Introduction

Family Aeshnidae (Anisoptera) includes large and vigorous dragonflies known as hawkers or darners or aeshnids. Aeshnids (Greek: ‘aeschna’ means ‘ugly’) are homogeneous in shape and their markings are nonmetallic with variable colors.  Taxonomically, family Aeshnidae includes 54 genera, 480 species all over world, while 13 genera representing 49 species are available in India (Subramanian and Babu, 2017). Cytogenetic data pertaining to 59 species under the genera Aeshna, Anaciaeschna, Anax, Andaeschna, Austroaeschna, Basiaeschna, Boyeria, Caliaeschna, Castoraeschna, Cephalaeschna, Coryphaeschna, Gynacantha, Gynacanthaeschna, Oplonaeschna, Planaeschna, Remartinia, Rhionaeschna and Staurophlebia have been reviewed which also includes 11 species from India (Table 2). Presently, cytogenetic investigations on Anax ephippiger, Anax immaculifrons, Anax indicus, Anax nigrofasciatus nigrolineatus, Anax parthenope and Gynacantha subinterrupta by carbol fuchsin staining and C – banding have been attempted. All the species posses 2n = 27m with X0 – XX sex determination except Anax ephippiger with 2n = 14 + neo XY sex determination. Distribution of C – heterochromatin has been observed and compared among the species. Chromosome complement of Anax indicus (Lieftinck, 1942) and C – banding on Anax ephippiger (Burmeister, 1839) and Anax indicus (Lieftinck, 1942) has been analysed for the first time. List of cytologically studied species of family Aeshnidae has been updated to 60 species.

Materials and methods

Male specimens of Anax ephippiger, Anax immaculifrons, Anax indicus, Anax nigrofasciatus nigrolineatus, Anax parthenope and Gynacantha subinterrupta were captured from different localities from India (Table 1). Alive specimens were dissected in 0.67 % saline solution (Sodium chloride in distilled water) in the field and testes were removed from the abdomen. Subsequently, the testes were put in sodium citrate (0.9 %) for 45 minutes and then fixed in freshly prepared Carnoy’s fixative (3 parts absolute alcohol : 1 part glacial acetic acid) and tapped on grease – free slides. Slides were proceeded for carbol fuchsin staining (Carr and Walker, 1961) and C – banding (Sumner, 1972). Relevant meiotic stages were micro- photographed for further cytogenetical investigations.

Table: 1: Collection details of species of family Aeshnidae.

Results

Carbol fuchsin staining

Chromosome complement of Anax immaculifrons (Fig. 1c), Anax indicus (Fig. 1e), Anax nigrofasciatus nigrolineatus (Fig. 1g), Anax parthenope (Fig. 2a), Gynacantha subinterrupta (Fig. 2c) show 2n (♂) = 27 (24A+2m+X0), while the only exception is Anax ephippiger with 2n (♂) = 14 (10A + 2m + neo – XY) resulted by the 13 simultaneous fusions in the complement (Fig. 1a). Moreover, variation in the size of sex chromosome and m chromosomes is observed. X chromosome is 2^nd smallest in Anax nigrofasciatus nigrolineatus, Gynacantha subinterrupta, while it is medium sized in Anax immaculifrons, Anax parthenope and is of large sized in Anax indicus and Anax ephippiger (neo – XY). The size of m bivalent is slightly smaller than X chromosome in Anax parthenope (Fig. 2a), minute in Anax immaculifrons (Fig. 1c), Anax indicus (Fig. 1e), Anax nigrofasciatus nigrolineatus (Fig. 1g), Gynacantha subinterrupta (Fig. 2c) and small in Anax ephippiger (Fig. 1a) (Table 3).

C banding

During diplotene, in Anax ephippiger, cross shaped autosomal bivalent are showing the terminal, sub – terminal and interstitial C – bands, m bivalent possesses less amount of C – heterochromatin, while neo – XY bivalent is cross shaped and showing interspersed C – bands in (Fig. 1b). Similarly, in Anax parthenope, bivalents show terminal and interstitial C – bands, while m bivalent possesses less amount of C – heterochromatin and X chromosome reveals terminal C – bands (Fig. 2b). During the diakinesis, Anax indicus (Fig. 1f), Anax nigrofasciatus nigrolineatus (Fig. 1h) and Gynacantha subinterrupta (Fig. 2d) and metaphase – I, Anax immaculifrons (Fig. 1d) autosomal bivalents possess terminal C – bands, while m bivalent is C – negative  except in Anax nigrofasciatus nigrolineatus (Fig. 1h) with less amount of C – heterochromatin and X chromosome is entirely C – positive in all the species (Table 4).

Figure 1: Anax ephippiger Normal complement 1a. Diakinesis, C – banding 1b Diplotene. Anax immaculifrons Normal complement 1c. Diakinesis, C – banding 1d Metaphase – I. Anax indicus Normal complement Click here to view figure

Figure 2: Anax parthenope Normal complement 2a. Diakinesis, C – banding 2b Diakinesis. Gynacantha subinterrupta Normal complement 2c. Diakinesis, C – banding 2d. Diplotene.  Click here to view figure

Discussion

Cytogenetic data pertaining to 60 species (including Anax indicus of present study) of family Aeshnidae have been reviewed. Chromosome number in males varies from 2n = 14 – 27, resulted by the fusion of chromosomes (Fig. 3). The species are differentiated on the basis of chromosome numbers as 2n = 14, 15, 19, 21, 23, 24 (1species each); 2n = 16 (2 species each); 2n = 25 (4 species each); 2n = 26 (4 species each) and 2n = 27 (44 species). Most frequent chromosome number is 2n = 27 which is present in 73.3% of the species and considered as the type number of the family (Table 2).

Table 2: List of cytogenetically examined species of the family Aeshnidae. Nomenclature is based on ‘World Odonata List’ by Paulson et al. (2022).

Kiauta (1967a – d) discussed the evolution of chromosome number in odonate species. He considered n = 9 as ancestral chromosome number and divided dragonflies’ chromosomes into two groups, one with high – n complements (n = 9 to 15) and second with low – n complements (n = 3 to 7). He explained that size of chromosomes of high – n species is smaller than the size of low – n species. He found the low – n complement in tropical species, while high – n complement in temperate region species based on geographical distribution. He also explained that in Odonata breaks lead to haploid numbers 10 to 15 and fusions lead to haploid numbers 3 to 8 from the ancestral chromosome number n = 9.

During the present study, all the species of family Aeshnidae show 2n = 27, (26A+X) which is the type number of the family as earlier reported (Table – 2). The only exception is Anax ephippiger with 2n = 14, (10A+2m+neo – XY) which originated by the 13 successful fusions between autosomes and autosome with sex chromosome. The complement of the species is stable because same complement in the species has been reported by Seshachar and Bagga (1962).

 Micro chromosomes and their size

Micro chromosomes (m) are considered as cytogenetic marker of the order Odonata. Absence or presence of m chromosomes depicts the taxonomic status of a species. McGill (1904) observed the presence of m chromosomes in chromosome complement of Anax junius for the first time in the family Aeshnidae. Later, Oguma (1930) proposed the “m chromosome theory” and considered m chromosome as an autosome which undergoes gradual diminution in volume until they disappear. Later, Dasgupta (1957) and Cumming (1964) supported the theory, but Kiauta (1968a) discarded the theory and considered m chromosomes as fragment of autosome which is present in 80% of the odonate species. He further explained that accidental breaks can occur at any time in the holocentric chromosomes which is responsible for the variation in the size of m chromosomes.

In the family Aeshnidae, out of 60 cytogenetically studied species, m chromosomes are present in 45 species, while they are absent in 15 species. Presently, all the 6 species of the family show the presence of m chromosomes in the complement (Table 2). Variation in the size of m chromosomes can serve as the identifying feature of odonate species and to differentiate closely related species of the same genus. Presently, variation in the size of m chromosomes has been recorded. Size of m bivalent is slightly smaller than X chromosome in Anax parthenope (Fig. 2a), minute in Anax immaculifrons (Fig. 1c), Anax indicus (Fig. 1e) Anax nigrofasciatus nigrolineatus (Fig. 1g), Gynacantha subinterrupta (Fig. 2c) and small in Anax ephippiger (Fig. 1a) (Table – 3).

Size and behaviour of sex chromosomes

In Odonata, majority of the species possess the XX(♀)/X0(♂) sex determining mechanism (Fig. 4). In the family Aeshnidae, Out of 60 species, 50 species possess XX(♀)/X0(♂) sex mechanism, while 10 species show neo – XY sex determining mechanism, originated by the fusion of sex chromosome with an autosome (Fig. 4). 16.6% species possess neo – XY sex determining mechanism which is very high in the family Aeshnidae as compared to other families of the order.

Figure 4: Sex determining mechanism in species of family Aeshnidae. Click here to view figure

During the present study, Anax immaculifrons (Fig. 1c), Anax indicus (Fig. 1e), Anax nigrofasciatus nigrolineatus (Fig. 1g), Anax parthenope (Fig. 2a) and Gynacantha subinterrupta (Fig. 2c) show XX/X0 sex mechanism, while Anax ephippiger possesses neo – XY (Fig. 1a) as earlier reported (Seshachar and Bagga, 1962). They observed largest X chromosome and medium sized Y chromosome in the chromosome complement and explained that almost 13 centric fusions occurred in Hemianax ephippiger, which decreased the chromosome number from 27 to 14 and one fusion occurred between X chromosome and an autosome. Similar results have been found in the species. Schematic presentations for the evolution of chromosome number and sex determining mechanism in Anax ephippiger (Burmeister, 1839): 2n = 14, (10A+2m+neo – XY) is established (Fig. 5).

Figure 5: Schematic presentations for the evolution of chromosome number and sex determining mechanism in Anax ephippiger Click here to view figure

Size of the X chromosome is peculiar feature of the species and is variable in different species. Majority of the researcher are silent as to the size of X chromosome, but few reports have recorded the size of X chromosome as smallest element in Anax amazili (Mola et al., 1999) and in Aeshna nigroflava (Pereplov and Bugrov, 2002), while as second largest element  in Aeshna crenata (Pereplov and Bugrov, 2002). Presently, X chromosome is 2^nd smallest in the complement of Anax nigrofasciatus nigrolineatus, Gynacantha subinterrupta, while it is medium sized in Anax immaculifrons, Anax parthenope and large sized in Anax indicus and neo – XY of Anax ephippiger (Table 3).

Table 3: Morphological characterization of chromosome complements in the species of families Aeshnidae.

C – banding

In the family Aeshnidae, C – banding has been reported on 11 species (Thomas and Prasad, 1986; Perepelov et al., 1998; Perepelov and Bugrov, 2002; Nokkala et al., 2002; Walia et al., 2016, 2018; Walia and Somal, 2019). They found terminal C – bands on autosomal bivalents and X chromosome is mostly C – positive. Presently, C – banding on 6 species of family Aeshnidae have been under taken. C – bands are mostly present at the terminal regions, while amount of C – heterochromatin varies in the species. Moreover, distribution of C – heterochromatin in m bivalent and X chromosome shows variations. The m bivalent is C – negative in Anax immaculifrons, Anax indicus and Gynacantha subinterrupta, while possesses less amount of C – heterochromatin in Anax ephippiger, Anax nigrofasciatus nigrolineatus and Anax parthenope. On the other hand, X chromosome is C – positive in Anax immaculifrons, Anax nigrofasciatus nigrolineatus, Anax indicus and Gynacantha subinterrupta, while shows terminal C – bands in Anax parthenope.  Anax ephippiger and Anax parthenope possess different C – banding pattern as C – bands are present on the sub-terminal and interstitial regions of autosomal bivalents, while cross shaped neo – XY bivalent in Anax ephippiger shows interspersed C – bands (Table 4).

Table 4: Distribution of C – heterochromatin in the species of family Aeshnidae.

Presence of C-heterochromatin on the terminal regions is due to the localization of centromeric activity at the terminal regions of the bivalents which is necessary for the segregation of chromosomes during division and is peculiar feature of holocentric chromosomes present in Odonata and in other insect groups. Cytogenetic analysis on Anax indicus (Lieftinck, 1942) has been attempted for the first time and C – banding of Anax ephippiger (Burmeister, 1839) and Anax indicus (Lieftinck, 1942) has been studied for the first time. List of cytologically studied species of family Aeshnidae has been updated to 60 species. 

Conclusion

Chromosome complement and C – banding of six species of family Aeshnidae have been done and list of cytologically studied species of family has been updated to 60 species. All species have 2n = 27m with X0 – XX sex determination except Anax ephippiger with 2n = 14 + neo XY resulted by the 13 simultaneous fusions between the autosomes and  autosome with sex chromosome. C-heterochromatin distribution has been compared among the species. C – bands are primarily seen at the terminal regions of autosomal bivalents, while Anax ephippiger and Anax parthenope also have C – bands in the interstitial and sub-terminal sections of the bivalents. Additionally, the distribution of C-heterochromatin for sex chromosome and m bivalent varies in the species.

Acknowledgements

We thankful to the Department of Zoology and Environmental Sciences, Punjabi University, Patiala for providing all the lab facilities, to UGC, New Delhi for financial support.

Conflict of Interest

There are no conflict of interest.

Funding Sources

There is no funding source.

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