Naturwissenschaften

, Volume 92, Issue 6, pp 282–286 | Cite as

Fertility signaling—the proximate mechanism of worker policing in a clonal ant

  • Anne Hartmann
  • Patrizia D’Ettorre
  • Graeme R. Jones
  • Jürgen Heinze
Short Communication

Abstract

In eusocial insects, the ability to regulate reproduction relies on cues that signal the presence of fertile individuals. We investigated the variation of cuticular hydrocarbons (CHCs) with reproductive status in Platythyrea punctata, an ant, in which all workers are capable of producing daughters from unfertilized eggs (thelytoky). Who reproduces is determined through dominance and worker policing. New reproductives, which developed their ovaries after separation from an old reproductive for a short period of time, were attacked by nonreproductives upon reintroduction into their colony. In contrast, aggression against new reproductives with fully developed ovaries, which had been separated over a longer period, was initiated by fights between old and new reproductives. CHC profiles varied with ovarian development. New reproductives were only attacked when they expressed a CHC profile similar to old reproductives, but not when it was similar to that of nonreproductives. CHCs appear to signal the fertility of individuals and induce policing behavior towards surplus reproductive workers.

Introduction

Cuticular hydrocarbons (CHCs) appear to play a fundamental role in signaling fertility in eusocial insects. CHC profiles vary consistently with ovarian activity (Monnin et al. 1998; Liebig et al. 2000; Cuvillier-Hot et al. 2001, 2004; Heinze et al. 2002), and colony members may utilize this to recognize reproductives and to regulate reproduction in a way that maximizes their inclusive fitness (Keller and Nonacs 1993).

Platythyrea punctata is an unusual ant in that workers can produce diploid females from unfertilized eggs by thelytokous parthenogenesis, which results in a clonal colony structure (Heinze and Hölldobler 1995; Schilder et al. 1999a, b). All individuals are morphologically identical and equally capable of laying eggs, but each colony nevertheless contains only one, rarely two egg-layers (Schilder et al. 1999b). “Monogyny” apparently enhances colony efficiency and is maintained by self-restraint of nonreproductives as long as a reproductive is present (Hartmann et al. 2003). Workers, who become fertile after separation from an old reproductive, are attacked and even killed by their nestmates when returned to the nest (Hartmann et al. 2003). Such “worker policing” (Monnin and Ratnieks 2001) requires that workers can distinguish “old” and “new” reproductives. Here, we examine whether CHC profiles might provide the proximate cues for such discrimination.

Methods

P. punctata colonies from Puerto Rico were reared in the laboratory and behavioral and reproductive status of workers was determined as previously described (Hartmann et al. 2003).

One to 2 weeks after collection, CHC profiles of one reproductive and 10–13 foragers and inside workers from three colonies were analyzed. The possible role of CHCs in worker policing was examined by inducing the development of new reproductives (NRs) by dividing eight laboratory colonies (A–H) in half, part I with old reproductives (OR) and part II with only nonreproductives. Parts I and II of colonies A, B, and C were reunited after NRs had laid eggs for less than 27 days. In colonies D to H only the NRs were reintroduced into part I after having laid eggs for a much longer time (D: 78 days; E: 99 days; F: 47 days; G: 99 days; H: 57 days). Colonies A, B, and C were observed six times per day for 10 min each over a period of 7 days before and after reunification (Hartmann et al. 2003). In colonies D to H, we noted all interactions involving NRs during five sessions of 10 min each over a period of 4–24 h after reintroduction. As a control, in colonies D to H we similarly noted interactions involving a nonreproductive from part II, who was introduced into part I after the NR had been removed.

Before colony division, we analyzed CHCs from all ORs, 2–3 inside workers, and 2–3 foragers from each colony. In colonies A, B, and C, CHC profiles from ORs and NRs and 2–3 nonreproductives from parts I and II each were analyzed 1–3 days before reunification and again 3–11 days after reunification. In colonies D to H, CHCs were extracted from ORs, NRs, and 4–13 nonreproductives from parts I and II, 2–13 days before NRs were reintroduced. After the experiment, all individuals were dissected and the status of their ovaries determined.

For chemical analyses, CHCs were extracted from live ants by rubbing their gasters for 5 min with a 7 μm polymethylsiloxane fiber (Supelco, Bellefonte, USA) (Solid Phase Microextraction, SPME, Arthur and Pawliszyn 1990). The fiber was injected into an Agilent Technologies 6890N gas chromatograph with split/splitless inlet, flame ionization detector: Rtx-5 capillary column (30 m × 0.25 mm × 0.50 μm, Restek, Bellefonte, PA, USA); Helium (1 ml/min): 100–180°C, 30°C/min, 180–280°C, 3°C/min, 280°C for 10 min.

Compounds were identified on the basis of their mass spectra, which were produced using an Agilent 5973N MSD/DS system with a 6890N GC with split/splitless inlet and MS interface, and a 5973N MS system (70 eV electron impact ionization) and confirmed with reference to synthetic standards. Double bond positions were located using a DMDS reaction followed by GC-MS.

Groups of workers (foragers, inside workers, old and new reproductives) were separated on the basis of their CHC profiles by discriminant analysis (DA). The relative proportions of 30 peaks (Table 1, excluding the irregularly occurring peaks 23 and 28) were subjected to a principal components analysis (PCA) to reduce the number of variables for DA (minimum eigenvalue of extracted factors was equal to 1). To determine, which substances differ between reproductives and nonreproductives, we calculated for each compound the correlation ratio (an index of correlation between the proportion of each compound and behavioral/reproductive group). Compounds were ranked in descending order according to their correlation ratios and the difference between groups was tested by Mann–Whitney U-test.
Table 1

Cuticular hydrocarbons of the ant Platythyrea punctata. The proportions of peaks marked with the symbols ▴ and ▾ were significantly higher and lower, respectively in reproductive than non-reproductive workers (Mann–Whitney U-test, all P<0.05)

Peak

Compound

Peak

Compound

1:

C21

▾17:

2,9-C25:2, 9-C25:1

2:

2-MeC21

18:

7-C25:1

3:

C22

▴19:

C25

4:

2-MeC22

▾20:

9-pentacosyne

5:

2,9-C23:2

21:

9-MeC25, 11-MeC25, 13-MeC25

▾6:

9-C23:1

▴22:

7-MeC25

7:

7-C23:1

23:

5-MeC25

8:

C23

▴24:

2-MeC25

▴9:

9-MeC23, 11-MeC23

25:

3-MeC25

10:

7-MeC23

26:

C26

▴11:

5-MeC23

27:

11-MeC26, 12-MeC26, 13-MeC26

▴12:

2-MeC23

28:

8,16-C27:2

▴13:

3-MeC23

▾29:

9-C27:1, x, x-C27:2, 7-C27:1

14:

C24

30:

C27

15:

10-MeC24, 11-MeC24, 12-MeC24, 13-MeC24

31:

9-MeC27, 11-MeC27, 13-MeC27

▾16:

8,16-C25:2

32:

2-MeC27

Results

CHCs are linear alkanes, alkenes, dienes and methyl-branched alkanes with chain lengths from C21 to C27 (Table 1). Of particular interest is the occurrence of 9-pentacosyne, the first alkyne to be observed on the cuticle of an ant. The CHC profiles of reproductives, foragers and inside workers from three natural colonies were completely separated by DA (function 1 accounted for 80% of the total variance; Wilks’ λ=0.043, F16,54=12.99, P<0.00001; correct classification: reproductives 100%; foragers 94.4%; inside workers 93.8%). CHC profiles differed in the same way in the laboratory colonies (Fig. 1a). Reproductives were characterized by higher amounts of methyl-branched C23 and C25 and lower proportions of alkenes with chain lengths of 23 to 27 as well as of one alkyne (Table 1).
Fig. 1

Discriminant analysis based on the proportions of cuticular hydrocarbons extracted from Platythyrea punctata colonies A to H (a) before separation of colony parts I and II (Wilks’ λ=0.09, F14,78=12.96, P<0.00001; 87.5% reproductives, 90.5% inside workers, and 78.9% foragers correctly classified) and (b) before reintroduction. The group of reproductives in (b) includes both old reproductives (ORs) and NRA3, NRB2 and NRC1 (Wilks’ λ=0.27, F16,188=10.81; P<0.00001; all reproductives, 51.3% inside workers, and 81.6% foragers correctly classified). ▴ old reproductive; ▵ new reproductive (attacked); ⋄ new reproductive (not attacked); ○ inside worker; • forager

After separation, two or three workers each began to reproduce in part II of colonies A, B, and C (Table 2). When parts I and II were reunited shortly after the onset of reproduction, nonreproductives attacked several but not all of these NRs by antennal boxing, biting, and dragging (Table 2). ORs rarely engaged in aggression (for details see Hartmann et al. 2003).
Table 2

Ovarian development of old reproductives (OR) and new reproductives (NR), amount of aggression received by reproductives after reunification of separated colony parts (0: none, ±: not more than before reunification; +/++: more/much more than before reunification), and the category individuals grouped with in a discriminant analysis (DA) based on their cuticular hydrocarbon profiles (see text). Ovarian status: I undeveloped, without any oocytes; II inactive without yolky oocytes, but with nurse cells; III elongated, with 1–3 yolky oocytes; IV fully developed, elongated, with more than three yolky oocytes

Colony

Ovariole length (mm)

Number of yolky oocytes

Ovarian status

Aggression received

Category in DA directly before reunification

Category in DA after reunification

A

OR

4.1

7

IV

±

OR

OR

NR1

0

II

±

IW

IW

NR2

1.48

3

III

±

IW

IW

NR3

1.52

>2

III

++

OR

OR

B

OR1

2.45

3

III–IV

++

OR

IW

OR2

2.43

4

IV

++

OR

OR

NR1

1.43

0

II

+

IW

IW

NR2

1.29

0

I

++

OR

OR

C

OR

3.47

6

IV

0

OR

OR

NR1

1.54

0

II

±

OR

IW

NR2

1.12

0

I

±

IW

IW

D

OR

3.12

6

IV

 

OR

NR

2.64

5

IV

++

OR

E

      

OR

3.65

5

IV

 

OR

NR

3.6

4

IV

++

OR

F

OR

a

   

a

NR

3.23

5

IV

++

OR

G

OR

3.26

6

IV

 

OR

NR

3.17

5

IV

++

OR

H

      

OR

3.57

5

IV

 

OR

NR

2.51

4

IV

++

OR

IW Inside worker

aDied before second analysis; not dissected

In contrast, attacks in colonies D to H, in which NRs were reintroduced after 2–3 months of separation, consistently started with fights between OR and NR. During these interactions, one ant would grab an opponent’s leg or antenna and rub it against the tip of her gaster. Gaster rubbing elicited attacks by nonreproductives against NRs from colonies D, E, and G, and against both OR and NR from colony F. In colony H, only the OR was attacking the NR, and neither the NR nor other workers were aggressive. Nonreproductive workers from part II, who were reintroduced into part I as a control, were never attacked.

All ORs had well-developed ovaries of type III or IV (Table 2). Ovaries of NRs in colonies A, B, and C were much less developed. Neither ovarian status nor the number of developing oocytes differed between attacked and tolerated NRs (Table 2). NRs of colonies D to H had fully developed type IV ovaries with slightly, but not significantly less oocytes than the respective OR (Wilcoxon matched pairs test, T=0.00, P=0.068).

Before reintroduction, the CHC profiles of eight NRs (NRA3, NRB2, NRC1, NRD – NRH) differed from those of nonreproductives and resembled the profiles of ORs. Seven of these NRs (all but NRC1) were strongly attacked after reintroduction, while four NRs with CHC profiles similar to nonreproductives received almost no aggression (Table 2). When the eight NRs were grouped with ORs, all were correctly classified by DA (Fig. 1b).

Shortly after reunification, the CHC profiles of NRC1 and ORB1 reverted to those of nonreproductives. Grouping NRA3 and NRB2 with ORs and the other NRs and ORB1 with inside workers resulted in a 100% correct classification (Wilks’ λ=0.05; F16,38=8.65; P<0.00001).

Changes in cuticular hydrocarbons occasionally lagged behind ovarian development. For example, while NRA2 had the profile of a nonreproductive, her ovaries were elongated and contained yolky oocytes. Workers obviously reacted more to the cuticular profile of NRs than to their reproductive status: NRA2 received almost no aggression.

Discussion

According to our comparison of behavioral, chemical, and dissection data, CHC profiles in P. punctata are correlated with ovarian activity and might provide the basis for the recognition of reproductive status and policing. Despite its unusual thelytokous reproduction, resulting in clonality and a lack of relatedness-driven conflict, P. punctata in this respect resembles ants with an ordinary biparental reproduction.

CHCs of P. punctata reproductives are characterized by the dominance of branched hydrocarbons, which also differentiate reproductives from nonreproductives in other social insects (Cuvillier-Hot et al. 2001; Dietemann et al. 2003; Hannonen et al. 2002; Heinze et al. 2002). Due to their structure, branched alkanes might be more informative than linear alkanes (Breed 1998; Dani et al. 2001).

P. punctata workers can distinguish between an OR and a not yet fully fertile NR and are aggressive towards the latter. Whilst aggression correlates with the CHC data, the ovarian status does not always fully correlate with the hydrocarbon profile, which sometimes lags behind and sometimes precedes the ovarian development. Time lags between CHCs and reproductive status are also known from paper wasps (Sledge et al. 2001) and ants, e.g., Dinoponera quadriceps workers need several weeks to develop the CHC profile characteristic of established α-workers after obtaining the top-position in the colony’s hierarchy (Peeters et al. 1999; see also Cuvillier-Hot et al. 2004).

When both reproductives are well-established egg-layers, aggression by workers starts only after fighting between OR and NR, indicating that they do not distinguish between mature reproductives. Worker aggression seems to be provoked by gaster rubbing, i.e., a reproductive rubs the opponent’s antenna or leg against the tip of her gaster, apparently marking her rival. Similar gaster rubbing with Dufour gland secretion initiates punishment of Dinoponera workers, who try to usurp the α-worker (Monnin et al. 2002). In P. punctata, workers apparently do not distinguish between established reproductives but attack those who are marked through gaster rubbing be they the old or the new reproductive. Gaster rubbing therefore constitutes no alliance between the old reproductive and nonreproductives against a pretender. Given that the colony members are clones, such a preference for the old reproductive might also not be selected. Instead, individuals should benefit most from choosing the more fecund of the two reproductives. The origin of the secretion applied during gaster rubbing is presently not known and the Dufour gland seems to be absent in P. punctata (Morgan et al. 2003).

The lack of a discrimination between fully fertile OR and NR match observations in the ant Harpegnathos saltator, where workers attack nestmates who had been isolated for only a few weeks (Liebig et al. 1999) but tolerate those separated for 90 days. As in P. punctata, only the reproductives start dueling, but in contrast both continue to lay eggs and are permanently accepted by other workers (Liebig 1998). Our discriminant analysis does not separate new and established NRs, suggesting that workers here use other cues than CHCs or respond only to certain compounds of the CHC profile (see also Dietemann et al. 2003).

Notes

Acknowledgements

We were supported by the EU through the ‘INSECTS’ research network (HPRN-CT-2000-00052) and DFG (He 1623). J. Korb and J. Torres helped in the field, M. Roux with statistics, and F. Drijfhout with chemical analysis. DNRA provided a collection permit in Puerto Rico

References

  1. Arthur CL, Pawliszyn J (1990) Solid phase microextraction with thermal desorption using fused silica optic fibers. Anal Chem 62:2145–2148Google Scholar
  2. Breed MD (1998) Chemical cues in kin recognition: criteria for identification, experimental approaches, and the honey bee as an example. In: Vander Meer RK, Breed KE, Espelie KE, Winston ML (eds) Pheromone communication in social insects. Ants, wasps, bees and termites. Westview Press, Boulder, pp 57–78Google Scholar
  3. Cuvillier-Hot V, Cobb M, Malosse C, Peeters C (2001) Sex, age and ovarian activity affect cuticular hydrocarbons in Diacamma ceylonense, a queenless ant. J Insect Phys 47:485–493Google Scholar
  4. Cuvillier-Hot V, Lenoir A, Crewe R, Malosse C, Peeters C (2004) Fertility signalling and reproductive skew in queenless ants. Anim Behav 68:1209–1219Google Scholar
  5. Dani FR, Jones GR, Destri S, Spencer SH, Turillazzi S (2001) Deciphering the recognition signature within the cuticular chemical profile of paper wasps. Anim Behav 62:165–171Google Scholar
  6. Dietemann V, Peeters C, Liebig J, Thivet V, Hölldobler B (2003) Cuticular hydrocarbons mediate discrimination of reproductives and nonreproductives in the ant Myrmecia gulosa. Proc Natl Acad Sci USA 100:10341–10346Google Scholar
  7. Hannonen M, Sledge MF, Turillazzi S, Sundström L (2002) Queen reproduction, chemical signalling and worker behaviour in polygyne colonies of the ant Formica fusca. Anim Behav 64:477–485CrossRefGoogle Scholar
  8. Hartmann A, Wantia J, Torres JA, Heinze J (2003) Worker policing without genetic conflicts in a clonal ant species. Proc Natl Acad Sci USA 100:12836–12840Google Scholar
  9. Heinze J, Hölldobler B (1995) Thelytokous parthenogenesis and dominance hierarchies in the ponerine ant, Platythyrea punctata (F. Smith). Naturwissenschaften 82:40–41Google Scholar
  10. Heinze J, Stengl B, Sledge MF (2002) Worker rank, reproductive status and cuticular hydrocarbon signature in the ant, Pachycondyla cf. inversa. Behav Ecol Sociobiol 52:59–65Google Scholar
  11. Keller L, Nonacs P (1993) The role of queen pheromones in social insects: queen control or queen signal. Anim Behav 45:787–794Google Scholar
  12. Liebig J (1998) Eusociality, female caste dimorphism, and regulation of reproduction in the ponerine ant Harpegnthos saltator Jerdon. PhD thesis. Wissenschaft und Technik Verlag, BerlinGoogle Scholar
  13. Liebig J, Peeters C, Hölldobler B (1999) Worker policing limits the number of reproductives in a ponerine ant. Proc R Soc Lond B 266:1865–1870Google Scholar
  14. Liebig J, Peeters C, Oldham NJ, Markstädter C, Hölldobler B (2000) Are variations in cuticular hydrocarbons of queens and workers a reliable signal of fertility in the ant Harpegnathos saltator?: Proc Natl Acad Sci USA 97:4124–4131Google Scholar
  15. Monnin T, Malosse C, Peeters C (1998) Solid-phase microextraction and cuticular hydrocarbon differences related to reproductive activity in queenless ant Dinoponera quadriceps. J Chem Ecol 24:473–490Google Scholar
  16. Monnin T, Ratnieks FLW (2001) Policing in queenless ponerine ants. Behav Ecol Sociobiol 50:97–108CrossRefGoogle Scholar
  17. Monnin T, Ratnieks FLW, Jones GR, Beard R (2002) Pretender punishment induced by chemical signalling in a queenless ant. Nature 419:61–65PubMedGoogle Scholar
  18. Morgan ED, Jungnickel H, Keegans SJ, Do Nascimento RR, Billen J, Gobin B, Ito F (2003) Comparative survey of abdominal gland secretions of the ant subfamily Ponerinae. J Chem Ecol 29:95–114CrossRefPubMedGoogle Scholar
  19. Peeters C, Monnin T, Malosse C (1999) Cuticular hydrocarbons correlated with reproductive status in a queenless ant. Proc R Soc Lond B 266:1323–1327CrossRefGoogle Scholar
  20. Schilder K, Heinze J, Gross R, Hölldobler B (1999a) Microsatellites reveal clonal structure of populations of the thelytokous ant Platythyrea punctata (F. Smith) (Hymenoptera; Formicidae). Mol Ecol 8:1497–1507Google Scholar
  21. Schilder K, Heinze J, Hölldobler B (1999b) Colony structure and reproduction in the thelytokous parthenogenetic ant Platythyrea punctata (F. Smith) (Hymenoptera; Formicidae). Insectes Soc 46:150–158Google Scholar
  22. Sledge MF, Boscaro F, Turillazzi S (2001) Cuticular hydrocarbons and reproductive status in the social wasp Polistes dominulus. Behav Ecol Sociobiol 49:401–409CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • Anne Hartmann
    • 1
  • Patrizia D’Ettorre
    • 1
  • Graeme R. Jones
    • 2
  • Jürgen Heinze
    • 1
  1. 1.Biologie IUniversität RegensburgRegensburgGermany
  2. 2.School of Chemistry and Physics, Lennard-Jones LaboratoriesKeele UniversityStaffordshireUK

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