Corresponding author: Vladimir E. Gokhman ( firstname.lastname@example.org )
Academic editor: Danon Clemes Cardoso
© 2017 Vladimir E. Gokhman, Fabricio Fagundes Pereira, Marco Antonio Costa.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Gokhman VE, Pereira FF, Costa MA (2017) A cytogenetic study of three parasitic wasp species (Hymenoptera, Chalcidoidea, Eulophidae, Trichogrammatidae) from Brazil using chromosome morphometrics and base-specific fluorochrome staining. Comparative Cytogenetics 11(1): 179-188. https://doi.org/10.3897/CompCytogen.v11i1.11706
Chromosomes of three chalcid wasp species from Brazil, Palmistichus elaeisis Delvare et LaSalle, 1993, Trichospilus diatraeae Cherian et Margabandhu, 1942 (both belonging to the family Eulophidae) and Trichogramma pretiosum Riley, 1879 (Trichogrammatidae), were studied using chromosome morphometrics and base-specific fluorochrome staining. The present study confirmed that these species respectively have 2n = 12, 14 and 10. Chromomycin A3 / 4’, 6-diamidino-2-phenylindole (CMA3/DAPI) staining revealed a single CMA3-positive and DAPI-negative band within haploid karyotypes of both P. elaeisis and T. pretiosum. This CG-rich band clearly corresponds to the nucleolus organizing region (NOR). Moreover, analogous multiple telomeric bands found on all chromosomes of T. diatraeae may also represent NORs. Certain features of karyotype evolution of the phylogenetic lineage comprising both Eulophidae and Trichogrammatidae are discussed. The results obtained during the present study demonstrate the importance of chromosome research on tropical parasitoids that remain poorly known in this respect.
Parasitoids, chromosomes, karyotypes, NORs, CMA3, DAPI
Parasitoid Hymenoptera are one of the most species-rich, taxonomically complicated and economically important insect groups (
Parasitoid strains were kept as lab stocks in the Laboratory of Biological Control of Insects of the Faculdade de Ciências Biológicas e Ambientais of the Universidade Federal da Grande Dourados, Dourados, Mato Grosso do Sul, Brazil, in climate-controlled chambers at 25 ± 1°C, 70 ± 10% relative humidity, and 14-hour photoperiod. All examined strains were maintained on lepidopteran hosts. Specifically, both studied members of the family Eulophidae and T. pretiosum were respectively bred on the pupae of Diatraea saccharalis (Fabricius, 1794) (Crambidae) and eggs of Ephestia kuehniella (Zeller, 1879) (Pyralidae). Parasitoids were identified by Marcelo Teixeira Tavares (Eulophidae) and Ranyse Barbosa Querino da Silva (Trichogrammatidae).
Chromosomal preparations were obtained from cerebral ganglia of parasitoid prepupae taken from dissected hosts generally following the protocol developed by
For conventional staining, preparations were stained with freshly prepared 3% Giemsa solution in 0.05M Sørensen’s phosphate buffer (Na2HPO4 + KH2PO4, pH 6.8) for about 15 min. Fluorochrome staining with chromomycin A3 and 4’, 6-diamidino-2-phenylindole (CMA3/DAPI) was performed according to
Metaphase plates were analyzed under an Olympus BX51 microscope. Images of chromosomes were taken with an Olympus DP72 camera using ImageProPlus software. To prepare illustrations, the resulting images were arranged and enhanced using Adobe Photoshop 8.0. The same software was also used for taking measurements from selected metaphase plates with good chromosome morphology. Statistical analysis was performed using STATISTICA 5.5. The chromosomes were classified following guidelines provided by
Five pairs of large metacentric chromosomes of similar size and a much smaller pair of acrocentrics were found in this species with 2n = 12 (Fig.
The karyotype of this species contains four pairs of large metacentrics of approximately the same size (the first pair is slightly longer than the remaining ones) and three considerably smaller pairs of acrocentric chromosomes; the chromosome number in T. diatraeae is thus 2n = 14 (Fig.
CMA3/DAPI-stained metaphase plates of parasitoids. a–c P. elaeisis, female d–f T. diatraeae, female g–i T. pretiosum, male a, d, g CMA3 staining b, e, h DAPI staining c, f, i merged CMA3/DAPI staining. Arrows and arrowheads respectively indicate CMA3-positive and DAPI-negative bands (NORs) as well as both CMA3- and DAPI-positive chromosome arms. Bar = 10 μm.
The largest chromosome pair in the karyotype of this species is metacentric, whereas the remaining four pairs are clearly smaller; three of them are somewhat similar in size, and the fourth one is substantially shorter (2n = 10). Most these chromosomes are acrocentric (pairs no. 2, 4, and 5) except for the third pair of metacentric chromosomes which is more or less equal in length to the fourth pair of acrocentrics (Fig.
Relative lengths (RL) and centromeric indices CI) of parasitoid chromosomes (mean ± SD). For each species, numbers of analyzed diploid metaphase plates are given in brackets.
|Chr. no.||P. elaeisis (6)||T. diatraeae (4)||T. pretiosum (15)|
|1||20.44 ± 0.78||48.43 ± 2.42||20.28 ± 0.72||44.28 ± 4.08||25.94 ± 1.48||42.32 ± 2.92|
|2||19.47 ± 0.42||47.57 ± 1.67||18.38 ± 0.72||45.17 ± 3.72||20.55 ± 0.83||0|
|3||18.68 ± 0.30||47.64 ± 1.86||16.75 ± 0.91||44.44 ± 3.75||18.97 ± 0.78||44.89 ± 2.73|
|4||17.88 ± 0.32||47.61 ± 1.59||14.88 ± 0.68||44.51 ± 4.45||18.66 ± 0.84||0|
|5||16.98 ± 0.45||46.61 ± 1.97||11.20 ± 0.72||0||15.88 ± 0.95||0|
|6||6.55 ± 0.39||0||10.18 ± 0.66||0||-||-|
|7||-||-||8.33 ± 0.66||0||-||-|
Chromosome sets of P. elaeisis and T. diatraeae were first studied by
The chromosome set of T. pretiosum was previously studied by
Staining of P. elaeisis karyotype with certain base-specific fluorochromes was apparently performed for the first time by
Karyotypes of Trichogramma species have never been examined using base-specific fluorochromes. Nevertheless, NORs in this genus were previously studied by
The chalcid families Eulophidae and Trichogrammatidae belong to the same phylogenetic lineage that also includes Aphelinidae and a few smaller groups of which the chromosomes are as yet unknown (
In conclusion, we would like to stress the importance of studying chromosome sets of tropical parasitic wasps for our understanding of the karyotype structure and evolution in parasitoid Hymenoptera in general. For example, the chromosome set of T. diatraeae demonstrates deviating karyotypic features in terms of both Giemsa and CMA3/DAPI staining. In particular, the presence of multiple CMA3-positive telomeric bands on all chromosomes of this species was not previously detected in any other parasitoid. Moreover, this study reveals for the first time considerable karyotypic differences within the speciose and practically important genus Trichogramma, again in terms of both chromosome morphometrics and the number of NORs, thus potentially contributing to our knowledge of its taxonomy and phylogeny. Our data therefore confirm that further research of parasitoid Hymenoptera from various geographical regions will undoubtedly demonstrate more variation in the overall karyotype structure and distribution of different chromosome segments in this group.
The authors are grateful to Marcelo Teixeira Tavares (Universidade Federal do Espírito Santo, Vitória, Espírito Santo, Brazil) and Ranyse Barbosa Querino da Silva (Embrapa Meio-Norte, Teresina, Piauí, Brazil) for identifying parasitoids as well as to Robert B. Angus (University of London, London, UK) for kindly checking the language of the paper. The present study was partly supported by research grants no. 15-04-07709 from the Russian Foundation for Basic Research (RFBR) to VEG and no. 310178/2015-0 from the Brazilian Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) to MAC.