Firenze University Press Caryologia www.fupress.com/caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Karyotype analysis of Trichogramma embryophagum Htg. (Hymenoptera: Citation: F. Farsi, H.E. Eroğlu, J. Nozari, V. Hosseininaveh (2020) Karyo- Trichogrammatidae) using a new method and type analysis of Trichogramma embry- ophagum Htg. (Hymenoptera: Tricho- estimate its karyotype symmetry grammatidae) using a new method and estimate its karyotype symmetry. Caryologia 73(3): 65-70. doi: 10.13128/ caryologia-203 Fatemeh Farsi1, Halil Erhan Eroğlu2, Jamasb Nozari1,*, Vahid Hossein- inaveh1 Received: April 03, 2019 1 Department of Plant Protection, College of Agriculture and Natural Resources, Univer- Accepted: June 02, 2020 sity of Tehran, Karaj, Iran 2 Published: December 31, 2020 Yozgat Bozok University, Faculty of Science and Art, Department of Biology, Yozgat, Turkey Copyright: © 2020 F. Farsi, H.E. Eroğlu, * Corresponding author. E-mail: [email protected] J. Nozari, V. Hosseininaveh. This is an open access, peer-reviewed article published by Firenze University Press Abstract. Different methods of chromosome preparation for insect are now being used (http://www.fupress.com/caryologia) across the world. Well-spread chromosomes with explicit morphology, in addition to and distributed under the terms of the no cell wall debris are required for karyotype investigations. Cytogenetic knowledge Creative Commons Attribution License, in Trichogramma is extremely limited. In the article, chromosome characters, karyo- which permits unrestricted use, distri- type and monoploid ideogram of Trichogramma embryophagum Htg. (Hymenoptera: bution, and reproduction in any medi- Trichogrammatidae) were evaluated using a new method. Also, karyotype symmetry/ um, provided the original author and source are credited. asymmetry of this species was calculated for the first time as one of the Trichogramma species. Our results showed that the diploid chromosome number of the wasp was 2n Data Availability Statement: All rel- = 10. The karyotype formula was 6m + 4a. The symmetry/asymmetry index value was evant data are within the paper and its 1.8. The new method resulted in higher quality metaphase plates spread and provided Supporting Information files. an ideal karyomorphology for this parasitoid which has small chromosomes. Competing Interests: The Author(s) declare(s) no conflict of interest. Keywords: ideogram, karyotype, 8-hydroxyquinoline, symmetric karyotype. INTRODUCTION A high-quality chromosome preparation is critical for cytogenetic stud- ies of Hymenoptera parasitoids. In the majority of parasitoid species, com- plications are caused by the minute size of the insect (Baldanza et al. 1993), the small size of their chromosomes (Paladino et al. 2013), and low number of chromosomes, 2n = 6 found in Brachymeria intermedia (Chalcididae) and Encarsia protransvena (Aphelinidae) (Hung 1986; Baldanza et al. 1999) which make their cytogenetic studies limited. Although modern molecular tech- niques such as Fluorescent in situ Hybridization (FISH) have already taken been on an important role (Yalcin and Kulduk 2018), they have never been able to be a complement replacement for classical methods. Some disadvan- tages including being costly and difficult (due to requiring probes and fluo- Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 73(3): 65-70, 2020 ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.13128/caryologia-203 66 Fatemeh Farsi, Halil Erhan Eroğlu, Jamasb Nozari, Vahid Hosseininaveh rescent microscope), requiring highly-skilled cytogeneti- (Gokhman et al. 2017). It should be noted that the prob- cists, and the inability to reveal chromosome inversions lems described for each of these methods also exist for (Di- Nizo et al. 2017) have caused cytogenetics to show a Trichogramma karyotype studies. The minute size and significant tendency towards using classical methods. lifestyle of Trichogramma species also intensify these Squash is a well-known and oldest method for flat- problems (Manickavasagam 1991; Hung 1982). In most tening metaphase plate in insects (Kocak and Okutaner studies, only the haploid number of these species has 2017). However, difficulty in achieving well-spread, dis- been raised, and the chromosomal details including the torted, and stretched chromosome are the main dis- length of the arms that could provide many comparisons advantages of this method (Chirino et al. 2014; Kocak were not possible (Hung 1982; Laurent et al. 1998). One and Okutaner 2017). During squashing, producing well- parameter that can be obtained from the chromosomal spread plates are often not desirable due to the concen- information is karyotype symmetry/asymmetry (Peruzzi tration of cells in a small area. Over time, spread (hot- and Eroğlu 2013). Karyotype symmetry/asymmetry is plate spread) method conducted by various researchers obtained based on the availability of chromosome detail, flourished (Bressa et al. 2009; Sadilek et al. 2016). Its including relative chromosome size and position of cen- major disadvantage is the attendance of cellular debris tromere. Until now, this parameter which can help in around the chromosomes (Chirino et al. 2014), making the evaluation of enter- species relationship seldom has their morphology unpleasant. Considering the men- calculated in class of insect. In the cytogenetic studies, tioned constraints, there is always a need for a simple species that are more similar in the terms of chromo- and efficient method for the preparation of metaphase somal parameters such as karyotype symmetry/asymme- plates in parasitoids. try will have more affinity. Despite the vast amount of research on insect kar- In this research, the karyotype, ideogram, and chro- yotype, only about 500 species of parasitoid wasps have mosomal detail of T. embryophagum were investigated available data (Gokhman 2009). Among these, only for the first time with the introduction of a new method more than 10 are related to Trichogramma. So far, 239 considering all steps of Hymenoptera parasitoids chro- species of Trichogramma have been reported through- mosome preparation. These chromosome analyses were out the world (Khan and Yousuf 2017). There are eleven complemented by an estimation of its karyotype symme- species of this parasitoid in Iran (Nazeri et al. 2015) that try as one of the species of Trichogrammatidae. Trichogramma embryophagum Htg. is one of them. T. embryophagum distributed in some of regions of Iran, including the provinces of West and East Azerbaijan, MATERIAL AND METHOD Yazd, Khorasan (Poorjavad et al. 2011). Cytogenetic studies and karyotype investigation have not yet been Insect done on this species. Because of the substantial posi- tion that Trichogramma occupy in the biological control T. embryophagum Htg. individuals originated from programs (Parra 2009), investigations on their chromo- parasitized eggs of carob moth, Ectomyelois ceratoniae, some details have provided precious information for the (Zeller) (Lepidoptera: Pyralidae), were collected on pome- understanding various perspectives such as mechanisms granate in the center of Iran (Yazd Region, 32.1006° N, of sex determination, evaluation of sex chromosome, 54.4342° E). The wasps were reared on Ephestia kuehniel- existence of accessory chromosomes and phylogenetic la Zeller (Lepidoptera; Pyralidae) eggs in a climate-con- relationships. trolled chamber at 25 ± 1 °C, 70 ± 5% relative humidity, An evaluation of existing literature shows that dif- L16: D8 photoperiod. E. kuehniella eggs were obtained ferent methods was used in karyotype studies of Tricho- from a culture maintained in the Biosystematics Labora- gramma. Studies about Trichogramma were derived from tory at The University of Tehran, Karaj, Iran. routine chromosomal preparation methods, including the use of various percentages of colchicine as pretreat- Preparation chromosome ment, staining by different colors, and then squashed (Hung 1982; Liu and Xiong 1998). Interestingly, no Sample from egg host were Dissected out in physi- banding staining on the Trichogramma species has ever ological solution for Ephestia (Glaser, 1917, cited by been reported. Molecular cytogenetic including Flores- Lockwood 1961). Two kinds of pre-treatment were used, cence in situ hybridization (FISH) has been applied for including hypotonic and other is combined it. First, sam- only the two species of T. kaykai Pinto & Stoutham- ples were transferred into a drop of 0.075 M KCl for 8 er (van Vugt et al. 2005) and T. pretiosum Riley minutes [0.075 M KCl: 0.5592 g KCl/100 ml redistill. Karyotype analysis of Trichogramma embryophagum 67 H2O] on a shaker at 20 °C and 30 rpm. After wise, the S/A I = (1 × M) + (2 × SM) + (3 × A) + (4 × T) / 2n samples were excised and pretreated in solution mix- (Eroğlu 2015). ture of 8-hydroxiquinoline (0.002 w.v): colchicine (0.05 Eroğlu (2015) reported the new classification model w.v) contains low concentration of dimethyl sulfoxide from full symmetric to full asymmetric with five differ- (DMSO) at about 4° C for 30 minutes on shaker 30 rpm; ent types. The chromosomes, are all metacentric, form and were washed in hypotonic solution (NaCl 0.9% + KCl full symmetric karyotype; unlike those, are all telocen- 0.042% + CaCl2 0.025% in distilled water) for 3 times. tric, form full asymmetric karyotype. Then they were Fix in freshly prepared Carnoy`s fixative (6:3:1 - ethanol: chloroform: acetic acid) for 20-30 min- utes. They were Transferred