Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 73(3): 65-70, 2020 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.13128/caryologia-203 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: F. Farsi, H.E. Eroğlu, J. Nozari, V. Hosseininaveh (2020) Karyo- type analysis of Trichogramma embry- ophagum Htg. (Hymenoptera: Tricho- grammatidae) using a new method and estimate its karyotype symmetry. Caryologia 73(3): 65-70. doi: 10.13128/ caryologia-203 Received: April 03, 2019 Accepted: June 02, 2020 Published: December 31, 2020 Copyright: © 2020 F. Farsi, H.E. Eroğlu, J. Nozari, V. Hosseininaveh. This is an open access, peer-reviewed article published by Firenze University Press (http://www.fupress.com/caryologia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distri- bution, and reproduction in any medi- um, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. Karyotype analysis of Trichogramma embryophagum Htg. (Hymenoptera: Trichogrammatidae) using a new method and estimate its karyotype symmetry Fatemeh Farsi1, Halil Erhan Eroğlu2, Jamasb Nozari1,*, Vahid Hossein- inaveh1 1 Department of Plant Protection, College of Agriculture and Natural Resources, Univer- sity of Tehran, Karaj, Iran 2 Yozgat Bozok University, Faculty of Science and Art, Department of Biology, Yozgat, Turkey * Corresponding author. E-mail: nozari@ut.ac.ir Abstract. Different methods of chromosome preparation for insect are now being used across the world. Well-spread chromosomes with explicit morphology, in addition to no cell wall debris are required for karyotype investigations. Cytogenetic knowledge in Trichogramma is extremely limited. In the article, chromosome characters, karyo- type and monoploid ideogram of Trichogramma embryophagum Htg. (Hymenoptera: Trichogrammatidae) were evaluated using a new method. Also, karyotype symmetry/ 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 = 10. The karyotype formula was 6m + 4a. The symmetry/asymmetry index value was 1.8. The new method resulted in higher quality metaphase plates spread and provided an ideal karyomorphology for this parasitoid which has small chromosomes. 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- 66 Fatemeh Farsi, Halil Erhan Eroğlu, Jamasb Nozari, Vahid Hosseininaveh rescent microscope), requiring highly-skilled cytogeneti- cists, and the inability to reveal chromosome inversions (Di- Nizo et al. 2017) have caused cytogenetics to show a significant tendency towards using classical methods. Squash is a well-known and oldest method for flat- tening metaphase plate in insects (Kocak and Okutaner 2017). However, difficulty in achieving well-spread, dis- torted, and stretched chromosome are the main dis- advantages of this method (Chirino et al. 2014; Kocak and Okutaner 2017). During squashing, producing well- spread plates are often not desirable due to the concen- tration of cells in a small area. Over time, spread (hot- plate spread) method conducted by various researchers flourished (Bressa et al. 2009; Sadilek et al. 2016). Its major disadvantage is the attendance of cellular debris around the chromosomes (Chirino et al. 2014), making their morphology unpleasant. Considering the men- tioned constraints, there is always a need for a simple and efficient method for the preparation of metaphase plates in parasitoids. Despite the vast amount of research on insect kar- yotype, only about 500 species of parasitoid wasps have available data (Gokhman 2009). Among these, only more than 10 are related to Trichogramma. So far, 239 species of Trichogramma have been reported through- out the world (Khan and Yousuf 2017). There are eleven species of this parasitoid in Iran (Nazeri et al. 2015) that Trichogramma embryophagum Htg. is one of them. T. embryophagum distributed in some of regions of Iran, including the provinces of West and East Azerbaijan, Yazd, Khorasan (Poorjavad et al. 2011). Cytogenetic studies and karyotype investigation have not yet been done on this species. Because of the substantial posi- tion that Trichogramma occupy in the biological control programs (Parra 2009), investigations on their chromo- some details have provided precious information for the understanding various perspectives such as mechanisms of sex determination, evaluation of sex chromosome, existence of accessory chromosomes and phylogenetic relationships. An evaluation of existing literature shows that dif- ferent methods was used in karyotype studies of Tricho- gramma. Studies about Trichogramma were derived from routine chromosomal preparation methods, including the use of various percentages of colchicine as pretreat- ment, staining by different colors, and then squashed (Hung 1982; Liu and Xiong 1998). Interestingly, no banding staining on the Trichogramma species has ever been reported. Molecular cytogenetic including Flores- cence in situ hybridization (FISH) has been applied for only the two species of T. kaykai Pinto & Stoutham- er (van Vugt et al. 2005) and T. pretiosum Riley (Gokhman et al. 2017). It should be noted that the prob- lems described for each of these methods also exist for Trichogramma karyotype studies. The minute size and lifestyle of Trichogramma species also intensify these problems (Manickavasagam 1991; Hung 1982). In most studies, only the haploid number of these species has been raised, and the chromosomal details including the length of the arms that could provide many comparisons were not possible (Hung 1982; Laurent et al. 1998). One parameter that can be obtained from the chromosomal information is karyotype symmetry/asymmetry (Peruzzi and Eroğlu 2013). Karyotype symmetry/asymmetry is obtained based on the availability of chromosome detail, including relative chromosome size and position of cen- tromere. Until now, this parameter which can help in the evaluation of enter- species relationship seldom has calculated in class of insect. In the cytogenetic studies, species that are more similar in the terms of chromo- somal parameters such as karyotype symmetry/asymme- try will have more affinity. In this research, the karyotype, ideogram, and chro- mosomal detail of T. embryophagum were investigated for the first time with the introduction of a new method considering all steps of Hymenoptera parasitoids chro- mosome preparation. These chromosome analyses were complemented by an estimation of its karyotype symme- try as one of the species of Trichogrammatidae. MATERIAL AND METHOD Insect T. embryophagum Htg. individuals originated from parasitized eggs of carob moth, Ectomyelois ceratoniae, (Zeller) (Lepidoptera: Pyralidae), were collected on pome- granate in the center of Iran (Yazd Region, 32.1006° N, 54.4342° E). The wasps were reared on Ephestia kuehniel- la Zeller (Lepidoptera; Pyralidae) eggs in a climate-con- trolled chamber at 25 ± 1 °C, 70 ± 5% relative humidity, L16: D8 photoperiod. E. kuehniella eggs were obtained from a culture maintained in the Biosystematics Labora- tory at The University of Tehran, Karaj, Iran. Preparation chromosome Sample from egg host were Dissected out in physi- ological solution for Ephestia (Glaser, 1917, cited by Lockwood 1961). Two kinds of pre-treatment were used, including hypotonic and other is combined it. First, sam- ples were transferred into a drop of 0.075 M KCl for 8 minutes [0.075 M KCl: 0.5592 g KCl/100 ml redistill. 67Karyotype analysis of Trichogramma embryophagum H2O] on a shaker at 20 °C and 30 rpm. After wise, the samples were excised and pretreated in solution mix- ture of 8-hydroxiquinoline (0.002 w.v): colchicine (0.05 w.v) contains low concentration of dimethyl sulfoxide (DMSO) at about 4° C for 30 minutes on shaker 30 rpm; and were washed in hypotonic solution (NaCl 0.9% + KCl 0.042% + CaCl2 0.025% in distilled water) for 3 times. Then they were Fix in freshly prepared Carnoy s̀ fixative (6:3:1 - ethanol: chloroform: acetic acid) for 20-30 min- utes. They were Transferred on a clean slide into a drop (5-10μl) of 60% acetic acid and their head was cut off from other parts of the body with tungsten needles, after a while, 5-10μl of 60 % acetic acid was added again. The slides were put on a heating plate at 45 °C until the ace- tic acid almost evaporates. Water was removed bypassing the slides through an ethanol series (70 %, 80 %, 96 %; for 30 seconds each of it, respectively) and the samples were let air-dried. The slides were stained immediately with 5 % Giemsa in phosphate buffer (pH 6.8). Microscopic photograph and analysis The chromosome slides were examined under Olym- pus BX53 microscope and chromosomes were photo- graphed using an Olympus DP72 camera. Chromosome measurements were made on 10 metaphase plates by application of KaryoType software (Altınordu et al. 2016). Arm rations, average lengths, and centromeric index were calculated and then were classified according to Levan et al. (1964) considering their centromere position. The karyotype symmetry/asymmetry was calculated. The formula for symmetry/asymmetry index is given below. S/AI = (1 × M) + (2 × SM) + (3 × A) + (4 × T) / 2n (Eroğlu 2015). Eroğlu (2015) reported the new classification model from full symmetric to full asymmetric with five differ- ent types. The chromosomes, are all metacentric, form full symmetric karyotype; unlike those, are all telocen- tric, form full asymmetric karyotype. RESULT The karyotype of T. embryophagum Htg. contained three pairs of large metacentric and two considerably smaller pairs of acrocentric chromosomes. Two of the metacentric chromosomes were of similar size whereas the third pair was close to submetacentric. Thus, the diploid chromosome number was 2n = 10, and the kar- yotype formula was 6m + 4a (Figure 1a, b). The detailed chromosomal data are given in Table 1, and monoploid ideogram is given in Figure 2. In T. embryophagum, haploid chromosome length and mean chromosome length are 6.38 and 1.28 μm. The rates of relative length and centromeric index range from 9.40 to 28.06 and from 18.33 to 48.04, respectively. The symmetry/asymmetry index value is 1.8. DISCUSSION In this research, the main issues were to have a regular cell layer without overlapping and the absence of qualitative damages to the morphology of chromo- somes while allowing the possible spread of chromo- Figure 1. a, b) Mitotic metaphase plate of T. embryophagum. Giemsa staining. Magnification 100x. 68 Fatemeh Farsi, Halil Erhan Eroğlu, Jamasb Nozari, Vahid Hosseininaveh somes within the cell. Th e method explained here has aimed at achieving both goals. Th e chromosome struc- ture had a high resolution, which is related to how the chromosomes was prepared. Th e process of karyotyping is completed through some steps such as pretreatment, fi xation and staining. On both sides of the cell, outside and inside, the concentration of water is the same, and Potassium and sodium ions are the most osmotically solutions inside and outside of the cell, respectively (Leaf 1973). In this case, the cell is in an isotonic state. Th e KCl changed the osmosis of the cell. Pre-treatment by KCl aff ects both cellular swelling and better chromo- somes spread. About KCl, hypotonic rate and duration of treatment are important. Rupturing the cell mem- brane and the damages of chromosome (Earley 1975) can be caused by the lack of attention to these cases. Our result indicated that the hypotonic rate and dura- tion of KCl reported here overcome protoplast damage. Sadilek et al. (2016) stated that KCl causes the osmosis of the cell due to receiving additional water that result- ed in a larger cell. Th is process eff ects more identifi ed of the chromosome. Th e next step aft er KCl was using combined pretreatment. Colchicine is a substance that aff ects the metaphase stage of the dividing cell. Guo et al. (2018) stated that colchicine increased the yield of the metaphase plate. Th e 8-hydroxyquinoline is eff ec- tive in prolonging the metaphase and further compres- sion of the chromosomes. It is noteworthy that, until the present time, this substance has not been used as pre-treatment in insect karyotype studies but has been used extensively in plant karyotype. This substance can maintain the shape of the chromosome during the preparation process. Th is feature has been used in some plant studies (Fernandez et al. 2009; Zarifi and Güloğlu 2016). We found that 8-hydroxyquinoline pre- vented the distortion and stretch of the chromosome, which are the main drawbacks of squash and spreading methods. Use of colchicine alone showed each chromo- some clumped exceedingly and their centromeres were not distinctly in our pre-tests while using 8-hydrox- yquinoline alone, the metaphase plates were few. As a result, a combination of two substances as pretreat- ment improved chromosome spreading. Ma et al. (1996) reported that the effects of these materials together make this as improvement whereas colchicine depo- lymerized microtubules, 8-hydroxyquinoline decreased the rate of progression among mitotic stages and also resulted in a disorderliness in chromosome movement. In combined pretreatment, dimethyl sulfoxide (DMSO) aff ected the better penetration of compounds of pre- treatment into the cell. DMSO has been reported as an eff ective penetration enhancer (Gurtovenko and Anwar 2007; Williams and Barry 2012). Notman et al. (2006) stated that DMSO induces pores in the membrane. Another study indicated that DMSO concentration is critical (Gurtovenko and Anwar 2007). We used a low concentration of DMSO in our method. In this concen- tration, DMSO induces membrane thinning (Gurtoven- ko and Anwar 2007).Th e approach used in the present study focused on providing a quick and easy method for those insects which have small chromosomes, especially Trichogramma wasps. Th e advantages of the new meth- od are as follows: 1. In the present method, the chromosomes are readily accessible through morphological features, including the centromere position. According to this, the evaluation of all of the parameters such as karyotype symmetry/asymmetry can be available. 2. Higher quality metaphase plates spread which is achieved through the use of a combined pre-treatment which its results are an ideal karyomorphology. Table 1. Th e detailed measurement data of chromosome pairs of Trichogramma embryophagum. Pair L (μm) S (μm) L + S (μm) RL (%) L / S CI (%) Type 1 0.93 0.86 1.79 28.06 1.08 48.04 m 2 0.91 0.73 1.64 25.71 1.25 44.51 m 3 1.02 0.61 1.63 25.55 1.67 37.42 m 4 0.58 0.14 0.72 11.29 4.14 19.44 a 5 0.49 0.11 0.60 9.40 4.45 18.33 a Abbreviations: long arm length (L), short arm length (S), total chro- mosome length (L + S), relative length (RL), arm ratio (L/S), cen- tromeric index (CI), metacentric (m), acrocentric (a). Figure 2. Ideogram of T. embryophagum. Chromosomes are num- bered from the longest (1) to shortest (5). 69Karyotype analysis of Trichogramma embryophagum 3. Providing a single layer of cell and decreasing the overlapping cells, therefore, a better selection of meta- phase plates is possible. The Trichogrammatidae species were less likely to be karyotypically studies in comparison with other parasi- toid families. The karyotype T. embryophagum Htg. con- sists of 10 chromosomes (n= 5, 2n = 10). Members of this family showed 10 chromosomes previously published (Hung 1982; Van Vugt et al. 2003; Gokhman et al. 2017), although there is an exception, T. kaykai, which has been diagnosed with one B chromosome that is termed psr (from Paternal Sex Ratio) (Stouthamer et al. 2001). The metaphase images indicated that chromosomes are metacentric (first, second and third chromosome pairs) and acrocentric (fourth and fifth chromosome pairs). According to these chromosome types, the S/AI value is 1.8, and the karyotype is symmetric type in T. embry- ophagum. Karyotype symmetry/asymmetry is one of the cheapest and most popular parameters that can be obtained from cytogenetic studies (Peruzzi and Eroğlu 2013). The symmetric karyotype is characterized by an excess of metacentric, submetacentric chromosomes. In T. embryophagum due to two pairs of acrocentric chro- mosomes, S/AI value is close to 2.0 (between symmet- ric and asymmetric). Gokhman (2009) expressed that two trends have occurred in evolution of karyotype in parasite Hymenoptera. First one is decreasing in chro- mosome number, and the other one is karyotype dis- symmetrisation, which happened as a result of the pro- portion acrocentric in a chromosome set. Despite the widespread use of various methods that are adopted to calculate the symmetry/asymmetry karyotype in plants, this parameter has not been taken into consideration in other organisms (Eroğlu 2015). So far, karyotype sym- metry/asymmetry has been seldom calculated in the class of insects and other species of parasitoids. Our result showed for the first time that in one of the spe- cies of Trichogramma wasps, the karyotype is symmetric type according to a new method. Karyotype symmetry/ asymmetry is applied on one hand to determination evolutionary relationship, and on the other hand, to compare different levels of the taxonomy (Eroğlu 2015). In the case of Trichogrammatidae, information obtained due to the lack of other information’s in this case is not possible to assess the issue mentioned above in this time but our results can be used as a basis for future studies in the above fields. In conclusion, the described method is cost-effec- tive and technically easier to make for the resolution of chromosome details. The morphological characteristics of each chromosome are better observed. Also, no spe- cial equipment is required as compared to molecular methods. According to the described method prepara- tion of chromosome detail, karyotype and also ideogram of T. embryophagum were provided for the first time. We adduce as other species of Trichogramma are also evalu- ated in which the diploid number is 2n = 10. The karyo- type is a symmetric type. 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