Printer Friendly

Testes and spermatozoa as characters for distinguishing two ant species of the genus Neoponera (Hymenoptera: Formicidae).

The correct diagnosis of a species is often challenging, especially in the case of cryptic species. The misidentification can invalidate results already published, contribute negatively to the knowledge of the organism in question, and waste financial resources associated with research and control programs. Therefore, it is important to find easily visible traits that allow the diagnosis of the species under study. The external morphologies of the ant species Neoponera inversa (Smith) and Neoponera villosa (F.) (Hymenoptera: Formicidae) are very similar, and as a result, they were previously considered as the single species N. villosa. However, studies comparing petiole morphology and isozyme patterns (Lucas et al. 2002), chromosome number (Mariano et al. 2007), and external morphologies (Fernandes et al. 2014) revealed that they form, in fact, a complex of sympatric cryptic species. These characteristics make the distinction difficult between these species. Thus, to identify them requires a specialist and, in general, methods that are time consuming and expensive.

Morphological characteristics of the male reproductive system have been used in the systematics of insects particularly in Hymenoptera (Wheeler & Krutzsch 1992; Dias et al. 2013). In addition, spermatozoa have been used for phylogenetic analyses to determine taxonomic variations in insects (Jamieson et al. 1999; Dallai 2014). In Hymenoptera, the sperm may be free in the seminal vesicle, as reported for parasitic wasps (Lino-Neto et al. 1999) and most Aculeata (Moreira et al. 2004), or arranged in bundles as described for ants such as Crematogaster victima Smith (Oliveira et al. 2014) and Lasius pallitarsis (Provancher) (Burnett & Heinze 2014) and for sawflies (Schiff et al. 2001).

Morphological characteristics of testes and sperm may be useful in the differentiation of these sympatric, cryptic species of ants. In this study, we describe the numbers of follicles per testis and the morphometries of the spermatozoa of N. inversa and N. villosa to establish additional characters by which they can easily be distinguished.

Five colonies of N. inversa and 5 colonies of N. villosa were collected in Ilheus, state of Bahia, Brazil, and reared for 3 mo within artificial nests in the laboratory. These nests were kept at 25 [+ or -] 4 [degrees]C with about 50 to 80% relative humidity. All colonies were fed on a mixture of honey, apple, and Tenebrio molitor L. (Coleoptera: Tenebrionidae) larvae ad libitum. Our studies used in total 50 males consisting of 5 males from each colony.

To quantify the number of follicles per testis, 15 males of N. inversa and 15 of N. villosa (3 specimens per colony) were dissected, and the reproductive system was photographed with a stereoscopic microscope (Zeiss, Stemi 2000-C). For the morphometric analysis of sperm, the seminal vesicles of 2 males from each colony (totaling 10 specimens per species) were transferred to histology slides, dissected in pH 7.2 phosphate buffered saline (PBS), fixed in 4% paraformaldehyde in 0.1 M phosphate buffer for 20 min, and washed in distilled water for 20 min. The spermatozoa were observed and photographed under a light microscope (Olympus, BX-60). To visualize and measure the nuclei, some slides were stained for 15 min with 4,6-diamidin-2-phenylindole (DAPI) at a concentration of 0.2 mg/mL in PBS and observed under an epifluorescence microscope (Olympus, BX-60) equipped with a BP 360-370 nm filter. For the measurements, 50 nuclei and 50 flagella of each species (10 cells per colony of both species) were randomly measured using the program Image-Pro Plus Version 4.5. The mean values were estimated and compared using the Mann-Whitney test at 5% significance.

Results showed that in the 2 species N. inversa and N. villosa, the reproductive system consisted of a pair of testes surrounded by a thin peritoneal sheath that can be broken easily (Fig. lA, B). Each testis opened into a long and thin vas deferens, forming the seminal vesicle, and both seminal vesicles and paired accessory glands terminated into a short ejaculatory duct (Fig. lA, B). When the peritoneal sheath was broken, 3 follicles per testis were observed in N. inversa (Fig. 1C), whereas 4 follicles per testis were observed in N. villosa (Fig. ID).

In the seminal vesicle, the spermatozoa were individualized, long, and threadlike. The total lengths of the sperm of N. inversa and N. villosa were 155 [+ or -] 10.1 [micro]m and 175 [+ or -] 5.4 [micro]m, respectively (Fig. 2A, B). Lengths of flagella did not differ between the 2 species (P = 0.3734) and averaged 125 [micro]m. However, the nucleus of N. inversa sperm was smaller than the nucleus of N. villosa (P < 0.001), with a length of 3O [+ or -] 1.6 [micro]m and 50 [+ or -] 2.3 [micro]m, respectively (Fig. 2C, E).

The different numbers of follicles per testis in N. inversa (3) and N. villosa (4) support the proposition that they are distinct species within the genus Neoponera (Lucas et al. 2002) and facilitate greatly the distinction between them. The numbers of testicular follicles vary in different groups within Hymenoptera. Three follicles per testis have been observed in Vespidae (Brito et al. 2005) and Andrenidae, H al ictidae, and some species of Megachilidae (Ferreira et al. 2004). Four follicles per testis have been observed in Mellitidae, other species of Megachilidae, and Apidae sensu stricto (Roig-Alsina & Michener 1993; Ferreira et al. 2004). Interestingly, the number of testicular follicles can be up to 250 in Apis mellifera L. (Hymenoptera: Apidae) (Snodgrass 1984), whereas in ants, the number varies from 1 to 25 among different species (Wheeler & Krutzsch 1992).

In N. inversa and N. villosa, the spermatozoa were long, threadlike, linear, and free from each other in the seminal vesicle as also observed in the ant Acromyrmex subterraneus (Forel) (Hymenoptera: Formicidae) (Moreira et al. 2004). However, in C. victima (Oliveira et al. 2014) and L. pallitarsis (Burnett & Heinze 2014), these cells are maintained in bundles in the seminal vesicle in the mature male as reported for sawfly wasps (Schiff et al. 2001).

In Hymenoptera, the difference in lengths of the sperm can be used to differentiate cryptic species (Pereira et al. 2008). Total lengths of sperm are variable among species, for example, 13 to 577 [micro]m in Vespidae (Quicke et al. 1992) and 80 to 1,500 [micro]m in Apoidea (Quicke et al. 1992; Zama et al. 2004). In Formicidae, the mean length of the sperm in Solenopsis invicta Buren is about 70 [micro]m (Lino-Neto & Dolder 2002), whereas in Pseudomyrmex, it ranges from 53 to 70 [micro]m (Moya et al. 2007). Thus, our results showed that sperm lengths in N. inversa (155 [micro]m) and N. villosa (175 [micro]m) are the longest recorded to date in ants. Furthermore, we showed that the difference in the size of the sperm between N. inversa and N. villosa is due to a difference in the lengths of nuclei, with the nucleus being 40% longer in N. villosa. This result is consistent with a cytogenetic study on differences in chromosome number between these 2 species, with N. inversa showing fewer chromosomes (2n = 30) than N. villosa (2n = 34) (Mariano et al. 2007). Considering the importance of these reproductive cells, and that the amount of DNA in the same species is generally constant, the difference in nuclear size of the sperm of these 2 ant taxa supports that they are different species.

Although all colonies have been collected in the same region (Ilheus, Bahia State, Brazil), the way the data were sampled and their uniformity, as well as the sample size, provide strong evidence that the number of follicles per testis and the nuclear length of sperm are species specific. Therefore, the characters cited above can be used to distinguish easily these 2 cryptic species of ants.

We are grateful to the Brazilian research agencies FAPEMIG, CNPq, PRONEX SECTI-FAPESB/CNPq - PNX 0011/2009, to Jose Adade, Jose Raimundo Maia, and Jose Crispin (Laboratory of Myrmecology at the Cocoa Research Center, Ilheus, Bahia, Brazil) and to the 2 reviewers who greatly improved the manuscript with their suggestions.

References Cited

Brito P, Moreira J, Lino-Neto J. 2005. Morphology of male reproductive system and sperm of Mischocyttorus sp. (Hymenoptera: Vespidae: Polistinae). Brazilian Journal of Morphological Sciences 22: 175-176.

Burnett WE, Heinze J. 2014. Sperm bundles in the seminal vesicles of sexually mature Lasius ant males. PLoS One 9: e93383.

Dallai R. 2014. Overview on spermatogenesis and sperm structure of Hexapoda. Arthropod Structure and Development 43: 257-290.

Dias G, Oliveira CM, Lino-Neto J. 2013. Testicular and spermatogenic characteristics of Lagria villosa (Tenebrionidae: Lagriinae) with taxonomic inferences. Tissue and Cell 45: 227-230.

Fernandes IO, Oliveira ML, Delabie JHC. 2014. Description of two new species in the Neotropical Pochycondylo foetido complex (Hymenoptera: Formicidae: Ponerinae) and taxonomic notes on the genus. Myrmecological News 19: 133-163.

Ferreira A, Abdalla FC, Kerr WE, Cruz-Landim C. 2004. Comparative anatomy of the male reproductive internal organs of 51 species of bees. Neotropical Entomology 33: 569-576.

Jamieson BGM, Dallai R, Afzelius BA. 1999. Insects: Their Spermatozoa and Phylogeny, 1st edition. Science Publishers Inc., Enfield, New Hampshire, USA.

Lino-Neto J, Dolder H. 2002. Sperm structure and ultrastructure of the fire ant Solenopsis invicta (Buren) (Hymenoptera, Formicidae). Tissue and Cell 34: 124-128.

Lino-Neto J, Bao SN, Dolder H. 1999. Structure and ultrastructure of the spermatozoa of Bephratelloidespomorum (Fabricius) (Hymenoptera: Eurytomidae). International Journal of Insect Morphology and Embryology 28: 253-259.

Lucas C, Fresneau D, Kolmer K, Heinze J, Delabie JHC, Pho DB. 2002. A multidisciplinary approach to discriminating different taxa in the species complex Pachycondyla villosa (Formicidae). Biological Journal of the Linnean Society 75: 249-259.

Mariano CSF, Delabie JHC, Santos JRM, Pompolo SG. 2007. Evolucao cariotipica em Pachycondyla spp. (Ponerinae) neotropicais. Biologico 69: 409-412.

Moreira J, Zama U, Lino-Neto J. 2004. Release, behavior and phylogenetic significance of spermatozoa in bundles in the seminal vesicle during sexual maturation in Aculeata (Hymenoptera). Brazilian Journal of Morphological Sciences 21: 185-189.

Moya J, Mancini K, Lino-Neto J, Delabie J, Dolder H. 2007. Sperm ultrastructure of five species of the Neotropical ant genus Pseudomyrmex (Hymenoptera: Formicidae). Acta Zoologica 88: 181-187.

Oliveira CM, Moreira J, Gomes LF, Camargo-Mathias MI, Lino-Neto J. 2014. Sperm bundles in the seminal vesicle of the Cremotogoster victimo (Smith) adult males (Hymenoptera: Formicidae). Neotropical Entomology 43: 201-208.

Pereira RAS, Lino-Neto J, Prado AP. 2008. Sperm morphology: a novel way to associate female-males of highly sexual dimorphic fig wasp species. Symbiosis 45: 143-147.

Quicke DLJ, Ingram SN, Baillie HS, Gaitens PV 1992. Sperm structure and ultrastructure in the Hymenoptera (Insecta). Zoologica Scripta 21: 381-402.

Roig-Alsina A, Michener CD. 1993. Studies of the phylogeny and classification of long-tong bees (Hymenoptera: Apoidea). University of Kansas Science Bulletin 55: 123-162.

Schiff N, Flemminc AJ, Quicke DLJ. 2001. Spermatodesmata of the sawflies (Hymenoptera: Symphyta): evidence for multiple increases in sperm bundle size. Journal of Hymenoptera Research 10: 119-125.

Snodgrass RE. 1984. Anatomy of the Honey Bee, 1st edition. Cornell University Press, Ithaca, New York, USA.

Wheeler DE, Krutzsch PH. 1992. Internal reproductive system in adult males of the genus Camponotus (Hymenoptera: Formicidae: Formicinae). Journal of Morphology 211: 307-317.

Zama U, Lino-Neto J, Dolder H. 2004. Structure and ultrastructure of spermatozoa in Meliponini (stingless bees) (Hymenoptera: Apidae). Tissue and Cell 36: 29-41.

Marcelo S. Barcellos (1), Luiza C. B. Martins (2), Jamile F. S. Cossolin (1), Jose Eduardo Serrao (1), Jacques H. C. Delabie (3), and Jose Lino-Neto (1)*

(1) Departamento de Biologia Geral, Universidade Federal de Vicosa, 36570-900, Vicosa, Minas Gerais, Brazil

(2) Departamento de Quimica e Biologia, Universidade Estadual do Maranhao--UEMA, 65604-375, Caxias, Maranhao, Brazil

(3) Laboratorio de Mirmecologia, Convenio CEPLAC/UESC, CEPEC - 45600-000, Itabuna, Bahia, Brazil

* Corresponding author; E-mail:

Caption: Fig. 1. Male reproductive system of (A) Neoponera inversa and (B) Neoponera villosa, showing the testes (t) united by peritoneal sheath. Seminal vesicle (sv); vas deferens (vd); accessory glands (ag). Testes of (C) N. inversa and (D) N. villosa without peritoneal sheath, showing 3 and 4 follicles (arrows), respectively.

Caption: Fig. 2. Sperm of (A) Neoponera inversa and (B) Neoponera villosa in phase contrast, showing the transition (arrows) between the nucleus (n) and the flagellum (f). Nuclei stained with DARI of (C and D) N. inversa and (E and F) N. villosa sperm.


Please note: Some tables or figures were omitted from this article.
COPYRIGHT 2015 Florida Entomological Society
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2015 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Title Annotation:Scientific Notes
Author:Barcellos, Marcelo S.; Martins, Luiza C.B.; Cossolin, Jamile F.S.; Serrao, Jose Eduardo; Delabie, Ja
Publication:Florida Entomologist
Article Type:Report
Geographic Code:3BRAZ
Date:Dec 1, 2015
Previous Article:Seasonal abundance of Thysanoptera species in Tillandsia usneoides (Poales: Bromeliaceae).
Next Article:Morphological and genetic variation in Mexican wild populations of Tamarixia radiata (Hymenoptera: Eulophidae).

Terms of use | Privacy policy | Copyright © 2020 Farlex, Inc. | Feedback | For webmasters