The X chromosome still has a lot to reveal – revisiting Hermann Henking's work on firebugs

In the second half of the 19th century, biologists used newly formulated synthetic dyes to stain fixed specimens obtained from many different organisms. As an example, the staining of salamander embryonic tissues by Walther Flemming in 1879 revealed the characteristic chromosome positions during the stages of mitosis (Flemming, 1879). With the dyes Flemming used, brightly stained colored bodies were observed and later called ‘chromosomes’. In 1876, Oskar Hertwig then showed fertilization and some illustrations of oocyte meiosis in the sea urchin (Hertwig, 1876), while fertilization and oocyte meiosis in the roundworm Ascaris was described by Édouard Van Beneden in 1883 (Van Beneden, 1883). As early as 1887, Friedrich Leopold August Weismann noted that the aim of the two meiotic divisions in both male and female germ tissue is to divide the chromosome number by two (Weismann, 1887). In the same year, it was Walther Flemming again who observed meiosis in salamander spermatocytes and also noted differences between the two cell divisions (Flemming, 1887). In all of the systems mentioned above, there were no chromosomes with obvious outlier behavior from all of the others, and there were no readily apparent differences in chromosome number or structure between sexes.

Hermann Henking then explored a new system in 1891, the firebug Pyrrhocoris apterus; he prepared fixed, stained samples of firebug testes and observed a very obvious difference in the behavior of one chromosome compared to the rest, calling that chromosome the ‘Element x’ (Henking, 1891). He realized that the X element (later called the X chromosome) of Pyrrhocoris is the largest chromosome in the cell, and is easy to follow in both meiotic divisions. Henking thoroughly described his observations of the X element (Fig. 1A), not knowing that his work was critical to explaining sex determination (Henking, 1891).

A few years later, Clarence Erwin McClung observed a similar chromosomal behavior in Xiphidium fasciatum (Conocephalus fasciatus), the slender meadow katydid (McClung, 1899). He subsequently postulated that this X element, which he now termed the ‘accessory chromosome’, is important for determining the sex of an individual (McClung, 1901, 1902). Meiosis in Pyrrhocoris was further detailed first by Julius Gross (Gross, 1907), and then by Edmund B. Wilson (Wilson, 1909), who, at that point, knew that the X chromosome was associated with sex determination. Both Gross and Wilson recapitulated the results of Henking, reporting the distinctive behavior of the X chromosome. Wilson also made note of the fact that the X chromosome was a univalent chromosome surrounded by bivalent autosomes, and he also described the distinctive condensation patterns of the X chromosome throughout meiosis (Wilson, 1909). Only much later, were firebugs studied cytogenetically (Messthaler and Traut, 1975; Grozeva et al., 2011), but little new information about the behavior of the X chromosome has been reported beyond what we describe below.

Henking, Gross and Wilson all analyzed multiple fixed and stained testes content specimens of P. apterus (Henking, 1891; Gross, 1907; Wilson, 1909). In metaphase I, they observed that the univalent X chromosome is aligned with all of the autosomal bivalents. In anaphase I, the sister chromatids of the X chromosome separate from one another, while all of the autosomes separate their homologs and maintain connected sister chromatids. In metaphase II, the single X chromatid again aligns with all of the autosomes on the metaphase plate (Fig. 1Ai). In anaphase II, however, when all of the autosomal sister chromatids separate from one another, the single X chromatid lags at the spindle midzone, apparently stretching and eventually moving towards one of the two spindle poles (Fig. 1Aii–Aiv). Interestingly, the bug Protenor belfragei (Schrader, 1935) and the nematode Auanema rhodensis (Shakes et al., 2011; Winter et al., 2017) show exactly the same X chromosome behavior in male meiosis (Fig. 1B). In another nematode system, Caenorhabditis elegans, as well as in many hemipteran insects of the suborder Auchenorrhyncha, a lagging of the X chromosome is also observed. In these species, however, lagging occurs in anaphase of the first meiotic division (Fig. 1C), thus illustrating the fact that X chromosome segregation can follow different patterns (Fabig et al., 2016; Felt et al., 2017, 2022).

The lagging X chromosome in P. apterus remains interesting, as it has the potential to contribute to our knowledge about general chromosome behavior with possible links to human health. In particular, both the development of univalent chromosomes and the separation of sister chromatids in meiosis I are linked to aneuploidy and congenital developmental delay in humans. Characterizing the behaviors of a univalent X chromosome might offer insight into the behavior of pathological univalents and the mechanisms of early sister chromatid separation in human meiosis. Along this line, merotelic attachments (i.e. situations in which single chromatids attach to microtubules coming from both spindle poles) experience a lag in anaphase separation (Cimini, 2007). This is also true for the P. apterus X chromatid, which appears to form a merotelic attachment every meiosis II, and then lags in anaphase II (Henking, 1891; Wilson, 1909). Thus, a closer observation and a manipulation of this interesting chromosome could expand our understanding of the development of aneuploidy and the process of error correction. In the firebug P. apterus, it will be possible to observe the attachment, re-orientation and subsequent anaphase movements of a large merotelic chromosome. Such observations should illuminate the chromosome behaviors that can lead to human health conditions like developmental delay or cancer, which can be caused by or exacerbated by aneuploidy.

The P. apterus X chromosome can also offer insight into the behavior of holocentric chromosomes (i.e. chromosomes which have spindle-attachment sites along their entire length, such as in the above-mentioned nematode C. elegans (Albertson and Thomson, 1993, Oegema et al., 2001). Insects of the order Hemiptera, to which P. apterus belongs, have holocentric chromosomes in mitosis (Melters et al., 2012). Interestingly, as observed both by Henking and Wilson, P. apterus has a particularly long X chromosome (Henking, 1891; Wilson, 1909). The large size of this chromosome with its extended kinetochore region along the length of this chromosome offers an opportunity to understand how long chromosomes with large attachment sites form correct attachments to the spindle in both meiosis and mitosis. Using P. apterus as a model system, would also allow researchers to determine how errors are corrected in living cells. This could be followed by applying phase-contrast microscopy without the need of imaging tagged cells, as required in nematode C. elegans (Fabig et al., 2020).

The P. apterus univalent X chromosome also offers insights into evolutionary aspects as well. Important outstanding questions in this regard are how do chromosomes without a pairing partner behave when surrounded by chromosomes that have a pairing partner and what characteristics of cells lead to particular univalent behaviors. In organisms with heteromorphic sex chromosomes, the chromosome that only appears in the heterogametic sex often degenerates and could potentially be lost (Steinemann and Steinemann, 2005). Studying univalent X chromosome behavior might give insight into our own distant evolutionary future, after the possible loss of the human Y chromosome.

All conclusions drawn about the behavior of the Pyrrhocoris apterus X chromosome that we describe above were obtained in fixed and stained specimens. The fixation methods used (typically a solution of methanol or ethanol and acetic acid) might not reveal chromosome position or status of chromosomes as they occur in living cells or could alternatively exaggerate behaviors that are subtle in living cells. To truly understand and manipulate the behavior of this fascinating univalent X chromosome throughout meiosis and mitosis, one must observe cell division in living cells. Important for this, P. apterus is abundant outdoors during warm weather. This insect can also be raised in a laboratory environment for many generations. The testes, in which meiosis and mitosis occur, can easily be located and their contents spread to form primary cultures, and the cells in those primary cultures are easily observed through phase-contrast or DIC microscopy. Such live-cell observations will not simply capture fixed moments in time, but will reveal the continuum of chromosome movements through division. Moreover, Hemipteran insects of the suborder Heteroptera (to which P. apterus belongs) can be manipulated with molecular techniques like RNAi.

With all of the questions about which the X chromosome of Pyrrhocoris apterus can offer insights, and with the ease of study of the organism and the potential techniques currently available, one wonders why it has taken so long for anyone to return to the study of this interesting chromosome. In the light of current 3D reconstruction and possibilities to quantitatively analyze data on chromosome dynamics and ultrastructure, the X chromosome in P. apterus has a lot to reveal, and it is thus timely to revisit this interesting system.