Introduction
Cytogenetic studies are important for assessing the genetic variation and for inferring the karyoevolution of species (King Reference King1993; Schubert Reference Schubert2007), being useful for cytotaxonomy and phylogenetics (Bitencourt et al. Reference Bitencourt, Affonso, Giuliano-Caetano and Dias2011; Miao et al. Reference Miao, Wang and Hua2018; König et al. Reference König, Paschke, Pollmann, Reinisch, Gantert and Weber2019). In addition, the methodological advances in cytogenetics have allowed the development of refined investigations into the microrearrangements in the karyotype of certain groups (Bitencourt et al. Reference Bitencourt, Sampaio, Ramos and Affonso2014; Gokhman et al. Reference Gokhman, Bolsheva, Govind and Muravenko2016). In particular, analyses about heterochromatin composition have revealed unique karyotypic traits at the species and population levels of many organisms, including insects (Lorite et al. Reference Lorite, Garcia, Carrillo and Palomeque1999; Gokhman Reference Gokhman2021).
A large number of cytogenetic reports in the order Hymenoptera are available. They range from chromosomal analyses to genome sequencing, but most are restricted to ants, social wasps, and bees (Pompolo and Takahashi Reference Pompolo and Takahashi1990; Brito et al. Reference Brito, Pompolo, Magalhães, Barros and Sakamoto-hojo2005; Lorite and Palomeque Reference Lorite and Palomeque2010; Carvalho and Costa Reference Carvalho and Costa2011; Menezes et al. Reference Menezes, Silva, Carvalho, Andrade-Souza, Silva and Costa2013, Reference Menezes, Carvalho, Correia, Silva, Somavilla and Costa2014; Cristiano et al. Reference Cristiano, Simões, Lopes and Pompolo2014; Barros et al. Reference Barros, de Aguiar, Mariano, Andrade-Souza, Costa, Delabie and Pompolo2016). For parasitoid wasps, cytogenetic information has been reported in about 500 species, which is considered a small number when compared to the remarkable richness of this group of insects (Gokhman Reference Gokhman2009).
Accordingly, the parasitoid wasps of the superfamily Chalcidoidea comprise nearly 23 000 species distributed into 22 families (Huber Reference Huber, Foottit and Adler2017). Amongst them, the Chalcididae represent a family characterised by wide variation in colour and size of species recognised as solitary endoparasitoids of several orders of holometabolous insects, mainly larvae of Diptera and pupae of Lepidoptera. Other species in this family have been recorded as parasitoids of Coleoptera, Hymenoptera, and Strepsiptera, and others yet are obligatory hyperparasitoids of Ichneumonoidea and Tachinidae (Tavares and Araújo Reference Tavares and Araújo2007). In addition, gregarious and ectoparasitoid species are reported within Chalcididae (Universal Chalcidoidea Database Community 2023). Currently, this family is divided into 87 genera and 1464 valid species, including 28 genera and 439 taxa in Neotropical regions. Sixteen genera and 219 species are reported in Brazil (Universal Chalcidoidea Database Community 2023).
Considering the wide diversity of Chalcidoidea, this superfamily remains underrepresented in cytogenetic studies: karyotype information is available for only 1% (about 240) of valid species (Gokhman Reference Gokhman2020). These data are even scarcer in the family Chalcididae, for which chromosomal studies are restricted to karyotyping based on traditional methods of five species from three genera (Hung Reference Hung1986; Amalin et al. Reference Amalin, Rueda and Barrion1988; Johnson et al. Reference Johnson, Grissell, Gokhman and Valero2001). Brachymeria is the most studied genus inasmuch as karyotypic data are available for B. lasus Walker (2n = 10), B. ovata Say (2n = 10), and B. intermedia Nees (2n = 6) (Hymenopetera: Chalcididae) (Hung Reference Hung1986), the latter being characterised by the lowest haploid number (n = 3) ever recorded for Chalcidoidea (Gokhman Reference Gokhman2020).
The goal of the present study is to provide the first chromosomal data in the parasitoid wasp, Brachymeria (Pseudobrachymeria) vesparum Bouček, 1992 (Hymenoptera: Chalcididae). We also report novel information about C-banding and base-specific fluorochrome staining for species in the Chalcididae family – information that can be used to infer the species’ karyoevolutionary pathways.
Materials and methods
Adults and immatures of B. vesparum (Fig. 1A) were obtained from nests of the social wasp, Polistes canadensis Linnaeus (Hymenoptera: Vespidae), found as hyperparasites of immature forms of the parasitoid wasp, Pachysomoides sp. (Hymenoptera: Ichneumonidae) (Fig. 1B), at Universidade Estadual do Sudoeste da Bahia (UESB) – Campus II, Jequié, Bahia, Brazil (13° 51′ 4″ S and 40° 4′ 52″ W). About 10 nests containing parasitised cells were collected. The adults that emerged were stored in 70% alcohol and were identified by taxonomist Dr. Marcelo Tavares, Universidade Federal do Espírito Santo (UFES; Vitória, Espirito Santo, Brazil).
Figure 1. A, Representative specimen of Brachymeria (Pseudobrachymeria) vesparum obtained from nests of the social wasp Polistes canadensis (Hymenoptera: Vespidae) and B, hyperparasiting immatures of the parasitoid wasp Pachysomoides sp. (Hymenoptera: Ichneumonidae). Approximate size, Fig. 1A: 1 mm.
The mitotic metaphases were obtained from the cerebral ganglia of larvae in the prepupal stage according to Imai et al.’s (Reference Imai, Taylor, Crosland and Crozier1988) air-drying technique. Chromosomes were stained in 10% Giemsa solution (Merck KGaA, Damstadt, Germany) in Sörensen buffer (0.06 M; pH 6.8, according to the protocol of Guerra and Souza Reference Guerra and Souza2002) for determining chromosomal number and morphology.
The pattern of heterochromatin distribution was determined by C-banding (Sumner Reference Sumner1972), with slight modifications (Siqueira et al. Reference Siqueira, Aguiar, Strüssmann, Del-Grande and Recco-Pimentel2008). The regions in the pairs of bases rich in AT (adenine and thymine) and GC (guanine and cytosine) were mapped on chromosomes by base-specific fluorochrome staining, using chromomycin A3 (CMA3), distamycin (DA), and 4,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich–Merck KGaA, Damstadt, Germany), as reported by Schmid (Reference Schmid1980).
The best metaphases were analysed and photographed using a Solaris-T microscope (BEL Engineering, Monza, Italy) fitted with a portable digital camera SCMOS mini USB2.0 SCMOS00350KPA model (MEKEY, Chongqing, China). The karyotypes were arranged by pairing chromosomes in decreasing order of size using Adobe Photoshop CS6 (Adobe, Inc., San Jose, California, United States of America), and the haploid number was determined from the modal frequencies. The chromosomal morphology was classified based on their arm ratio (Levan et al. Reference Levan, Fredga and Sandberg1964), using the software Image Pro Plus (https://mediacy.com/image-pro/). The ideograms representing the karyotype of Brachymeria species were established using the software EasyIdio, version 3.0 (Diniz and Xavier Reference Diniz and Xavier2006), based on chromosomal measurements obtained in the present study and on data available in the literature for the genus (Hung Reference Hung1986).
Results
All specimens of B. vesparum showed 2n = 10 metacentric chromosomes (fundamental arm number or FN = 20) with a karyotype formula of 2K = 10 m (Fig. 2A). The C-banding revealed pericentromeric heterochromatic blocks at most chromosomes and at the terminal regions on pairs 3 and 4 and nearly the entire short arms of pair 2 (Fig. 2B). After base-specific fluorochrome staining (CMA3/DA/DAPI), most heterochromatic regions were AT-rich (AT+). However, GC-rich (GC+) sites were identified at interstitial regions on short and long arms of pairs 1 and 4, respectively (Fig. 2C). Heteromorphic GC+ signals were also observed between homologous chromosomes from pair 4.
Figure 2. Metaphases of specimens of Brachymeria (Pseudobrachymeria) vesparum: A, after conventional Giemsa staining; B, C-banding; and C, base-specific fluorochrome staining. The scale bar corresponds to 10 µm.
Discussion
About 53 species of Brachymeria are described for the Neotropical region (Tavares and Araújo Reference Tavares and Araújo2007), but to date, cytogenetic data are available for only three species. The diploid values reported in this genus range from 2n = 6 in B. intermedia (Hung Reference Hung1986) to 2n = 10 in B. lasus, B. ovata (Hung Reference Hung1986), and B. vesparum (present study).
In general, the chromosomal information in parasitoid wasps of the superfamily Chalcidoidea, with few exceptions, indicates the species might be divided into two groups: (1) families characterised by low chromosomal numbers (n = 3–7) and (2) families with higher chromosomal numbers ranging from n = 8 to n = 11 (Gokhman Reference Gokhman2020). The putative ancestor haploid number of parasitoid hymenopterans ranges from 14 to 17, indicating multiple reduction events of chromosomal numbers within some lineages (Gokhman Reference Gokhman2004), such as Pteromalidae and Eulophidae (Gokhman Reference Gokhman2009, Reference Gokhman2020). Conversely, the basal haploid values in Chalcidoidea vary from n = 3 to n = 11, with a predominance of n = 5 and n = 6. In this case, centric fusions are regarded as the main rearrangements involved in the karyoevolution of this group (Gokhman Reference Gokhman2013, Reference Gokhman2022).
The lowest chromosome number among Chalcidoidea, n = 3, has been found in Brachymeria intermedia and in certain members of the families Aphelinidae and Perilampidae (Hung Reference Hung1986; Baldanza et al. Reference Baldanza, Gaudio and Viggiani1999; Gokhman Reference Gokhman2005). Despite the apparent conservativism of the diploid number in Brachymeria, variation in the karyotype formulae has been reported in this genus (Hung Reference Hung1986). In general, congeneric species share karyotypes with a predominance of metacentric pairs, but submetacentric and acrocentric chromosomes have also been reported in the three species cytogenetically analysed (B. intermedia, B. ovata, and B. lasus; Hung Reference Hung1986), except for B. vesparum from the present study (Table 1).
Table 1. Cytogenetic data available in species of the family Chalcididae with their respective haploid (n) and diploid (2n) numbers, karyotype formulae, and origin of samples
The unique karyotype formula herein reported for B. vesparum, when compared to the other congeneric species, suggests that structural rearrangements, such as pericentric inversions and centric fusions, played a key role in the karyotype evolution of these insects (Fig. 3). Even if the chromosome pairs are grouped into one-armed (acrocentric) and biarmed (meta/submetacentric) classes, the karyotype structure informs how the Brachymeria species have differentiated, given that B. vesparum, B. lasus, B. ovata, and B. intermedia present FN = 20, FN = 18, FN = 16, and FN = 12, respectively.
Figure 3. Ideograms representing the karyotypes of Brachymeria species (B. intermedia, B. lasus, and B. ovata by Hung (Reference Hung1986) and B. (Pseudobrachymeria) vesparum, from the present study), indicating the putative structural rearrangements that took place along the karyoevolution of the genus.
Genome architecture, including the chromosomal structure, is directly related to the transmission of genetic traits within and among populations and therefore is capable of promoting species diversification (Feder et al. Reference Feder, Gejji, Powell and Nosil2011). Differences related to chromosomal rearrangements have been related directly to speciation events (Potter et al. Reference Potter, Bragg, Blom, Deakin, Kirkpatrick, Eldridge and Moritz2017). For example, heterozygous inversions or translocations might affect gametogenesis, leading to infertility or affecting the survival of hybrid forms (Livingstone and Rieseberg Reference Livingstone and Rieseberg2003). In this way, the unique chromosomal features observed across Brachymeria species are species-specific markers for diagnosing congeners, thereby reinforcing their reproductive isolation.
Despite the major role of heterochromatin in the karyotype diversification of several animal groups (Bitencourt et al. Reference Bitencourt, Affonso, Giuliano-Caetano and Dias2011; Tavares et al. Reference Tavares, Ferreira, Travenzoli and Lopes2021), the analysis of heterochromatin distribution in the present study was poorly informative. C-bands at pericentromeric regions are commonly reported in many species of the superfamily Chalcidoidea (Gokhman and Westendorff Reference Gokhman and Westendorff2000; Gokhman Reference Gokhman2022), and the presence of heterochromatic chromosomal arms has also been observed in some parasitoid wasps (Baldanza et al. Reference Baldanza, Gaudio and Viggiani1999; Gokhman and Westendorff Reference Gokhman and Westendorff2000). Such conspicuous C-bands most likely represent remnants of heterochromatin from chromosomes after fusion events, thereby supporting the role of chromosomal rearrangements in the evolution of this group of insects that previous studies (e.g., Gokhman Reference Gokhman2013) have proposed.
As for the composition of heterochromatin revealed by fluorochrome staining, the species examined in the present study revealed both AT- and GC-rich regions in two chromosome pairs, which Baldanza et al. (Reference Baldanza, Gaudio and Viggiani1999) reported in other parasitoids. Furthermore, the GC+ sites on pair 4 were heteromorphic, possibly as a result of a heterozygous paracentric inversion, similar to what Tavares and Teixeira (Reference Tavares and Teixeira2021) reported in Pachodynerus nasidens (Eumeninae). Gokhman et al. (Reference Gokhman, Pereira and Costa2017, Reference Gokhman, Nugnes and Bernardo2019) reported positive CMA3 signals at the interstitial region in two parasitoid wasps, Palmistichus elaeisis Delvare and LaSalle, 1993 (Hymenoptera: Eulophidae) and Baryscapus silvestrii Viggiani and Bernardo, 2007 (Hymenoptera: Eulophidae), as well as at the terminal regions of all chromosomes in the karyotype of Trichospilus diatraeae (Cherian and Margabandhu, 1942) (Hymenoptera: Eulophidae).
The mapping of nucleolus organiser regions (NORs) in karyotypic studies has also been carried out in cytogenetic studies of parasitoids (Baldanza et al. Reference Baldanza, Gaudio and Viggiani1999; van Vugt et al. Reference van Vugt, Nooijer, Stouthamer and Jong2005), usually revealing single or double NORs in Chalcidoidea (Gokhman Reference Gokhman2022). These regions can be identified by several methods, including base-specific fluorochrome staining because NORs are usually interspersed with GC-rich sites (CMA3 + signals; Schweizer Reference Schweizer1980). However, it should be pointed out that additional fluorochrome signals unrelated to NORs might be present throughout the chromosomal DNA (Gokhman Reference Gokhman2022). Accordingly, caution is recommended before concluding that the interstitial CMA3 + signals on pairs 1 and 4 of B. vesparum refer to NORs. For this reason, other techniques, such as silver nitrate staining and fluorescent in situ hybridisation with rDNA probes, should also be carried out to confirm the location of ribosomal cistrons.
Even though refined methods of chromosomal analyses have been performed on insects (van Vugt et al. Reference van Vugt, Nooijer, Stouthamer and Jong2005, Reference van Vugt, Jong and Stouthamer2009; Bolsheva et al. Reference Bolsheva, Gokhman, Muravenko, Gumovsky and Zelenin2012; Gokhman et al. Reference Gokhman, Pereira and Costa2017), these reports remain scarce in parasitoid wasps (Gokhman Reference Gokhman2010; Gebiola et al. Reference Gebiola, Giorgini, Navone and Bernardo2012), and several taxonomic uncertainties in these insects have yet to be resolved. The karyotypic data described in the present study for Brachymeria advance the understanding of evolutionary mechanisms and cytogenetic patterns for the examined species. Moreover, the karyotype structure and the distribution of GC-rich heterochromatic sites provide useful diagnostic characters for cytotaxonomy. We therefore recommend that similar studies examine other members of the family Chalcididae to reveal and refine understanding of the evolutionary and systematic inferences in these insects, including the potential identification of cryptic forms or species complexes.
Acknowledgements
The authors thank Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brazil, for financial support (code 001), Dr. Marcelo Tavares from the Universidade Federal do Espírito Santo (UFES) for the identification of the analysed species, and Universidade Estadual do Sudoeste da Bahia and the Graduate Program in Genetics, Biodiversity and Conservation for supporting the present study.
Competing interests
The authors declare they have no competing interest.
