Current Microbiology https://doi.org/10.1007/s00284-018-1459-3 Transovarian Transmission of Blochmannia and Wolbachia Endosymbionts in the Neotropical Weaver Ant Camponotus textor (Hymenoptera, Formicidae) Manuela Oliveira Ramalho1,2 · Alexsandro Santana Vieira1 · Mayara Cristina Pereira1 · Corrie Saux Moreau2 · Odair Correa Bueno1 Received: 7 December 2017 / Accepted: 19 February 2018 © Springer Science+Business Media, LLC, part of Springer Nature 2018 Abstract Camponotus is a hyper-diverse ant genus that is associated with the obligate endosymbiont Blochmannia, and often also with Wolbachia, but morphological studies on the location of these bacteria in the queen’s ovaries during oogenesis remain limited. In the present study, we used the Neotropical weaver ant Camponotus textor to characterize the ovary using histol- ogy (HE) techniques, and to document the location of Blochmannia and Wolbachia during oogenesis through fluorescence in situ hybridization (FISH). This is the first morphological report of these two bacteria in the same host with polytrophic meroistic ovaries and reveals that Blochmannia is found inside late-stage oocytes and Wolbachia is associated with the nuclei of the nurse cells. Our results provide insights into the developmental sequence of when these bacteria reach the egg, with Blochmannia establishing itself in the egg first, and Wolbachia only reaching the egg shortly before completing egg development. Studies such as this provide understanding about the mechanisms and timing of the establishment of these endosymbionts in the host. Introduction The ant genus Camponotus Mayr, 1861 is one of the most diverse and has a worldwide distribution [2, 6]. They have Electronic supplementary material The online version of this generalist diets and can nest in cavities in trees, in hollow article (https ://doi.org/10.1007/s0028 4-018-1459-3) contains or rotten twigs, or in the ground [13, 23], and some species supplementary material, which is available to authorized users. construct nests in trees with silk produced by larvae, as in the case of Camponotus textor Forel, 1899 [27, 31]. This * Manuela Oliveira Ramalho [email protected] group of ants is also known to have obligatory symbiotic relationships with bacteria [10, 11, 16, 39] and studies have Alexsandro Santana Vieira Blochmannia [email protected] shown that the main associated taxa are and Wolbachia, representing about 95–98% of all sequencing Mayara Cristina Pereira Camponotus chromaiodes [email protected] reads of Bolton, 1995 [7]. These symbiotic bacteria may have positive or negative Corrie Saux Moreau Blochmannia [email protected] effects on the host. , for example, is known for its beneficial effects because it provides a number of amino Odair Correa Bueno [email protected] acids to the host, thus it has a nutritional role, especially in the early developmental stages of host life [10, 11, 16, 1 Departament of Biology e Center for Studies on Social 41]. For many arthropod hosts, Wolbachia is known for its Insects, Biosciense Institute, São Paulo State University negative effect in manipulating host reproduction, such as (UNESP), Campus Rio Claro, Avenida 24A, 1515, Bela Vista, Rio Claro, SP 13506-900, Brazil parthenogenesis, death of males, feminization, and cyto- plasmic incompatibility (CI) [4, 12, 28, 35]. For bedbugs, it 2 Field Museum of Natural History, Department of Science and Education, Integrative Research Center, 1400 South Lake may aid in nutrition with vitamin B supplementation [18]. Shore Drive, Chicago, IL 60605, USA However, its function in ants is not known, especially in the Vol.:(0123456789)1 3 M. O. Ramalho et al. sterile workers, who are not able to reproduce [1, 29, 30]. the nucleus of the nurse cells, and it is believed that this Both bacteria can be transmitted vertically (maternal inherit- bacterium likely reaches the egg through the cytoplasmic ance), with Blochmannia acting as a primary and obligatory bridges that exist between the nurse chamber and the egg endosymbiont and Wolbachia as secondary and facultative chamber [3, 42]. But this bacterium has not been localized in [1, 11, 39]. insects that exhibit polytrophic meroistic ovaries. For Bloch- A previous study by Ramalho et al. [24] using next-gen- mannia, in Camponotus floridanus, it is already known that eration sequencing techniques surveyed the bacterial com- it is present in the young and mature oocytes, via follicular munity present across all stages of development of multiple cells in polytrophic meroistic ovaries [22]. This leads to the Camponotus colonies. The main bacteria found in the egg question of whether other species of Camponotus have the and queen were Blochmannia and Wolbachia, reinforcing same pattern of distribution for Blochmannia and what is the idea that the route of acquisition of these endosymbionts the location of Wolbachia within polytrophic meroistic ovar- occurs through maternal inheritance. Thus, morphological ian tissues during dual infections. In this study, we used studies of the reproductive organs of Camponotus ant queens fluorescence in situ hybridization (FISH) to document the may inform the strategies of the establishment of these endo- distributions of Blochmannia and Wolbachia symbionts in symbiotic bacteria in the host. detail during the oogenesis of the queen of Camponotus tex- The acquisition of endosymbionts in oviparous insects tor and provide a possible developmental mechanism of how can occur either at the beginning or in the late stages of these bacteria reach the egg. oogenesis, although there are few studies in Hymenoptera (bees, wasps, and ants) that exhibit polytrophic meroistic ovaries [22]. Insects with polytrophic meroistic ovaries have Materials and Methods nurse cells (grouped inside the nurse chamber) and oocytes that alternate along the length of ovariole. This set of nurse The silk nest of Camponotus textor containing adult, imma- chambers (containing the nurse cells) plus the egg chamber ture individuals, breeding and several queens (polygeny), (containing the oocytes) is called the ovarian follicle. To was collected in August 2016 in Araraquara São Paulo, investigate transmission of Wolbachia, several studies have Brazil (Lat. -21.8262, Long. -48.2001). Eight workers were been carried out in Drosophila and tsetse flies, which have collected and stored in 95% ethanol for screening of Wol- meroistic telotrophic ovaries, in which the nurse chamber bachia and Blochmannia infections. Total DNA extraction is located basally and the apical region is where the germa- was performed following the same parameters described by rium (containing the stem cells) is found [3, 9, 14]. Frydman Ramalho et al. [26]. For the confirmation of the presence of et al. [14] were able to experimentally add Wolbachia to these bacteria in the colony, the primers Bloch 16S-462F the Drosophila melanogaster abdomen and to monitor their and Bloch 16S-1299R [39] and Wsp81f and Wsp691r [43], tissue distribution. They found that only 15 days after the for Blochmannia and Wolbachia, respectively, were used to infection, Wolbachia was detected in the germ line and the amplify this target region through PCR, following the same transmission route was through the somatic stem cell in the parameters of Ramalho et al. [25]. Three C. textor queens germarium. from the same colony were dissected in 1X PBS (Fig. 1a). Blochmannia has been detected using fluorescence Some ovarioles were submitted to the histological technique in situ hybridization (FISH) during oogenesis of Campono- Hematoxylin and Eosin (HE) and to the total assembly tech- tus floridanus, a species commonly found in the Nearctic nique for FISH. region [22]. These authors found that the bacterium was not present in the germarium, nor in the nurse cells, but they Morphology—Hematoxylin and Eosin were located within the oocyte and believed to have been transferred via follicular cells [22]. As both bacteria, Bloch- For this technique, the protocol described by Junqueira and mannia and Wolbachia, were found in large numbers infect- Junqueira [19] was followed. The extracted ovarioles were ing workers of Camponotus textor, an exclusively Neotropi- fixed in 4% paraformaldehyde (w/v) for 24 h (h), and then cal species [25], this presents the opportunity to investigate transferred to buffer solution (Sodium Phosphate pH 7.4) for the localization of these bacteria in the reproductive tract of 24 h. Subsequently, they were dehydrated in an increasing this species. As the genus Camponotus is very species rich, series of alcohols (50–95%) over fifteen-minute intervals. it is unclear if this observed pattern is conserved across the At the end of the dehydration process, the material was group. transferred to the embedding historesin and held for 5 days. There are several studies addressing the specific location Subsequently, the organs were included in plastic molds of these bacteria in host tissues [1, 11, 14, 22, 34, 44] and a containing historesin (Leica Historesin) and polymerizer few studies have included insects with telotrophic meroistic 3–6 µm thick. The blocks were sectioned on a LEICA RM ovaries. These studies have found Wolbachia associated with 2255 microtome. The histological sections were placed on 1 3 Transovarian Transmission of Blochmannia and Wolbachia Endosymbionts in the Neotropical… Fig. 1 Schematic representation and photomicrography of the ova- by follicular cells (HE). f Young oocyte and nurse cells without the ries of a Camponotus textor