Morphology of ovaries and spermatheca of Atta sexdens (Hymenoptera: Formicidae) queens: implications for oviposition rates

Morfologia dos ovários e da espermateca de rainhas de Atta sexdens (Hymenoptera: Formicidae): implicações para as taxas de oviposição

Abstract

The nest of leaf-cutting ants is founded by a newly mated queen, which lays the first eggs, giving rise to workers responsible for nest construction and colony maintenance. Bioecological and physiological aspects of queens may help explain variations in the size of newly founded nests of species in the genus Atta. This study aimed to evaluate the relationship between ovary and spermatheca morphology and the oviposition rate of Atta sexdens Linnaeus (Hymenoptera: Formicidae) queens. Forty-one newly founded nests of A. sexdens were collected, and the volume of each fungus garden was measured. The queens from these nests were isolated and fed daily with the colony’s symbiotic fungus to determine their daily egg production over four days. At the end of this period, the ovaries and spermathecae of the queens were dissected and analyzed under light microscopy. The oviposition rate of A. sexdens queens ranged from 0.5 to 90.5 eggs per day. Ovaries showing signs of degeneration, including yolk reabsorption in oocytes, were observed in queens with the lowest oviposition rates. Spermatheca morphology was similar among all queens. The relationship between ovarian morphology and oviposition rate in young A. sexdens queens supports the hypothesis that differences in ovarian condition contribute to variations in egg production. Such differences may ultimately influence colony growth in the field.

Keywords:
Atta sexdens; spermatheca; leaf-cutting ant; ovary

Resumo

O ninho de formigas cortadeiras é fundado por uma rainha recém-acasalada, que deposita os primeiros ovos, dando origem às operárias responsáveis pela construção do ninho e manutenção da colônia. Aspectos bioecológicos e fisiológicos das rainhas podem ajudar a explicar variações no tamanho de ninhos recém-fundados de espécies do gênero Atta. Este estudo teve como objetivo avaliar a relação entre a morfologia dos ovários e da espermateca e a taxa de oviposição de rainhas de Atta sexdens Linnaeus (Hymenoptera: Formicidae). Foram coletados 41 ninhos recém-fundados de A. sexdens, sendo medido o volume de cada jardim de fungo. As rainhas desses ninhos foram isoladas e alimentadas diariamente com o fungo simbionte da colônia para determinar sua produção diária de ovos durante quatro dias. Ao final desse período, os ovários e as espermatecas das rainhas foram dissecados e analisados em microscopia de luz. A taxa de oviposição das rainhas de A. sexdens variou de 0,5 a 90,5 ovos por dia. Ovários apresentando sinais de degeneração, incluindo reabsorção de vitelo nos oócitos, foram observados em rainhas com as menores taxas de oviposição. A morfologia da espermateca foi semelhante entre todas as rainhas. A relação entre a morfologia ovariana e a taxa de oviposição em rainhas jovens de A. sexdens sustenta a hipótese de que diferenças na condição ovariana contribuem para variações na produção de ovos. Tais diferenças podem, em última instância, influenciar o crescimento da colônia em campo.

Palavras-chave:
Atta sexdens; espermateca; formiga cortadeira; ovário

1. Introduction

Leaf-cutting ants of the genus Atta use fresh plant material to cultivate and maintain their symbiotic fungus (Sousa et al., 2022). These ants cultivate hyphal swellings (gongylidia), cultivated with freshly cut plant material, is the main food source for their larvae and adults (Swanson et al., 2019;Santos and Sousa-Souto, 2021;Lutinski et al., 2021;Oliveira et al., 2024). During the nuptial flight, a newly mated female (queen) descends to the ground, detaches the wings, and digging for several hours a vertical tunnel 8.5 to 15 cm deep and 9 to 12 mm in diameter, as well as a small chamber measuring 2.2 to 2.5 cm in height and 3 to 4.5 mm in diameter, thereby initiating the leaf-cutting ant nest (Camargo et al., 2011). The queen then seals the tunnel entrance with soil removed during excavation, encloses itself, and deposits a small portion of the symbiotic fungus that was transported in its infrabuccal cavity. The depth of the initial chamber is crucial for colony success (Camargo et al., 2011; Fröhle and Roces, 2012). After the first workers emerge, the queen remains as the reproductive individual, whereas the workers perform excavation of new tunnels and chambers (Camargo et al., 2011; Sousa et al., 2022).

The queen’s egg-laying rate and the production of viable eggs during the early stages of colony establishment vary among Atta species (Araujo and Della Lucia, 1993; Moreira et al., 2019), likely reflecting differences in the morphology of the queens’ reproductive system. The reproductive system of leaf-cutting ant queens consists of a pair of meroistic polytrophic ovaries, typically with ca. 200 ovarioles, two lateral oviducts that converge into a common oviduct, and a genital chamber into which the spermatheca, containing stored spermatozoa, opens (Cardoso et al., 2008).

The number of ovarioles, which is associated with the oviposition rate, varies according to the species, with 28 per ovary in Acromyrmex subterraneus subterraneus Forel (Hymenoptera: Formicidae), 12 in A. rugosus Smith (Hymenoptera: Formicidae) (Farder-Gomes et al., 2019), 13 to 15 in A. ameliae De Souza, Soares & Della Lucia (Hymenoptera: Formicidae) (Soares et al., 2010), five to six in A. octospinosus Reich (Hymenoptera: Formicidae) and A. echinatior Forel (Hymenoptera: Formicidae) (Dijkstra et al., 2005), two to four in A. subterraneus brunneus Forel (Camargo et al., 2007), and more than 300 in the representatives of the genus Atta (Tschinkel, 1987; Farder-Gomes et al., 2019).

The lone queen of Atta sexdens, after nest foundation, regurgitates the symbiotic fungus stored in its infrabuccal cavity and lays the first eggs after five days (Camargo et al., 2011). The relative duration of egg hatching, larvae and pupae stages are 25, 22, and 10 days, respectively (Camargo et al., 2011). Queen activities also include fungus care (licking and depositing fecal fluid), self-feeding (oophagy), oviposition and brood care (Camargo et al., 2011).

This study tested the hypothesis that the oviposition rate of A. sexdens queens in newly founded nests is associated with the morphology of their ovaries and spermatheca.

2. Material and Methods

Forty-one newly founded nests of A. sexdens were excavated at the Lajeado Experimental Farm, Faculty of Agricultural Sciences (FCA/UNESP) (22° 50' 38.724" S 48° 25' 38.395"), in Botucatu, São Paulo state, Brazil. The queens and the contents of each nest were placed in 250 mL plastic containers, each containing a thin layer of plaster at the bottom to reduce moisture loss and transferred to the Social Insect Pest Laboratory.

Each queen, along with 50 workers and 10 larvae, was fed daily with the symbiotic fungus to record the number of eggs laid per queen over a four-day period at LISP. The mass of the fungus provided, and the initial body mass of each queen were weighed with an analytical balance.

2.1. Light microscopy

Following the oviposition period, the A. sexdens queens were immobilized at –4 °C for two minutes and dissected in 125 mM NaCl using fine-tipped forceps and microsurgical scissors. The spermathecae and ovaries were transferred to 4% paraformaldehyde for 24 h, dehydrated in a graded ethanol series (70 80, 90, and 95%) for 15 min at each concentration, and embedded in Leica resin following manufacturer´s instructions. Serial sections (4 µm) were obtained using a Leica RM 2145 rotary microtome, stained with Harris’s hematoxylin for 10 min and eosin for 5 min, mounted in Canada balsam, and examined under a light microscope in the Histology Laboratory at the Institute of Biosciences, UNESP in Rio Claro, São Paulo, Brazil.

2.2. Statistical analysis

Data on the number of eggs (over four days), fungus mass, and the initial body mass of each queen (g) were analyzed using correlation and simple linear regression. Results were expressed as correlation coefficient (r), F-value, P-value, and coefficient of determination (R²). All statistical analyses were performed using AgroEstat software, version 1.1.0712 (2010).

3. Results

3.1. Reproductive parameters

The oviposition rate of A. sexdens queens ranged from 0.5 to 90.5 eggs per day, while the fungus mass per nest varied between 0.2 and 29.2 g (Table 1).

Table 1
Total number of eggs (NE) per queen after four days, fungus mass (FM), and initial queen mass (QM) of Atta sexdens (Hymenoptera: Formicidae) evaluated in 41 newly founded nests.

Nest	NE	FM (g)	QM (g)
1	90.5	29.2	0.32
2	88.0	18.8	0.34
3	27.2	9.8	0.23
4	21.0	10.6	0.27
5	52.2	24.6	0.28
6	20.7	8.2	0.21
8	8.2	0.6	0.32
9	37.2	15.6	0.34
10	44.0	11.2	0.23
11	25.0	2.5	0.27
12	47.7	23.0	0.28
13	19.0	6.0	0.21
14	14.2	5.1	0.32
15	38.7	7.0	0.34
16	19.2	2.9	0.23
17	59.5	0.0	0.0
18	27.5	0.0	0.0
19	51.0	2.0	0.24
20	19.2	5.6	0.22
21	4.2	0.0	0.0
22	18.0	6.0	0.22
25	21.0	7.9	0.27
26	4.5	3.5	0.20
27	13.0	2.0	0.24
30	25.0	4.8	0.27
31	10.5	5.2	0.25
32	0.5	3.9	0.31
33	8.25	0.8	0.24
38	51.0	20.1	0.32
39	30.2	3.4	0.31
41	25.5	3.2	0.32
Mean	29.7	7.9	0.3
Standard error	4.00	1.41	0.02

A positive correlation was observed between the oviposition rate per queen and the fungus garden mass r = 0.792 (p < 0.0001, 79.22%) (Table 2). The coefficient of determination between the average number of eggs and fungus mass was 0.71, whereas the relationship between the average number of eggs and the initial body queen mass was weaker (R²= 0.14) (Table 2).

Table 2
Linear regression (R²) and linear correlation coefficient (r) between the number of eggs and the mass (g) of the fungus and the initial queen mass of Atta sexdens (Hymenoptera: Formicidae) after four days.

Variables	F	P	R2	r
Eggs vs. Fungus Mass	63.30**	< 0.0001	0.71	0.0001 (0.79**)
Eggs vs. Queen Mass	4.19ns	0.15	0.14	0.22 (0.42*)

• ^ns
  correlation not significant at 5%.
• ^*,**
  significant at 5% probability (Agroestat, 2010).

3.2. Morphology

The ovarioles of A. sexdens queens with a high oviposition rate (approximately 90 eggs/day) were well developed with follicles showing the terminal filament, oocytes and nurse cells (Figures 1A, 1B). Germ cell clusters were present in the germarium, and oocytes at different stages of maturation exhibited a regular rounded shape in cross section (Figures 1A, 1C). The young oocytes present homogeneous content and evident germinal vesicles (Figure 1C). The nurse cells are well-developed with round-shaped nucleus rich in decondensed chromatin (Figure 1C). Following oocyte growth there ae some yolk granules (Figure 1D) and the chorion deposition (Figure 1E).

Figure 1
Light micrographs of the ovaries of Atta sexdens queen (Hymenoptera: Formicidae) with an average oviposition rate of 90 eggs per day. A) General aspect showing long ovarioles (ovl) with follicles (fo). B) Detail of the follicles (fol) with the terminal filament (tf). C) Follicles with well-developed nurse cells (nc) with nucleus (n) rich in decondensed chromatin and developing oocytes (oo) with germinal vesicles (gv). D) Oocyte (oo) with some yolk granules (yo) and the follicular cells (fc). E) Detail of a more developed oocyte with yolk granules (yo) and chorion (co) deposition.

In queens laying an average of 47 eggs/day, oocytes presented irregular shapes from the early to advanced stages of maturation (Figure 2A). The oocyte content was disorganized, with a spongy or fragmented aspect with some large vacuolize areas (Figures 2B, 1C). Oocytes in advanced developmental stage presented irregular chorion deposition (Figure 2D). The nurse cells had well developed nucleus and nucleolus and the cytoplasm rich in vacuoles (Figure 2E).

Figure 2
Light micrographs of ovaries of Atta sexdens queen (Hymenoptera: Formicidae) with an average oviposition rate of 47 eggs per day. A) General aspect of the follicle (fol) showing nurse cells (nc) and oocytes (oo) with some disorganized content. B) Oocytes (oo) with some vacuolized (va) content. C) Oocyte (oo) with irregular chorion (co), vacuolized (va) and disorganized (circle) content. D) Oocytes (oo) with different yolk granules (yo). E) Nurse cells (nc) with well-developed nucleus (n) and nucleolus (nu) and vacuolized (va) cytoplasm. fc – follicular cells.

Nurse cells with cytoplasm rich in vacuoles and oocytes undergoing resorption were more frequent observed in queens with an oviposition rate averaging 27 eggs/day, characterized by disorganized ooplasm (Figure 3A) with many vacuoles and irregular chorion (Figure 3B).

Figure 3
Light micrographs of the ovary of an Atta sexdens queen (Hymenoptera: Formicidae) with an average oviposition rate of 27 eggs per day. A) Oocyte (oo) with extensive vacuolated content (va) and irregular chorion (co). B) Detail of the oocyte with vacuoles (va) and irregular chorion (co).

In queens with an average oviposition rate of 16 eggs/day, the ovaries presented disorganized ovarian follicles (Figure 4A). Follicles showed severe structural alterations, including nurse cells (Figure 4A). Most oocytes were in resorption, with extensive cytoplasmic vacuolization, fused yolk granules with liquefied aspect and irregular chorion (Figures 4A, 4B).

Figure 4
Light micrographs of the ovaries of an Atta sexdens queen (Hymenoptera: Formicidae) with an average oviposition rate of 16 eggs per day. A) General view showing disorganization of the ovarian follicle (fol) and oocyte (oo) with irregular chorion (co). B) Detail of the oocyte (oo) with disorganized content with vacuoles (va) and yolk granules (yo). tf – terminal filament.

Queens laying four eggs per day exhibited the most pronounced ovarian alterations. Oocytes at early stages of development were irregular in shape with structural disorganization characterized by extensive content vacuolization (Figure 5A). The nurse cells apparently undergoing death presented cytoplasm rich in vacuoles (Figure 5B). Some mature oocytes presented disorganized ooplasm and irregular chorion (Figure 5C).

Figure 5
Light micrographs of the ovary of an Atta sexdens queen (Hymenoptera: Formicidae) with an average oviposition rate of four eggs per day. A) General aspect showing disorganized follicle (fol) and oocyte (oo) with vacuolized (va) content. B) Nurse cells (nc) with cytoplasm rich in vacuoles (va). C) Derail of the oocyte showing irregular chorion (co), fused and disorganized yolk granules (yo). n – nucleus.

The spermatheca were similar in all queens of A. sexdens evaluated, presenting a sac-shaped, with multiple muscle layers, and the reservoir lumen was lined by a single-layered flattened epithelium (Figures 6A, 6B) with lumen storing spermatozoa (Figure 6B).

Figure 6
Light micrographs of the spermatheca of Atta sexdens queens (Hymenoptera: Formicidae). A) Spermathecal reservoir showing the lumen (lu), flattened epithelium (ep) and muscle layers (mu). B) Detail of the spermathecal reservoir showing spermatozoa (sz) in the lumen (lu).

4. Discussion

The oviposition rate of A. sexdens queens (0.5–90.5 eggs/day) and the fungus mass per nest (0.2–29.2 g) were, respectively, higher and lower than those reported for A. laevigata queens in colonies with fungus volumes of approximately 100 mL, which produced four to 11.86 eggs and maintained a fungus volume of 3200 mL (Araujo and Della Lucia, 1993).

The positive correlation between oviposition rate and fungus garden mass in A. sexdens is similar to reported for A. laevigata (Araujo and Della Lucia, 1993). This association likely reflects a functional relationship, in which increased egg production results in larger number of larvae, with high nutritional demand and necessitates the development of a more robust fungus garden. Field observations support this interpretation, as young A. sexdens nests with more productive queens consistently maintained larger fungus gardens (Moreira et al., 2019), reinforcing the direct link between reproductive output and symbiotic resource availability.

Well-developed ovarioles with preserved ovarian follicles in queens producing around 90 eggs/day indicate a high reproductive rate, consistent with the influence of environmental and genetic factors on ovarian activity and egg production in social insects (Bergland et al., 2008; Jervis et al., 2008; Green II and Extavour, 2012; Farder-Gomes et al., 2019).

In queens producing an average of 47 eggs/day, oocytes with irregular shapes and disorganized yolk were observed from early developmental stages. These alterations are likely the result of oocyte resorption, a strategy by which developing oocytes are degenerated under unfavorable conditions to conserve resources (Bell and Bohm, 1975; Souza et al., 2007; Van Oystaeyen et al., 2014).

Queens laying approximately 27 eggs/day exhibited nurse cells with cytoplasm rich in vacuoles, indicating structural degeneration and cell death, as reported in Acromyrmex rugosus, A. sexdens rubropilosa, and Diacamma sp., in which cytoplasmic material is transferred from nurse cells to the oocyte during oogenesis (Amaral and Machado-Santelli, 2009; Okada et al., 2010; Farder-Gomes et al., 2019). In these queens, oocytes undergoing resorption were more abundant than in those laying 47 eggs/day, suggesting that physiological and environmental factors, such as poor nutritional status, seasonal variations (temperature, humidity, photoperiod), and absence of reproductive partners, may trigger oocyte degeneration (Bell and Bohm, 1975; Kotaki, 2003; Souza et al., 2007; Kajita and Evans, 2009). Reduced egg production under these conditions likely compromises ovarian activity and activates resource-conserving mechanisms, with oocyte resorption acting as a key adaptive strategy under adverse reproductive scenarios.

High ovarian degeneration was observed in queens producing around 16 eggs/day, with follicles showing structural disorganization and complete loss of nurse chamber integrity. Similar findings have been reported in A. sexdens rubropilosa* (Amaral and Machado-Santelli, 2009), where nursing chamber disruption reduces the cytoplasmic material supplied to the oocyte, compromising its development (Mazurkiewicz and Kubrakiewicz, 2001).

The most severe ovarian deterioration was detected in queens laying four eggs/day. These queens exhibited widespread follicular disarray, loss of structural organization, and nurse chambers with cells apparently undergoing cell death even in early-stage oocytes. Similar findings were reported in A. sexdens rubropilosa (Amaral and Machado-Santelli, 2009). Such degenerative processes likely indicate a terminal stage of reproductive decline, where oogenesis is severely impaired.

Finally, the spermatheca of A. sexdens queens was found to be sac-shaped, composed of several muscular layers, and lined by simple squamous epithelium, a structural pattern commonly reported for insects (Cardoso et al., 2008; Amaral and Machado-Santelli, 2009).

5. Conclusion

Our findings support the hypothesis that variations in oviposition rate are associated with the morpho-histological condition of the ovaries of A. sexdens queens.

Data Availability Statement

The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.

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Edited by

• Editor:
  Takako Matsumura Tundisi

Publication Dates

• Publication in this collection
  29 May 2026
• Date of issue
  2026

History

• Received
  08 Nov 2025
• Accepted
  25 Mar 2026