Comparative reproductive biology of the social parasite Acromyrmex ameliae de Souza, Soares & Della Lucia and of its host Acromyrmex subterraneus subterraneus Forel (Hymenoptera: Formicidae)

ECOLOGY, BEHAVIOR AND BIONOMICS

Comparative reproductive biology of the social parasite Acromyrmex ameliae de Souza, Soares & Della Lucia and of its host Acromyrmex subterraneus subterraneus Forel (Hymenoptera: Formicidae)

Ilka M F Soares^{I, II}; Terezinha M C Della Lucia^II; Alice S Pereira^II; José E Serrão^III; Myriam M R Ribeiro^II; Danival J de SouzaII, IV, ^*

^IUniv do Estado da Bahia, Depto de Educação, Campus VIII, Av da Gangorra 503, Alves de Sousa, 48608-240 Paulo Afonso, BA, Brasil; [e-mail]

IIUniv Federal de Viçosa, Av P H Rolfs, Depto de Biologia Animal, 36570-000 Viçosa, MG, Brasil; [e-mail]; [e-mail]; [e-mail]; [e-mail]

^IIIUniv Federal de Viçosa, Av P H Rolfs, Depto de Biologia Geral, 36570-000 Viçosa, MG, Brasil

^IVactual address: Univ Federal do Tocantins, Campus de Gurupi, 77402-970, Gurupi, TO, Brasil; [e-mail]

ABSTRACT

Social parasites exhibit several characteristics that allow them to exploit their host species efficiently. The smaller size of parasite species is a trait commonly found in ants. In this work, we investigated several aspects of the reproductive biology of Acromyrmex ameliae De Souza, Soares & Della Lucia, a recently discovered parasite of Acromyrmex subterraneus subterraneus Forel. Sexuals of A. ameliae are substantially smaller than those from host species. Parasite queens laid significantly less worker eggs than host queens and inhibit sexual production of the host. The sex ratio of parasite species is highly female biased. Interestingly, we have observed parasite coupling on the laboratory, inside the nests and in the ground, opening the possibility to use controlled mating to study genetic approaches of parasitism in the ants.

Key words: Social parasitism, egg production, size reduction

One of the most remarkable traits of an insect society is the reproductive cooperation (Wilson 1971). However, social insects are vulnerable to reproductive parasitism, both interspecific and intraspecific. Host exploitation by parasitic species involves elaborated parasitic adaptations such as morphophysiological and behavioral alterations (Hölldobler & Wilson 1990). In the ants, sexuals of social parasite species are usually smaller than the queens of the host species. According to the miniaturisation hypothesis, size reduction might have evolved as a means of the species to take advantage in the system of host caste determination (Bourke & Franks 1991). Parasitic larvae may develop when the host does not produce sexuals; in addition, the parasite larvae develop into sexuals with lesser quantities of food than is required to produce workers (Bourke & Franks 1991, Nonacs & Tobin 1992). The smaller size reduces the capacity of host workers to distinguish between their immatures and those of the parasite as in Plagiolepis pygmaea (Latreille) (Aron et al 1999) and facilitates the subjection of the parasite to the host queen as in the extreme inquilines (those without the worker caste) such as Teleutomyrmex and Pseudomyrmex (Hölldobler &Wilson 1990).

Parasite queens and workers can also influence host reproduction. The workers of Acromyrmex insinuator Schultz, Bekkevold & Boomsma, the inquiline of Acromyrmex echinatior (Forel), are essential to the production of the parasite sexuals, indicating that this attribute is one of the main functions of the parasite workers (Sumner et al 2003). They are capable of inhibiting the reproduction of the host queen. This same behavior has been reported in P. pygmaea, the host of Plagiolepis xene Stärcke, in laboratory colonies (Passera et al 2001).

Internal differences of the reproductive tract are found between social parasites and their host species. In the ant Ectatomma tuberculatum (Olivier) the queens exhibit size dimorphism and differences in their oocyte numbers (Hora et al 2001, 2005). The microgynes - reproductive females of smaller sizes than macrogines in the same nest - are specialized in microgyne and male production and seldom produce workers; macrogynes produce large number of workers but never microgynes (Hora et al 2003). Using a terminology developed to parasitoid wasps (Flanders 1950), ants are synovigenic, i. e., queens continue to mature eggs throughout their reproductive life, including the parasitic queens.

Acromyrmex ameliae has recently been described by De Souza et al (2007). It is a social parasite of the inquiline type that has a few minor workers similar to those of its hosts, the leaf-cutting ants Acromyrmex subterraneus subterraneus Forel and Acromyrmex subterraneus brunneus (Forel), and appears to be closely related to these hosts.

This study investigated reproduction features of the social parasite A. ameliae, and compared some of these reproduction aspects with those of its host A. subterraneus subterraneus. Nuptial flight period in laboratory and size reduction in males and females were determined. Additionally, morphological and histological analyses of the female reproductive tract and comparative egg production by A. ameliae and its host A. subterraneus subterraneus were performed.

The following hypotheses were tested: 1) Females of A. ameliae have size reduction beyond the size observed in the major workers of the host; consequently 2) egg production in the parasite species is lower than in its host; 3) parasitic queens and/ or workers interfere in the egg production by the host queen.

Material and Methods

Parasitized colonies of A. subterraneus subterraneus were collected in Paraopeba, Minas Gerais State (MG), Brazil (19º 17'S; 44º 29' W) in 2003 and 2004. Each of these colonies had one host queen and variable numbers of parasite queens (2-6).

Nuptial flight in laboratory and sex proportion. Ten A. ameliae colonies produced alates in the laboratory and two (with the highest numbers) were used to calculate sex ratio in the species. Numbers of parasite alates (males and females) were counted in each nest and sex ratio was obtained by dividing the total number of males by that of females.

Size reduction of sexuals. To verify if A. ameliae has alate individuals significantly smaller than the largest workers of the host A. subterraneus subterraneus, the total body length (from head to gaster) of 44 males, 30 females and 50 major workers of the host and 150 males and 151 females of the parasite species were measured under a stereoscopic microscope using a micrometric ocular (Leica MZ 7.5; Göttingen, Germany). Major host workers were measured for size comparison with those of the parasite queen.

Description of the female reproductive tract. Females of A. ameliae were removed from two colonies and dissected for ovariole and mature oocytes counting. Ovarioles of three females were transferred to Bouin solution for 24h, dehydrated in an increasing series of ethanol solutions, and imbedded in JB-4 resin (Souza et al 2006). The 5 µm-thick sections were dyed with hematoxylin and eosin (Antunes et al 2002). Description of the reproductive tract was based on microscopic observation (100x) of the thin sections.

Comparison of A.subterraneus subterraneus and A.ameliae egg-laying and influence of parasite workers and queens on the oviposition of the host queen. Three colonies of A. subterraneus subterraneus with fungus garden volumes of 5000, 3100 and 2000 ml were used in this experiment. Twenty dealate parasite gynes were recovered from these colonies. The influence of the parasite species on the egg-laying of the host was tested by comparing the following treatments: 1) HQ - isolated host queen (n = 6); 2) HQ(HPW) - host queen with seven host and three parasite workers (n = 6); 3) PQ - isolated parasite queens (n = 20); 4) PQ(HPW) - parasite queens with eight hosts and two parasite workers (n = 16).

Oviposition of host and of parasite queens alone was evaluated and used for comparison of oviposition in the presence of workers of both host and parasite workers (n = 10). Test one consisted of evaluating oviposition of the host queen by itself and test 2 evaluated ovipositon of the host queen in the presence of seven host workers and three parasite workers. This also had the purpose of verifying if the presence of the parasite queen interfered in egg production by the host queen. Due to the small numbers of parasite workers and the larger number of A. ameliae queens, a small number of parasite workers was used in test number 4. The previous estimation of the proportion of A. ameliae workers in the parasitized colony of A. subterraneus subterraneus was around 3% (authors unpublished data).

For each test, the individuals were isolated in closed containers for a period of 16h and fed a diet of water and honey solution (Marinho & Della Lucia 1998). The use of the queens was randomized throughout the experiments and they were giving one week intervals between the tests for recovery. The queens were always removed from the colonies at 8:00h and returned at 00:00h. The numbers of eggs laid were counted at 4h intervals (12:00, 16:00, 20:00, and 00:00h). The ovipositing queens were marked in the gaster with non-toxic ink at the end of test 3 and later used in test 4.

Test 5 aimed to verify the effect of the parasite queens on the host queen egg-laying capability. Since we observed that host and parasite queens generally are not together physically in the fungus garden, it was assumed that if there was an effect of the parasite queen on oviposition, it would be of a chemical nature. Therefore, the host queen was physically isolated from the parasite queens but glass tubes connected the glass containers to allow volatile substances from parasite queens to reach host queens.

To determine if all the parasite egg-laying queens had been fertilized, 30 of these queens were dissected and their spermathecae were examined.

Results

Nuptial flight in laboratory and sex ratio. The 10 colonies of A. subterraneus subterraneus produced parasite alates in abundance from October to February resulting in drastic fungus reduction in all colonies and in subsequent death of two of these colonies. A smaller number of these alates occurred through May. Mating couples were observed. It is believed that some of these females were readopted by their colonies, since the observed number of female parasites in test 4, conducted a few weeks later, was higher, in some cases, than in test 3, conducted weeks before.

The sex ratio of alate males in relation to females was 1:5.3 (474 males and 2,514 females). In 2005 no mating flights were observed under laboratory conditions as occurred in 2003 and 2004, however, some couples were observed mating inside the colony. In November 2006, a large production of males was verified in some colonies. During that period host workers transported live parasite males from the fungus garden and the foraging arena to the colony waste.

Reduction in size of the sexuals.Acromyrmex ameliae sexuals were significantly smaller in size than those of its host A. subterraneus subterraneus. Male size differed between A. ameliae (< 7.0 mm) and A. subterraneus subterraneus (about 8.0 mm in length) (F_1,192 = 137.60, P< 0.001) (Fig 1a). Female parasites were also smaller about 8.0 mm in size, whereas females of the host were approximately 10 mm (F_1,179 = 1071.2, P< 0.001) (Fig 1b). Body size of A. ameliae females is smaller than that of the major workers of A. subterraneus subterraneus (~ 10.0 mm) (F_1,199 = 921.07, P < 0.001) (Fig 2).

Description of the female reproductive tract. The reproductive system of A. ameliae is composed by one pair of ovaries, formed by 13 to 15 ovarioles per ovary. The walls of the lateral and common oviducts are formed by a well developed musculature, layering a simple epithelium of flattened cells with nuclei having predominantly condensed chromatin. Internally, the center of the oviduct is coated by a thick cuticle which has projections in the shape of spines toward the center (Fig 3a).

A few ovarioles had developed oocytes. A single mature oocyte was observed per ovariole (Fig 3d). Common oviduct, vagina and spermatheca were also observed. The large spermatheca is transparent when empty but has a pearly colour when the female is fertilized. As in all the Hymenopterans, the ovaries are meroistic - when there are nurse cells associated with the developing egg - and polytrophic - where each oocyte is closely associated with nurse cells in its follicle. At the apical portion of the germarium, the oogonies can be observed and they later divide giving rise to cysts, in addition to the oocyte, the nursing cells with a developed nucleus and little condensed chromatin (Fig 3b, c).

In the vitelarium zone it is possible to identify the follicles formed by the nursing and oocytic chambers, the first being delimited by follicular and flat cells while in the oocytic chamber, these cells are column-like.

Comparison between egg-laying of A.subterraneus subterraneus and A.ameliae and effect of parasite workers on the oviposition of host and parasite queens. Females of A. subterraneus subterraneus laid significantly more eggs than those of A. ameliae (F_1,41= 149.04, P < 0.001) (Fig 4). While queens of host subspecies alone laid approximately 230 eggs when isolated for 16h, those of parasite queens laid less than six eggs under the same conditions. The presence of queens and workers of the parasite did not reduce significantly the oviposition of the host queen, although a considerable reduction has been observed. Furthermore, the presence of workers of either parasite or host did not affect A. ameliae oviposition (Fig 4).

Of the 30 dealate females of the parasite which oviposited, 11 were fertilized, i.e., their spermathecae were full of spermatozoa; 10 females, although showing characteristic of oviposition in the ovaries, had empty spermathecae (Fig 5). It is possible that these females produced trophic eggs, or eggs which gave rise to males. The remaining females (n = 9) did not have developed ovaries.

Discussion

The production of sexuals by A. ameliae during a few months of the year, especially from October to February, led to colony decline and, in some situations, to colony death, which is common in other Attini (Bekkevold & Boomsma 2000, Bueno et al 2002). It is likely that the removal of males from the fungus chamber and from the foraging arena is a strategy of the host to decrease the destruction caused by the males due to their excessive feeding. A similar behavior was observed in A. insinuator when the food sources of host colonies were reduced (Bekkevold & Boomsma 2000).

Mated females were readopted by their mother colonies; this explains the high number of parasite queens in some nests and the difference of this number in the experiments. Similar data were observed in the social parasite A. insinuator where dealate gynes remained in the mother-colony after stopping their production and food sources slowly declined (Bekkevold & Boomsma 2000).

The behavioral change in mating (initially they had a mating flight and later mated inside the nest) could promote endogamy. Intra-nest mating is common in some species of social parasites of the genera Epimyrma, Teleutomyrmex and Anergates; except for Epimyrma, the others are not endogamic. Such species have morphophysiological limitations such as: reduced wings and low dispersal ability (Dumpert 1981, Buschinger 1989), characteristics that together with nest aggregation are very common in social parasites. In A. ameliae, possible mating flight continues to occur in the field and there is less queen readoption since these nests occur very close to each other.

A female-biased population-wide sex ratio, that is, large production of alate females when compared to the number of males, as observed in A. ameliae is a common aspect of some species of social parasites, dulotic (species depending on workers of others species) or inquilines. In Epimyrma kraussei Emery and Solenopsis daguerrei (Santschi) the number of reproductive queens reaches three times that of males (Buschinger 1989, Calcaterra et al 1999); greater female production is also found in P. xene (Aron et al 1999). The larger number of females may lead to a competition between brothers (Bourke & Franks 1995) and may occur due to polyandry since, according to Calcaterra et al (1999), sexual ratio based on females is a characteristic originated from polyandry. This aspect was reported in P. xene (Aron et al 1999) and it seems to occur in S. daguerrei (Calcaterra et al 1999). However, the social parasite A. insinuator mates with only one non-brother male after leaving the nest for a short period of time (Sumner et al 2004). In A. ameliae the male has seminal vesicles about four times the size of the female spermatheca; from a biological stand point, this enables the male to fill the spermatheca and to mate with more than one female (Soares et al in preparation).

Sexual forms of A. ameliae are smaller than those of the host A. subterraneus subterraneus. It is likely that size reduction brings some benefit to the species, maybe allowing the immatures to develop more rapidly and with less food than the host immatures. In P. xene, whose male and female pupae are significantly smaller than the pupae of the host P. pygmaea, size reduction is important especially in the males because host workers identify and destroy all male immatures of their own species (Aron et al 2004). In A. ameliae, the sexuals are significantly smaller than the largest workers of the host subspecies.

The large difference in egg production between A. subterraneus subterraneus and A. ameliae is directly related to the morphology of the reproductive tract of the species. Whereas females of A. subterraneus subterraneus have approximately 28 ovarioles in each ovary containing about 24 mature oocytes each (Antunes et al 2002), females of the parasite had smaller numbers of both ovarioles and oocytes. Differences between the number of ovarioles of host and parasite were also found by Hora et al (2001). They observed that the microgynes of E. tuberculatum have smaller number of ovarioles than the macrogynes; however, in this case, the number of oocytes was not different among females, suggesting that both micro and macrogynes have similar individual fecundity (Hora et al 2005).

It is likely that the small number of ovarioles and oocytes in A. ameliae is associated with its life style of parasitism in which it needs worker force from the host colony because its number of workers is very small (approximately 3%) and has only minors.

The negative impact of reproductive females and workers of the parasite species on the host queen is related solely with the production of sexuals; colonies parasitized by A. ameliae under laboratory conditions did not produce sexuals of the host species. The worker caste of A. ameliae is essential to production of its sexuals, as in A. insinuator, and it suppresses host queen reproduction. This means that parasite queens that do not produce workers or fail to reach a certain reproduction threshold will have zero fitness.

The minimum proportion of workers necessary to produce sexuals is smaller if there is more than one parasite queen present (Sumner et al 2003). Negative interference of the parasite species (P. xene) on egg and worker production by host queens was also observed by Passera et al (2001). The presence of only workers of the host A. subterraneus subterraneus did not affect oviposition as already reported by Marinho & Della Lucia (1998), and the presence of parasite workers also did not interfere on the number of eggs laid by A. ameliae (Fig 4).

It can be concluded that A. ameliae has sexuals significantly smaller than those of A. subterraneus subterraneus and produces a significantly smaller number of eggs than the host. The presence of queens and/or parasite workers did not negatively affect host queen egg laying but inhibited the production of sexuals by the host queen.

Acknowledgments

We thank Dr Ricardo Della Lucia (Universidade Federal de Viçosa) and Dr Rosa Muchovej (University of Florida) for insightful comments on this manuscript, and Dr Leandro Souto (Universidade Federal de Sergipe) for his valuable help. We are also grateful to CNPq and Pronex/FAPESB (nº 158/03) for funding this project.

Received 05/IV/09.

Accepted 16/IX/09.

Edited by Fernando L Cônsoli - ESALQ/USP

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Publication Dates

• Publication in this collection
  19 Nov 2010
• Date of issue
  Oct 2010

History

• Received
  05 Apr 2009
• Accepted
  16 Sept 2009