Research Article |
Corresponding author: Snejana Grozeva ( sgrozeva@yahoo.com ) Academic editor: Seppo Nokkala
© 2015 Ana Maria Jauset, Eva Edo-Tena, Cristina Castañé, Nuria Agustí, Oscar Alomar, Snejana Grozeva.
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:
Jauset AM, Edo-Tena E, Castañé C, Agustí N, Alomar O, Grozeva S (2015) Comparative cytogenetic study of three Macrolophus species (Heteroptera, Miridae). Comparative Cytogenetics 9(4): 613-623. https://doi.org/10.3897/CompCytogen.v9i4.5530
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Macrolophus pygmaeus (Rambur, 1839) (Insecta, Heteroptera, Miridae) is a predator of key vegetable crop pests applied as a biocontrol agent in the Mediterranean region. M. pygmaeus and M. melanotoma (A. Costa, 1853) are cryptic species with great morphological similarity which results in their misidentification and negative consequences for the conservation of their populations on greenhouse and outdoor crops. In order to find out specific markers for their separation we studied the karyotype, male meiosis and heterochromatin composition of these species and additionally of a third species (as a reference one), M. costalis Fieber, 1858. We demonstrate here that all the three species share achiasmate male meiosis and sex chromosome pre-reduction. On the other hand, the species differ in karyotype, with 2n=28 (26+XY) in M. pygmaeus, 2n=27 (24+X1X2Y) in M. costalis, and 2n=34 (32+XY) in M. melanotoma, and heterochromatin distribution and composition. In addition, the species differ in sperm morphology: sperm cells of M. costalis are significantly longer with longer head and tail than those of M. melanotoma and M. pygmaeus, whereas sperm cells of M. melanotoma have a longer tail than those of M. pygmaeus. All these characters can be used as markers to identify the species, in particular the cryptic species M. melanotoma and M. pygmaeus.
Macrolophus , Miridae , Heteroptera , karyotype, sex chromosomes, achiasmate meiosis, sex chromosome pre-reduction, sperm morphology
The Miridae are the largest family of true bugs (Heteroptera, Cimicomorpha) with approximately 10000 species described (
The species from the present study, M. costalis, M. melanotoma (A. Costa, 1853), and M. pygmaeus, occur on a variety of plant species in the Mediterranean region. M. pygmaeus is an efficient predator of several key vegetable crop pests in Europe produced commercially and used widely as a biocontrol agent (
Sperm morphology is significant in fertilization (
With the aim of distinguishing between the cryptic Macrolophus species, both karyotype and male meiosis were studied for the first time in M. melanotoma and reinvestigated in M. pygmaeus and (as a reference species) in M. costalis using standard chromosome staining and fluorochromes DAPI and CMA3. In addition, morphology of sperm cells was examined in each of the three species.
Males and females of M. costalis, M. pygmaeus and M. melanotoma were collected in Catalonia, NE of Spain, in the vicinity of Mataró (Barcelona) (41.556 North, 2.475 East) from Cistus albidus Linnaeus, 1753, commercial tomato fields and Dittrichia viscosa (Linnaeus) Greuter, respectively. Colonies from collected individuals were set-up under controlled conditions (25 ± 1°C, 70 ± 10% RH and L16:D8 photoperiod) on tobacco plants (Nicotiana tabacum Linnaeus, 1753) with Ephestia kuehniella Zeller, 1879 (Lepidoptera, Pyralidae) eggs as a prey (
The abdomen of 20 M. pygmaeus, 23 M. melanotoma and 13 M. costalis males were placed in 3:1 fixative (96% ethanol-glacial acetic mixture) and the thorax in 70% ethanol for later species identification by DNA analysis (
Chromosomes were analyzed using light/fluorescent microscopy (Axio Scope A1 – Carl Zeiss Microscope) at 100× magnification and documented with a ProgRes MFcool – Jenoptik AG digital camera. All cytogenetic preparations and remains of the specimens are stored at the Institute of Biodiversity and Ecosystem Research, BAS in Sofia.
In every species, sperm cells from 10 males (other than those used for the karyotype analysis) were measured following
Published data: 2n=28 (24+X1X2X3Y) (
Spermatogonial metaphases consisted of 5 large (incl. Y) and 22 similar in size chromosomes (incl. two X) (Fig.
Male meiosis in Macrolophus costalis. (1–5 conventional staining 6 fluorochrome staining: 6a, c CMA36b, d DAPI) 1 spermatogonial metaphase 2 condensation stage 3a–c metaphase I 4a–c metaphase II 5 telophase 6a, b condensation stage 6c, d metaphase I. Sex-chromosomes are indicated by arrowheads. Heterochromatin blocks are indicated by arrows. Bar = 10 µm.
After staining with fluorochromes, bright DAPI- and CMA3- positive bands were observed in the same locations on the larger autosomal bivalents and sex chromosomes (Fig.
Published data: 2n=28 (26+XY) (
Spermatogonial metaphases consisted of 5 large (incl. X) and 23 similar size chromosomes (incl. Y) (Fig.
Male meiosis in Macrolophus pygmaeus. (7–10 conventional staining 11 fluorochrome staining: 11a, c CMA311b, d DAPI) 7 spermatogonial metaphase 8 condensation stage 9 metaphase I 10 metaphase II 11a, b metaphase I 11c, d anaphase. Sex-chromosomes are indicated by arrowheads. CMA3/DAPI signals are indicated by arrows. Bar = 10 µm.
After staining with fluorochromes, bright DAPI- and CMA3 -positive bands were observed on both sex chromosomes (Fig.
Published data: absent
At spermatogonial metaphase, there were 34 chromosomes (Fig.
Male meiosis in Macrolophus melanotoma. (12–16 conventional staining 17 fluorochrome staining: 17a CMA317b DAPI) 12 spermatogonial metaphase 13 condensation stage 14a–c metaphase I 15 anaphase I 16 metaphase II 17 condensation stage. Sex-chromosomes are indicated by arrowheads. Bar = 10 µm.
After staining with fluorochromes, bright DAPI- and CMA3 bands were observed on the sex chromosomes (Fig.
Sperm cells of the species studied were of similar shape, with a long and filiform head (Fig.
Sperm cells lenght (means ± standard error) of M. costalis, M. melanotoma and M. pygmaeus. Within each row, means followed by the same letter do not differ significantly (P≤0.005).
Sperm length (µm) | M. costalis | M. melanotoma | M. pygmaeus |
---|---|---|---|
Total | 236±0.9 a | 220.4±0.9 b | 215±1.4 c |
Head | 61.3±0.4 a | 51.3±0.4 b | 50.6±0.4 b |
Tail | 174.7±0.8 a | 169.1±1 b | 164.6±1.2 c |
The genus Macrolophus belongs to the tribe Dicyphini of the subfamily Bryocorinae (Heteroptera, Miridae). In Bryocorinae, besides the modal for the Miridae chromosome number 2n=34 (32+XY), some higher (2n=36+XY and 2n=46+XY/X1X2Y) and lower (2n=16-26+XY) chromosome numbers have also been described (
Differences in molecular organisation of chromatin revealed after fluorochrome staining, suggest an additional chromosome marker to differentiate Macrolophus species. In M. costalis, bright DAPI/CMA3 bands were observed in the same locations on the large autosomal bivalents and sex chromosomes whereas in two remaining species bright fluorescent bands were observed only on the sex chromosomes. In turn, M. pygmaeus differed from M. melanotoma in that it showed some additional weak DAPI-positive signals in a telomeric region of a larger bivalent.
As mentioned in Introduction, the cryptic species M. pygmaeus and M. melanotoma can be differentiated from each other based on the cuticular hydrocarbon profiles and specific molecular primers (
This study has been funded by the Spanish Ministry of Economy and Competitiveness (MINECO) (Project AGL2011-24349), and by a travel grant from the University of Lleida - Fundació “La Caixa” to Dr Grozeva. The chromosome analysis was performed using microscope Axio Scope A1 – Carl Zeiss Microscopy upgraded by the project WETLANET (FP7 CSA – SUPPORT ACTION, GA 229802). We thank cordially Prof. Dr V. Kuznetsova for the valuable advices to improve the manuscript.