and worker age

H. saltator workers eclose from their pupal cases containing nodiscernable venom, but were found to then fill their venom sacs toan average fullness of �118 nl by around 57 days of age (Fig. 5).Initial production rates, roughly calculated as the difference be-tween the average venom sac contents of adjacent ages dividedby the number of days between them, show the highest synthesisrate occurs just after eclosion (an average of �3 nl/day between 0and 15 days of age) and declines as workers age and their venomsacs fill (Fig. 5).

3.3. Venom replacement and worker age

The three different age groups examined in this study were cho-sen based on the data shown in Figs. 3–5. Thirty-day-old workerswere chosen to represent young workers as they have not yet filledtheir venom sacs to capacity (Fig. 5) and do not yet tend to ventureoutside the nest (Fig. 4). One hundred-day-old workers were cho-sen to represent mid-aged workers because their venom sacs arefull (Fig. 5) and they have only recently plateaued in their tendencyto be outside the nest (Fig. 4). Two hundred six-day-old workerswere chosen to represent old workers as they have long sinceplateaued in their tendency to be outside the nest (Fig. 4) and, atthe median observed lifespan, they have outlived half of their con-temporaries (Fig. 3).

Within each of the three age groups tested (30, 100, and206 days old), the D subgroup workers had lower venom sac vol-umes than those of the U subgroup, and the S subgroup workershad higher venom sac volumes than those of the D subgroup(Fig. 6). Thus, the depletion method was effective, and all threeof the tested age groups were able to replace spent venom. How-ever, the rate of replacement differed; the 100-day-old workers re-placed their venom at a higher rate (�5 nl/day on average) thanboth the 30-day-old and 206-day-old workers (�3 nl/day on aver-age each; Fig. 7).

4. Discussion

In order to investigate how the venom production and replace-ment abilities of H. saltator workers vary over lifetime, information

Fig. 5. Venom sac volume (means ± 95% C.I.s) of H. saltator workers by worker age.Sample sizes of each age group indicated. Venom sacs from ages 0 and 1 daycontained no venom; data shown for these ages represent the volumes of emptyvenom sacs. Worker size did not vary between age groups (ANOVA, F8,203 = 1.64,p = 0.12).

Fig. 7. Venom replacement rates (means ± 95% C.I.s) for three different age groupsof H. saltator workers. Rates calculated for each age group as (mean venom nl Ssubgroup workers – mean venom nl D subgroup workers)/14 days from eachcolony. Rates are those of venom replenishment after having subdued prey. Lettersindicate significant differences: Kruskal–Wallis test: H = 10.21, n1 = n2 = n3 = 15,p = 0.006. Nemenyi test (nonparametric Tukey-type multiple comparisons, family-wise a = 0.05): Age 30 vs. 100: q = 4.29, p < 0.01; Age 100 vs. 206: q = 3.38, p < 0.05;Age 206 vs. 30: q = 0.90, p > 0.50.

regarding worker longevity is required if given ages are to be eval-uated in a physiologically meaningful context. Though a workerlifespan of 1.5 years (�548 days) is reported in the literature(Schneider et al., 2011), it is a maximum, and as such lacks infor-mation regarding variation which is useful when planning experi-ments using age as a factor. Thus, worker longevity was assessed inthis study so discrete ages could be meaningfully assigned to rep-resent ‘‘young’’, ‘‘mid-aged’’, and ‘‘old’’ workers, and could be cho-sen with some amount of confidence that a sufficient number ofworkers would survive to those ages to obtain useful sample sizes.

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This longevity assessment showed H. saltator worker lifespan tovary between just over a week to nearly 2 years, with indicationsthat longer lifespans are possible. Of course, this assessment wasmade in the laboratory, were workers were sheltered from theusual sources of mortality present under natural conditions.

As the H. saltator workers progressed through their lives, theywere found to follow the typical pattern of age polyethism ob-served in other ants; workers tended to stay inside the nest whenyoung, and their propensity for exploration outside the nest grad-ually increased as they aged. This propensity for exploration isprobably associated with foraging behavior in H. saltator, since itexhibits low territoriality (J. Liebig, pers. comm.), and workerstherefore have little other reason to venture from the nest (H. sal-tator does not recruit nest mates to food; Maschwitz et al., 1979;Shivashankar et al., 1989). Venom production also followed a pat-tern similar to that seen in other ants (i.e., S. invicta, the other antspecies where data are currently available; Haight and Tschinkel,2003). Workers eclosed from their pupal cases containing no ve-nom, and then proceeded to fill their venom sacs. As in S. invicta,initial production rates observed in H. saltator workers were alsohighest just after eclosion.

The similarity to S. invicta is no longer apparent, however, whenvenom replacement is considered. No venom replacement wasfound to take place in S. invicta workers after 30 days of age (Haightand Tschinkel, 2003), but in H. saltator workers, all three agestested (30, 100, and 206 days of age) were able to replace spent ve-nom. So, the early-life limitation of venom-production ability seenin S. invicta does not appear to be a general physiological pattern inants, though it may be a common pattern in ant species which donot rely on venom for food acquisition.

Because of its reliance on venom for food acquisition and thelongevity and temporal polyethism pattern observed in its work-ers, the ability of H. saltator workers to replace spent venom is nec-essary if their effectiveness as foragers is to be maintained. With alifespan of 250 days and a transition to outside work (i.e. foraging)at 50 days, the foraging career of an average H. saltator worker con-stitutes 80% of its life (assuming it avoids becoming prey or other-wise lost in the field). Using the more conservative numbers of a212 day lifespan and a foraging career that starts at 74 days ofage (i.e., at the beginning of the location-score plateau), the poten-tial foraging career of an average H. saltator worker is still greaterthan half its life (�65%). If venom-production ability were limitedto early life in this ant species, workers would probably have totightly ration their non-renewable venom supply to remain effec-tive over these long foraging careers. However, the results of thisstudy show that even H. saltator workers which have aged to thepoint where half their contemporaries have died from old age havethe ability to replace spent venom, and thus have the potential tostill be effective foragers.

Though their ability to replace venom appears robust, H. saltatorworkers nonetheless exhibit venom-related behaviors that suggestselection pressure to optimize their investment. The age at whichworkers transition into their foraging careers, 50 days, coincideswell with the age at which worker venom sacs reach fullness(around 57 days), and the beginning of the location score plateau,74 days, coincides well with the age at which venom replacementrate measured highest (100 days). These coincidences of venomphysiology and exploratory behavior likely result in foraging effi-ciency benefits for H. saltator, as they lead to workers which arefully equipped (or nearly so) around the time they begin their for-aging careers, and the workers that are the most dedicated to for-aging are, at least initially, the most physiologically able to handlea higher prey encounter rate. A similar correlation between venomphysiology and defensive behavior was also found in the fire ant, S.invicta; defensiveness (number of stings delivered and venomdelivered per sting) peaked around the age workers finished ve-

nom production, i.e., when their venom sacs were full and theywere therefore the most physiologically suitable nest defenders(Haight, 2008).

Though this study was designed to quantify age-related pat-terns of venom replenishment, the resulting data provide insightinto possible patterns of venom use as well. When subduing thetwo crickets offered during the depletion treatment, the 30 and100 day olds, on average, spent similar proportions of their avail-able venom (63.7% and 72.8%, respectively), while the 206 day oldsspent much less (42.4%). This apparent difference could be due tosenescence, as perhaps the muscles used to expel venom are weak-er at this advanced age. Or, perhaps as workers age their venom be-comes more potent and the 206 day olds need less to successfullysubdue their prey. However, since the venom replacement ratealso declines as workers age past 100 days, senescence may bethe more likely cause. In the face of venom gland senescence, ve-nom conservation by old workers would also potentially extendtheir careers as effective foragers. But, given that longevity underfield conditions is most likely lower than found here, it is unclearwhether any such benefit is actually realized. Further research isneeded to determine how, if at all, these or other factors contributeto venom use in H. saltator.

Acknowledgements

I thank Juergen Liebig and Bert Hölldobler for the use of H. sal-tator colonies, lab space, and equipment, Adrian A. Smith for assis-tance with photography, Clint Penick for assistance with graphicdesign, Anne A. Madden and Kaustubh Gokhale for providinghard-to-find references, and Anne A. Madden, Kaustubh Gokhale,Rick Overson, Clint Penick, Neale Copple, Barbara Birtcil, LotharWissler, Juergen Liebig, and two anonymous reviewers for discus-sion and/or constructive criticism of the manuscript.

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