CYTOLOGY AND GENETICS OF T(9,l l) IN THE GERMAN COCKROACH, AND ITS RELATIONSHIP TO OTHER

CHROMOSOME 9 TRAITS

Department ofEntomology, Virginia Polytechnic Institute and State University, Blacksburg, Virigina, 24061, U . S . A .

A new chromosome translocation in the German cockroach, Blattella germanica, was studied. Cytologically, it involves chromosomes 9 and 11 and was designated T(9, l l ) . The approximate positions of the breakpoints were determined. In chromosome 9, the distance between the breakpoint and Df(9)Pw was measured. Genetically, linkage of T(9.11) with the chromosome 9 marker, ru, was estimated at 1.1 5 0.6%. The new chromosome 9 breakpoint and available linkage data were used to approximate the location of the linkage group on the chromosome. An attempt to correlate map and chromosome distances indicated chromosome 9 probably has a total of at least 50 crossover units.

Crosses between T(9, 11) heterozygotes and wild type showed significant sex differences in oothecal size and percent hatch. In males, the hatch data suggested preferential alternate disjunction. This was confirmed cytologically by examination of metaphase I and prophase I1 cells. In females, the hatch data suggested random disjunction. Similar hatch and cytological data are included for T(9,lO)Pw and T(2,ll)Cu. Intercrosses of T(9 , l l ) were largely sterile, precluding the attempt to test for a viable homozygote.

Introduction The best studied chromosome in the German cockroach, Blattella germanica (L.),

is chromosome 9 in the Cochran and Ross (1969) classification. It is known to carry linkage group 8. The approximate location of breakpoints for a reciprocal translocation, T(9,10)Pw, and a terminal deficiency, Df(9)Pw, as well as the loci for ruby-eye (ru) and notch sternite (st), have been tentatively mapped in the central portion of the chromosome. Based on circumstantial evidence, this is also presumed to be the general area where the centromere is located (Ross and Cochran, 1971).

The discovery of a new translocation, T(9,l l ) , offered the opportunity to advance the study of chromosome 9, as well as to ascertain pertinent cytological and biological characteristics of the new stock. Cytological data showed the T(9, l l ) breakpoint to lie some distance from the prowing breakpoint(s). Thus, it appeared that T(9, l l ) would make possible a first attempt at correlating chromosome distance with genetic map distance in B. germanica. Experimental results accomplishing this objective are reported herein. In addition, data on the frequencies of alternate vs. adjacent disjunc- tion, and their relationship with hatch estimates, are present for T(9, l l ) and two other translocation bearing stocks.

Materials and Methods Cytogenetic: The techniques used for chromosome preparation have been described

previously (Cochran and Ross, 1969; Ross and Cochran, 1971). As in the study of T(9,lO)Pw and T(2,l l)Cu, length measurements of the normal and translocated chromosomes were used to identify the chromosomes involved in T(9,ll) . The translocation was maintained by repeated outcrossing of progeny from translocation- bearing crosses to wild type (Ross and Cochran, 1973).

Data on alternate vs adjacent disjunction were obtained by making counts of cells at metaphase I and prophase 11. For comparative purposes, two other translocations,

Manuscript received May 7, 1974.

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640 D. G . COCHRAN AND M. H . ROSS

T(9,lO)Pw and T(2,11)Cu, were also studied. Ring and zig-zag configurations at metaphase I served to adequately distinguish adjacent and alternate disjunction, respectively (White, 1973). In these particular translocations, remnants of the ring configuration remain bound together as end-to-end associations at prophase I1 (Cochran and Ross, 1969), and can be readily distinguished from cells in which this association is lacking.

Genetic: Linkage data for group 8 (chromosome 9) were obtained by crossing T(9, l l ) heterozygotes to a double stock, homozygous for ru and heterozygous for Df(9)Pw (ru Pwlru +). F, hybrids were backcrossed to rulru. The latter stock was also homozygous for rose-eye (ro). Previous studies suggested the presence of ro would enhance chances of crossingover between ru and ~ f ( 9 ) P w . Backcross progeny were scored for phenotype. All males were examined cytologically for translocation heterozygosity. Estimates of map distances between the chromosome breakpoint and genetic loci are based on these data.

Due to close linkage, most phenotypically wild-type progeny from the backcrosses were doubly heterozygous for ru and T(9, l l ) (+ T/ru +). The females were used to obtain additional linkage data by crossing to ru Pwlru +. This also provided male progeny with the simultaneous presence of the translocation and the deficiency. These individuals were used for study of the lineup of Df(9)Pw in the T(9, l l ) configuration, and to estimate the distance between the twobreakpoints in chromosome 9.

Viability of the translocation homozygote (TIT) was tested by crosses between phenotypically wild-type cockroaches from ru backcrosses (+ Tlru +). If TIT was present, a 3+:1 ru segreation would be expected in the offspring. Lethality should produce a ratio of 2 + : 1 ru.

Egg hatch and mortality: Percent hatch is routinely estimated by dividing the total number of progeny by the number of eggs/ootheca for a given mating series (Ross, 1971, 1972; Ross and Cochran, 1970). This procedure was used in analyzing the data from the first set of backcrosses to rulru. Records were also made of the numbers of dead embryoslootheca. These were used to estimate percent mortality (avg. numbers of dead embryos + avg. eggs/ootheca). Additional mortality estimates were obtained in the course of maintaining T(9,ll) (Ross and Cochran, 1973).

Results Description of T(9 , l l ) : T(9, l l ) was one of approximately two dozen translocation

stocks isolated following an irradiation project (Ross and Cochran, 1973). Length comparisons of paired non-translocated chromosomes with wild type indicated numbers 9 and 11 are involved in the translocation (Table I, lines 1 and 2). This was confirmed by measurements of the translocation configuration (Table I, line 3). The slightly smaller values in the T(9,Il) data are probably due to the restriction placed on the length of chromosome 12 in deciding which cells to measure. The smaller mean value of the T(9,ll) chromosome 12 is reflected in the remaining data.

Figure l a shows an example of this translocation at late pachytene. Fig. Ib is a stylized drawing of the translocation figure indicating the approximate location of the breakpoints as determined from measurement of cells where the translocation figures had small areas of non-alignment. It appears that the breakpoint on chromosome No. 9 is very near the midpoint, while on No. 11 it is nearer one end (Fig. Ib). Based on a graphic interpretation of these data, tentative breakpoints are indicated for No. 9 as 4.6 pm from the left side of Fig. lb , and for No. 1 1 as 6.3 pm from the top of the figure.

Orientation of Df(9)Pw with respect to T(9,ll): Crosses were made between Df(9)Pw and T(9, l l ) to establish their cytogenetic relationship. The results of studying

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CYTOLOGY A N D GENETICS OF THE GERMAN COCKROACH 64 1

cells containing both traits are presented in Fig. lc-f. The Pw trait causes a lack of synapsis in that chromosome arm bearing the deficiency (Fig. lc-e). This allows interpretation of the data as shown in Fig If. The most probable location of the T(9,ll) breakpoint is removed from the Pw loci by about 2.0 pm.

From our earlier work with Pw traits, it was proposed that chromosome 9 is metacentric, while No. 10 is submetacentric (Ross and Cochran, 1971). If so, the T(9,ll) breakpoint on chromosome 9 is near its centromere. This raises the question of how the centromere orients with respect to Fig. l b and f. From the work of Cohen and Roth (1970) on mitotic chromosomes, it appears that of the four longest pairs two are metacentric and two are submetacentric. The longest pair (No. 12) is definitely submetacentric. The assumption that No. 10 is also submetacentric (Ross and Cochran, 1971) means that chromosomes 9 and 11 are metacentric. This would place the NO. 9 centromere between the T(9, l l ) and Df(9)Pw breakpoints (Fig. l b arrow), leaving a very small interstitial region.

Translocation homozygosity: In tests for a viable translocation homozygote (TIT), there was only one productive mating from about 30 pairs. Fourteen progeny (9 + and 5 ru) hatched from an ootheca with 43 eggs. No meaningful test of ru segregation could be made with such minute numbers. Oothecae from the non-productive matings were either filled with dead embryos in various stages of development, or the compartments contained whitish material with large areas of apparent air space in their upper portions. In those eggs showing embryonic development, the embryos were not as fully developed as in corresponding wild type oothecae. Thus, the absence of hatch may be related in part to premature deposition of the oothecae. In view of these results, the likelihood of obtaining a viable TIT from T(9, l l ) seemed remote. Accordingly, further efforts in this direction were abandoned.

Linkage: Only four backcrosses involving both ru and Df(9)Pw were found which also carried T(9,ll) . There was no crossingover between ru and Df(9)Pw. Therefore, data from the male progeny were pooled with those from the simple rulru backcrosses. The results were as follows: 139 T+/+ ru: 1 T ru/+ ru; 2 + +I+ ru: 142 + ru/+ ru. Recombination is estimated at 1.1 + 0.6%. This is largely a measure of crossingover in females, since only 20 of the 284 progeny were from backcrosses of hybrid males. From earlier crossover data for ru with Df(9)Pw, male crossingover averaged about 1.3x higher (Ross and Cochran, 1971). Thus, male crossingover between T(9,ll) and ru should not exceed 1.5%. An average value of 1.3% is used in comparing chromosome and map distances (See below).

Although the absence of crossingover between ru and Pw in backcrosses involving T(9, l l ) prohibited the definitive establishment of locus sequence, the combined genetic and cytogenetic data found here and published previously (Ross and Cochran, 1971) leave little doubt as to the following order: T(9,ll)-ru-Df(Pw). However, a problem in estimating the map distance separating the two chromosome breakpoints arises from variable estimates for linkage of ru with Df(9)Pw (Ross and Cochran, 1971). The largest discrepancy was in backcross data which also included ro. Recombination in the presence of ro averaged 6.8% for males and 4.3% for females. Excluding the ro data gives an overall estimate of 2.0 crossover units between ru and Df(9)Pw. This latter figure appears to be more usual for this portion of the chromosome. Paradoxically, the ro data may provide the best estimate of crossingover for comparison with measure- ments of this segment of the chromosome (See below).

Correlation of map distances with chromosome measurements: Figure 2 is an attempt to correlate the known genetic map with a specific chromosome region. The lower line indicates the placement of the breakpoints determined from chromosome

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CYTOLOGY AND GENETICS OF THE GERMAN COCKROACH 643

measurements. The breakpoint for the deficiency is reasonably precise, while those for the translocations are shown as a line corresponding to the region of nonalignment at

Fig. 1 . a. Photograph of the T(9, l l ) translocation at late pachytene. b. A stylized drawing of same. c . Photograph showing the chromosomal configuration of a double heterozygote T(9.11) and Df(9)Pw. d. A drawing of same. e. The configuration shown in c and d redrawn for clarity. f . A stylized drawing of same to correspond with b.

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644 D. G . COCHRAN AND M . H. ROSS

pachytene plus the calculated median point of this region. The latter was used to place the traits in the genetic map. The upper line represents the genetic map, and the values linkage distances. Linkage data are averages for the sexes, since no statistically significant sex differences in recombination have been found in group 8.

The approximate position of the known linkage map within 3.9-6.4 pm from the left end of chromosome 9 is reasonably certain (Fig. 2). More precise placement of the translocation breakpoints is not possible at present, but presumably they will be within the area of nonalignment. For T(9,lO)Pw this variation may already be somewhat restricted by the placement of Df(9)Pw within this region. New data for linkage group 8 support the previous findings with respect to gene sequences and a total map distance of less than 6 units (Ross, unpublished data). The ru locus is placed slightly closer to T(9, l l ) than T(9,lO)Pw on the basis of total linkage data, even though test crosses of ru with T(9,lO)Pw showed complete linkage (Ross and Cochran, 1967).

The linkage data indicate there is ordinarily little crossingover between T(9, l l ) and Df(9)Pw, ie., about 3.3% (Fig. 2). The correlation of this distance with the chromo- some measurements gives approximately 2.3 map unitslpm. It is questionable whether this provides a reliable measure of the total map distance expected for the entire chromosome (see discussion). If so, the linkage map would consist of a mere 20-25 units. Therefore, a second estimate was made using data in which crossover suppression seemed to be less marked, ie., those noted above for the ru-Df(9)Pw backcrosses which included ro. This correlation gives a total map of about 52 units (ca. 5.5 map unitslpm of chromosome length).

Hatchability and mortality data: Data derived during repeated outcrossing of T(9,ll) heterozygotes to wild type are summarized in Table 11, lines 1 and 2. Similar data were obtained in the backcrosses of T +/+ ru females to rulru males (Table I, line 3). There is close agreement between the two series for T/+ females, but these differed from the results obtained from mating T/+ males. The average number of eggs/ootheca was significantly less for the T/+ males. A second significant difference was that oothecae of T/+ females contained more dead embryos than those from matings of T/+ males. Consequently, mortality estimates differed, i.e., 49% and 42% for females and

Fig. 2. A diagramatic representation of chromosome 9. The upper line represents the genetic map as it is currently understood. The lower line indicates the position of three breakpoints on the chromosome. The numbers above the upper line are genetic map distances. Those below the lower line are chromosome measurement units. Correlations between the two lines are described in the text.

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CYTOLOGY AND GENETICS OF THE GERMAN COCKROACH 645

males, respectively. This could indicate a favoring of metaphase I alternate chromo- some disjunction in T(9, l l ) males. Peculiarly, sex differences in fecundity and mortality tended to equalize the numbers of live progeny. The average live progeny from TI+ male matings was estimated at 24.6 (42.7 less 18. I) , as opposed to 23.7 for the females (46.3 less 22.6).

Most estimates of hatch were obtained indirectly, by subtracting the percent mortality from 100% (Table 11, last col.). However, in the ru back:ross data, it was possible to compare this type of estimate (Table 11, line 3) with a dirzct estimate based on counts of live progeny. These matings averaged 21.4 k 2.2 progenylootheca resulting in 47% hatch as compared to the indirect estimate of 52%. It is probable that hatch estimates from counts of live progeny will generally be slightly lower than those based on numbers of dead embryos/ootheca. The addition of dead embryos and live progeny accounts for 95% of the eggs in the T(9, l l ) backcrosses, and suggests a 5% postembryonic loss. This seems to give a fuller accounting of egg hatch than that indicated by a direct estimate of 90.1% for wild-type matings (Ross and Cochran, 1970).

Directed chromosome disjunction: As mentioned above, hatch data suggested preferential alternate disjunction in T(9,ll) males (Table 11, line 1). To examine this possibility, a large number of metaphase I cells were studied. The ring structure of adjacent disjunction and the zig-zag of alternate disjunction are shown in Fig. 3, a and b, respectively. A distinction was also made at prophase I1 by using end-to-end associations of chromatid pairs (Fig. 3c) to identify the products of adjacent disjunction. The results of the cell counts for T(9,ll) males, as well as corresponding counts for T(9,lO)Pw and T(2,11)Cu, are summarized in Table HI. It is evident that T(9,ll) and T(9,lO)Pw exhibit a marked preferential alternate disjunction. On the other hand, T(2,ll)Cu has a random disjunction. The counts for each of the three translocations show excellent agreement between metaphase I and prophase I1 cells.

Hatch estimates from crosses between T/+ males and wild-type females correspond closely to the proportions of viable chromosome combinations calculated from the cell counts (Table III). For T(9,ll) and T(9,10)Pw, lethality is about 4% less than expected, while the values agree almost exactly for T(2,ll)Cu. Some reduction in hatch from the cytological data is to be expected (see preceding section). Why this did not occur with T(2,ll)Cu is unclear.

Because of the difficulty in working with oocytes it is not possible to obtain cytological data on disjunction in females. However, the nymphal mortality data provide

Egg mortality data from crosses involving T(9, 1 I) heterozygotes

No. of Avg. Avg. dead Estimated oothecae eggs/ embryos/ Mortality hatch

Mating examined ootheca oothecaa ("/.) (%'.)b

TI+$ X +/+O 15 42.7k 1.4 18.1?1.1 42 58 TI+? X +/+$ 27 46.3k0.9" 22.6?0.9C 49 5 1 T +/+ ru9 X ru/ru$ 9 45 .4k0 .8 21.622.1 48 52

'No attempt was made to distinguish between dead embryos and unhatched eggs, although the majority of filled compartments clearly contained dead embryos. "Estimated by subtracting the percent mortality from 100%, since no progeny counts were made for either of the 2 larger series (see text). "Significantly different from estimates of matings involving T/+ males.

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646 D. G . COCHRAN AND M . H . ROSS

some insight into this situation (Table 111). It appears that disjunction in T(9, 10)Pw females is similar to that occurring in males. Contrarily, with T(9,l I), a discrepancy occurs between the sexes. The estimates for females do not differ significantly from random assortment. It appears that directed disjunction is pronounced in T(9,ll) males but is minimal or nonexistent in females. T(2,ll)Cu females are sterile, thus precluding the opportunity to collect data.

Discussion The known genetic map of linkage group 8 (chromosome 9) is about 6 map units

(Fig. 2), yet chromosome 9 is one of the longest autosomes of B. germanica (Cochran and Ross, 1969). It seems likely that both genetic loci and chromosome breakpoints are concentrated in the central portion of the chromosome (Ross and Cochran, 1971). The present data confirm this situation by demonstrating that all except the st locus lie within the relatively short chromosome region between the T(9,ll) and Df(9)Pw breakpoints. In addition, estimates of map units per pm of chromosome length permit a first approxima- tion of total map distance for this chromosome. One estimate indicated chromosome 9 has about 50-55 map units. This is relatively close to the average map distance of 60 units/autosome derived from an average chiasma frequency of 1.3 (Cochran, unpublished data). Nevertheless, this correlation utilized linkage data showing an unusually high frequency of crossingover between the ru locus and Df(9)Pw. The bulk of linkage data indicate a low crossover frequency for the region between the T(9,ll) and Df(9)Pw breakpoints. Here correlations with chromosome measurements seem to give an unrealis- tically low estimate for the total chromosome. The low crossover frequencies may be due

Fig. 3 . a. Metaphase I in a translocation bearing spermatocyte showing the ring configuration of adjacent disjunction (arrow). b. A similar cell with the V-shaped (zig-zag) configuration of alternate disimction (arrow). c . A late prophase Il cell depicting the end-to-end association of chromatid pairs resulting from adjacent disjunction at metaphase I.

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CYTOLOGY AND GENETICS OF THE GERMAN COCKROACH 647

to the presence of the centromere and/or the ~ t ruc tur~of this chromosome region. Possibly the frequency is higher in the more distal portions of the chromosome arms. Indeed, if chiasma, which consistently prevent breakup of the ring-formation in T(9,ll) cells, expressed their normal frequency for the arms of chromosome 9, the total map would be estimated at about 100 units. More probably, there is some compensatory increase in crossingover in the distal portions of the pairing complex.

In selecting linkage distances for correlation with chromosome measurements, it was necessary to consider the possible influence of translocations on these estimates. Crossingover within areas which lie relatively close to breakpoints does not seem to be appreciably changed, unless the locus happens to lie within the area of nonalignment at the intersection of the arms in the pachytene configuration. The latter situation probably accounts for the complete linkage of ru with T(9,lO)Pw (Ross and Cochran, 1967), since the suppressive effect of a translocation can arise from synaptic failure (Roberts, 1970). This estimate was not used in the genetic mapchromosome correlations. Other group 8 linkage estimates involving translocations were included since these were close to those derived from studies using mutant loci only, i.e., ru, st and sry. These markers show close linkage in the absence of translocations (Ross and Cochran, 1968; Ross, unpublished data), although the data are insufficient for the detection of minor crossover depression. Likewise, in chromosome 10, T(9,lO)Pw does not appear to influence crossingover between the closely linked loci, ro and r (Ross and Cochran, 1969).

In Drosophila and Zea mays the centromeric region is more susceptible to breakage than other portions of the chromosome (Jancey and Walden, 1972). This may also be true

Cytological and biological data on chromosome disjunction in certain cockroach translocation stocks

Cell Counts

Stock Adjacent disj. Alternate disj. Percent alternate

Metaphase I

Prophase I1

Nymphal Lethality

Dead nymphs Live nymphs Percent hatch

T ( 9 , l I ) g b 27 1 377' 58.2 T(9 , 11)0 61 1 639' 51.1 T (9, 10) Pw8' 399 587 59.7 T(9, 10)PwQ 257 413 61.6 T (2, 11) Cu8' 1202 1180 49.5

aNumber of cells counted to obtain figures. Five or more insects were used in every case. bProgeny counts from crosses between indicated translocation heterozygote and wild type. 'Live nymphs were estimated for T (9 , l l ) by subtracting the total number of dead nymphs from the total number of egg case compartments examined. See text for comparison of this method with actual counts of live nymphs.

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648 D. G. COCHRAN AND M . H . ROSS

in B. germanica. Most chromosomes in B . germanica are metacentric (Cohen and Roth, 1970). Thus, breakage near the centromere would lead to the exchange of relatively large portions of the original chromosomes, as seems to be the case in most of our translocation stocks. Several lines of evidence indicate that chromosome 9 is metacentric (Ross and Cochran, 197 1). All known breakpoints, i.e., those for T(9,10)Pw, T(9, 10)Pwb, T(9, 10Zpwe, Df(9)Pw and T(9,l I), also lie within the central portion of the chromosome.

Breakpoint location and the involvement of relatively long chromosomes result in comparatively long pairing arms in T(9,l I ) , as well as in the P w translocations. Presumably this facilitates chiasma formation and is a major factor in preventing breakup of the ring formation. This type of translocation, as found here for T(9,ll) males and T(9,10)Pw, is generally characterized by preferential alternate disjunction (White, 1973). There are, of course, a variety of factors known to influence frequencies of alternate vs. adjacent disjunction. We are not currently in a position to either evaluate them for B. germanica or explain the apparent sex difference in T(9,ll). Nevertheless, it may be significant that T(2,l l)Cu, which involves a very small autosome, shows random segregation. Interestingly, it does not break up into two bivalents, as seems to occur in most translocations characterized by random disjunction (White, 1973), although it does have a tendency to form chains-of-four.

To our knowledge, prophase I1 cells have not been used heretofore to measure frequencies of alternate vs. adjacent disjunction. The validity of the method depends on the maintenance of the ring structure by terminalized chiasmata. At anaphase I in this species, adjacent disjunction is characterized by rupturing of the ring at the lateral margins but not at the poleward migrating ring ends. The latter then maintain an end-to-end association in prophase 11. This finding favors a mechanical rupturing of 'metaphase chromosomes, as opposed to a chemical rupturing (White, 1973).

Ultimately, these studies of T(9,l l), and similar work with other translocations, will provide a foundation for experiments in genetic control of this species. The close linkage of T(9,ll) with ru has made it possible to set up a mass-maintenance system for T(9,ll) by repeated backcrossing to rulru, in which approximately 99% of the normal-eyed progeny are translocation heterozygotes. Additional experiments, including tests of male competitiveness and the effects of repeated releases of T(9,ll) males into a population, are underway. Results from such studies will be reported in separate publications.

Acknowledgement This work was partially supported by a grant from the National Science Foundation

(No. GB28954) for which we express our appreciation.

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