CYTOLOGY AND GENETICS OF A PRONOTAL-WING TRAIT IN THE GERMAN COCKROACH

CYTOLOGY AND GENETICS OF A PRONOTAL-WING TRAIT IN THE GERMAN COCKROACH

Department o f Entomology, Virginia Polytechnic lnstitrlte and Stcrte University, Blacksburg, Virginia

A new pronotal-win5 mutant of the Gcrman cockroach, Blnttelln p - n m i c a (L.), has bml described and studied. h4nsphologically, it is similar to previous- Iy described tramlocarinn prowing traits (1'['),10)Pw) in this spccies. Gmrrically ic is inherited as: an auco~omal scmidonlinant lcthal nit11 somcwhat rcduced viability rspcciaHy throuah the males. Cymlogicdly rhe n i t is &?so- ciated wirl~ a tcrminal ricficicncy of thc ninth pair of n~ciotic chromosomes, and has been namcd Df(g)I'w. I t is linked wirh rhe g n u p VIlI marlccrs m and st at averaye map distances of 3.4 and I .5 unia, rcsf)ecrivcly. thus allo\ving. for the first time, a positive linkage gruupaurosomc correlation for thc Ger- man cockraach. Cornparatire studies of che deficiency and rran~locarion PW traits 11nve producerl an approximatiun of translocation brcalcpoints ~ n d ccntro- mere locarinn on autosonlcs 9 and If) , mrabIishmcnt of the rclatirlnsliip betwcen the deficiency and the translocation, and 3 tcntarire asignment of specific traits to specific locarions on chromosome 9. Thc genetic nlakcup of the prowing locus is ako discussed.

Introduction Some of the most intriguing aspects of genetic and cytogenetic investiga-

tions of the German cockroach Blattella gennanica (L.) thus far reported invoIve the pronomI-wing mutants (Ross, 1964; Ross and Cochran, 1965; Cochran and Ross, 1969). In an insect characterized by a low mutation fre- quency (Ross. unpublished) the discovery of three such traits is in itself remarkable. morphologically the traits rcsemhlc the paranotal Iobes of an- cescral insects, as was noted in regard to the first described prowing T(9,lO) PW (Ross, 1964). Cytologically the three occurrences T (9,IO) P w , T(9-10) Pwg and T(9,10) Pw" appear to be associated with closely similar if not identical reciprocaI inrerchangcs betwcen chramosomes 9 and 10 (Cochran and Ross, 1969; Cochran, unpublished). This may indicate a marked tendency for these chromosomes to exchange pam, Recentlv, a fourth prowing-like trait was discovered which did not prove to be associated wirh a reciprocal translocation. A combined study of its cytology and genetics was undertaken in the hope of elucidating some of the unsolved probjerns associated with prowing traits. The results are presented in this paper. Ir was shown that the new trait involves a deficiency of the 9th chromosome, and consequently is designated "Df (9)Pw".

Materials and Methods Genetic: The procedures for genetic experiments used in our laboratories

have been described elsewhere (Ross and Cochran, 1965, 1966). Standard crosses with wild type were used to determine the inheritance mechanism. Linhge tests were conducted for group 111, using rose-eye (m), and for group VIII, using ruby-eye (m) and notcl~ed aernirc {n'), since these are the two groups invoIved in the prowing translocations (Ross and Cochran, 196Rb, 1969). In onc group of marings, Df(9) Pw individuals were crossed with Manuscript received June 14, 1971.

Can. J. Genet. Cytol. 13: 522-535. 1971.

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PRONOTAL-WING TRAIT OF THE COCKROACH 523

rulru homozygotes. The F, double hybrids were selected, i.e. those showing the prowing phenotype, and backcrossed reciprocally to m. In another and larger series, m and ro linkage was tested simultaneously. Df(9) Pw were mated with the double homozygotes mlm, ro/ro. The phenotypically prowing males and females in the offspring were then backcrossed to mlm, rolro. The phenotype of these double homozygotes is easily distinguished from the m and ro parental types (Ross and Cochran, 1968a), and the backcross progeny were scored accordingly. Re~uIsion test crosses were made with st: hut reciprocal crosses were not possible since st females are sterile. Crosses were aIso made between Df (9) P w and T(9,10) Pw. F1 progeny were scored phenocypicaFlv in order to determine wild tvpe : prowing segregation. I t was not possible to identify the double heteroivaote. In addition, male nvrnphs showing a growing phenowpe were examined cyrologically for the purpose of estimating the proportjc;ns of deficiencv and translocation tvpes, and in hopes of studying the chromosome a~ignrnkts if a double heterozygote could be found.

Egg hatchability data were obtained similarly to those used in studies of fs and c r s mutants (Ross and Cochran, 1970). The average hatch was estimated by dividing the total number of offspring by the total number of compartments in the oothecae in the particular matings under study.

Cytogenceic: The techniques used in this phase of the study have been fully described (Cochran and Ross, 1969). Briefly, they co-ted of slide

from testes of knnwn age nymphal cockroacha by the squash method. An otcein-tvpe srain was used. O\)servation and study of the slides were by means of a keiss phase-contrast microscope. Chromosome rneasure- rnents were done wirh an ocular micrometer placed in the lower end of the eye-piece lens. Photographs of chromosomes were taken through an auto- matic shutter device using Plus X-Pan 1)Iack and white film with printing on high conmast paper.

Results Description of Df ( 9 ) Pw

Origin: Df(9) Pw wa5 discovered by one of us (M.W.R.) in the course of scoring a rest cross involving yellow-hody (y) and curtv-wing (T2,ll) CPL). In a total of five progeny, two individuals were phenotypically prowing. One was a steriIe curly female, the other a curly, yellow malc. The latter was used to the trait which has now been freed of CZE, although some y remain in the stock.

P h e n o f y p ~ : Phenotvpically, Df(9) Pw might be described as a partial expression of the prowiig trait, as seen jn the prowing translocations. The tracheal partern and shape of the nymphal pronota are indistinguishable from those characteristic of rhe prowing translocations (Ross, 1964; Fig. 6D), and they resemble those of wing buds. However, In adults there is little if any expansion of the postero-larerat portions of rhe pronoturn (Fig. la). The typical expression associated with at least two of the translocations, T(9,lQ) Pw and T{9,10) PmE, consists of extensions about equal to 1/3 the length of the forewing with well formed veins and considerable cross venation (Fig. Ib). In the third prowing trait, T (9,103 PwUIR (Cochran and Ross 1969), the expansions are frequently shorter and quite similar to those of Df(9) Pw.

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524 MARY H. ROSS AND DONALD G. COCHRAN

Inheritafzce: Df (9) PTU is inherited as an autosomal semidominant lethal. Crosses with wild type fit a 1: 1 ratio (139+: 130 Pw, X" = 0.30, P > 0.50). Matings of known hybrids fit a 2: 1 ratio (201 Pw; 98 +, X" = 0.1 1, P > 0.70), as did phenotypically prowing pairs randomly selected from the breeder colony (132 Pw: 60 +, X' = 0.38, P > 0.50). Hatch from the latter two groups, which included first oothecae only, was 58.5%. If it is assumed that 90% hatch is normal, loss of one fourth of these (the homozygote) would give a 67% hatch. Thus, it appears that hatch was somewhat lower than expected merely from death of the homozygotes. The hatch reduction characteristic of hybrid males used in the linkage analyses may have been a contributing factor (see Hatchability Data).

Cytology: Male nymphs bearing the phenotypic expression of the Df(9) Pw trait were examined cytologically. Information from the preceeding section has shown them to be heterozygous for the trait. I t was immediately evident that a reciprocal chromosome translocation was not involved with this stock, since each pair of chromosomes was independent of all others. In addition, measurements cf the chromosome pairs individually at late pachytene (Table I) showed them to be almost identical with previously reported measurements for wild-type chromosomes (Cochran and Ross, 1969). How- ever, close examination revealed that chromosome pair number 9 has one very abbreviated member (Fig. 2 ) . From the figure it is clear that the mem- bers of this pair allign properly at one end, but not at the other. This shows that the trait is a terminal-deficiency. Measurements of the deficient pair a t late pachytene revealed lengths of 23.0 and 15.1 scale units. A measurement of 23.0 is well within the normal range for chromosome number 9. Thus, deficiency constitutes approximately one third of the total chromosome length. In view of the viability of the heterozygote, it is obvious that in wild-type

Fig. . I . a. Expression of the prowing trait in the deficiency stock, and b. in the translocation stock. The photographs show the right side of detached pronota.

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PRONOTAL-WING TRAIT OF THE COCKROACH 5 2 5

individuals no genetic material occurs on the lost portion of the chromosome for which there is a requirement of both sets of alleles.

Linkage From the cptogenctic analysis, it was expected that Df (9) PW would show

Iinkage with markers for eithcr group 111 or VlII, since these groups corres- porld to the two chromosomes (Nos, 9 and 10) involved in the prowing translocations (Cochran and Ross, 1969). It was also anticipated that if the locus of any simpIe recessive marker which might be crossed wit11 Df(9) Pw shouId lic within the deficiency, the trait would appear in the F,. The results showed that all F, progcnv from crosses with TO, nr and st were phenotypical- lv wild type with sespect'to these traits. Thus, from this evidence one would ronclude shat these loci are nor on the Iosc part unIess a requirement for two sets of recessive aIleIes exists for the expression of a particular trait (see Dis- cussion).

In the triple backcrosses to the double marker stock rolro, mrlru the data urcre analymd separatelv for possible linkage of nf{9) Pw with TO (group 111) and nc (group V I I I ~ . The former showed independence, as indicated by the good fit to a 1 : r : l : l ratio (116+, +: 122+, ro: 116 Pw, +: I 0 4 Pw, TO;

X' --- 1.49, P > 0.50). Cnnverselv, Df(P) Pw showed close linkage with nr. This was also evident in the simple backcrosses to KV Jnl. T h e average recom- biuntion percentage was calculated to he 3.4 f 0.8. However, there were differences between the matings of hvhrid males and females and hetween the two mating sets, one of which iicluded YO {Table 11). Crossing over rcnded to be more frequent in the matinps of both groups of hybrid males, u-hcreas in linkage g ~ o u p s VI and X the reverse tendency lzas- been noted (Ross and Cochran, 1970; Ross, 197 1 ). In addition, recombination was more frequent in the presence of ro, possihlv due to the diffcrcnce in genetic back- ground. Because of these internal d;screpancjes a range of values may be tllc best current estimate. A range of 3.4 - 4.5, based on the overall average and the weighted average of the triple backcrosses, respectively, is proposed.

Fig. 2. Pachytene chromoson~es of Df(9) Pw showing the deficiency (arrows). a. Late pachytene-early diplotene b. Mid-pachytene. The apparent association of the X chroniosome with chromosome No. 9 in b is not characteristic of the stock.

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Linkage of Df(9) Pw was also tested with the other group VIII marker, st. The results of repulsion test crosses from the hybrid females are as fol- lows: 4+, +:274 Pw, +:254+, st: 4 Pw, st. Recombination was estimated at 1.5 -c 0.7%. If i t is assumed that the deficiency prowing locus lies at, or close to, the broken end of the chromosome, then one possible sequence of group VIII loci is as shown in Fig. 3e. The most probable locations of these loci are considered in more detail in the Discussion Section of this paper.

From the above sesuIrs, it is evident that linkage group VIII lies on chromosome 9, thus giving the first such correlation for an autosomal g-roup in this species, Furthermore, because of the known involvement of linlcage graups 111 and VIII and chromosomes 9 and 10 in the translocation prowing traits, by elimination, the correlation of linkage group 111 with chromosome 10 is also established.

Hatchability Data

Egg harch data were obtained for the linkage study af Df(9) P w with m, as weil as in the basic inheritance studies. As already noted, two tests were induded: one, a simple nl: test cross; the other, a test cross for both nt and TO

linkage. The data are summarized in Table 111. In both cross groups, oozl~ecal size and hatch from hybrid females were essentially normal (TabIe m, 2 and 43. Although those in the ryr backcross produced oothecae which

TABLE I Measurements of chromosomes from the Df(9) P.w stock of German cockroaches

I Chromosome number*

*Chromosome nrtmber I iu the S chrommome. All others were numbered on the basis of in- creasing length (see Cochran and Ross, 1969). +Means were based on detailed measurements of 12 cells from several different individuals. Consistency was acliicved by restricting measurements ro cells whose longest chromosome was within the 30-35 scaEe un i t range. Cells were rnagnifted to 2500 X for measurements. *"l'he data were analqhzed by thc Duncan multiple range test. All measurements were significantly different at the 5% level.

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TABLE I1 Results of linkage tests of Df(9) Pw with the ruby-eye (ru) mutant of Blaltella germanica

I I I

were somewhat larger than the average for wild type (VPI Normal), the size did not differ significantly from the overall mean of 45.5' 2 1.2 cornpart- mentsloothecae used previously for comparisons of this type (Ross and Coch- ran, 1970).

The two mating sets of hybrid males produced similarly sized oothecae, each with significantly smaller numbers of compartments (eggs) than found in either wild type or their reciprocal crosses (Table 111, 1 and 3 ) . Pb males

Parental genotype

Pvta + + ru d X - Q + ru + ru

Pw + + ru -0 X - d + ru + ru P w + + + ru ro - , - d X - - O b + ru 70 + ru' ro

p w + + -- + ru ro 1 Q X - , - d b + ru' ro + ru 7 0

Totals

TABLE 111 Summary of oothecal size, progeny counts and hatch from matings of Df(9) P W in Blattella

germanua

to indicate the prowing locus as marked by the deficiency. bAnalyzed for segregation of Df(9) Pw from ru only, i.e., not for ro linkage (see text).

No. of crossm

6

5

6

15

32 I

'Ootheca significantly smaller than corresponding set from hybrid females and also from wild type. bSignificantly lower than corresponding set from hybrid females atid frnm wild type.

Hatch ((:-1 ,CI

79.9

93.6

34.2

88.0

90.1

Parental genotype

+ ru P w + d X - 1 ) ru Q + + ru

PW + + ru (2) +u

Q X - d + ru P w + + + ru ro ,-- 0'' X -,- O

(3) +u ro + ru ro P w + + + ru ro

( 4 ) ~ - 0 X-,-cF + ru' TO + ru ro Wild type

% recombi- natiou

3 . 2 f 1 .3

0 . 0

6 . 8 f 2 .9

4 .3 f0 .8

3 . 4 f 0 . 8

Phenotypes and nos, of progeny

Avg compts./ ootheca -

3 8 . 8 f 0 . e

4 7 . 0 f 0 . 4

3 6 . 0 f 1.5*

4 4 . 1 f 1 .1

45.5f 1 . 0

ru, PW

3

0

2

1 1

16

Avg oRspring/ ootheca - - - -

31.Of O.fb

4 4 . 0 f 1 .0

1 2 . 3 f 2.Sb

3 8 . 8 4 ~ 1 .2

4 0 . 0 f 1 . 0

PW ----

84

99

32

260

475

4-3-

3

0

3

14

20

ru

96

121

37

298

552

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5 2 8 MARY H. ROSS AND DONALD G. COCHRAN

have a similar influence on oviposition of their mates, and several possibilities for this type of effect were discussed in connection with that mutant (Ross, 1971).

The hatch data for the rnatings of hybrid males were dissimilar, aIthough each exhibited a significant reduction over that normally expected (Table 111, 1 and 3). The estreme reduction in the crosses involving ro results from monaliry, since many dead embryos were observed in the oothecae. Un- doubtedly this lethality is related to meiotic abnormalities found in several hybrid malcs (Pm+/+m, + / r o ) which wcre examined cytologically (Coch- ran, unpublished). Peculiarly, hatch from the reciprocal crosses was excel- lenr. This suggests thar, in females, there may have been a non-random move- ment of chrarnosnmes, with the lethal com6inations being eliminated in the polar bodies. In the case of the hybrid male matings in the simple ru backcross (Table 111, I ) , the hatch reduction cannot be definitely attributed to mortality. Although dead embryos were noted in several oothecae, others included enough unfertilized eggs to affect hatch.

In spite of the hatch reductions, in these hybrid male matings, there was a fairly good 1: 1 segscgation of Df (9) Pw ( 1 391: 12 f Pw, X' = 0.62, P > 0.30). Conversely, in matings of hybrid females, hatch was excellent (Table In, 2 and 4) but segregation of Df (9) Pw was disturbed (433+: 370 PW f+ = 4.54, P > 0.01). This type of segregation could result from a tendency of the chromosome bearing the deficiency to go into a polar body. However, it should be noted that the numbers in rhe matings of hybrid males are rather small. Thus, a slight: viability loss of Df(9) Pw individuals could account for the apparcnr difference in segregation, assuming tha t couna of several hun- dred are necessary to detect the loss.

Comparative Cytology of T(9,lO) Pw and Df(9) Pw

From the data already presented in this paper, it appeared that a corn- parative study of the translocation and deficiency Fw traits might be useful. No attempts have been madc previously to locate the brealt points in the translocations since cytological landmarks in the meiotic chromosomes of this species are uncommon, Indeed, no clear indication of centromere position exists a t present except those given from mitotic chromosomes (Suomalainen, 1946; Cohen and Roth, 1970). Nevertheless, observation of the translocation figure at late pachytene revealed consjdcrabb regalarity. Extensive regions in each arm of the star-shaped figure align perfectly. If we assume that the breakpoints occur in the regions where alignment is imperfect (Sybenga, 1910), then the location of the breakpoints is already qnire restricted. Measurements of well displayed pachytene figures have been made to quantitate these regions. T h e results are prcsented in Fig. 3a. If this approach is accurate, rhe breakpoint for chromowme 9 lies in the region benveen 12.8-16.0 scaIe units measuring from thc left side of the figure. In a corresponding manner the breakpoint for chromosome 10 Iies benveen 14.C18.3 scaIc units srardng a t the top of the figure.

With this information available, jr is possible to make a comparison between the m a traits. As previouslv shown Df(9) Pw is characterized by a terminal deficiency of chromosome 9. Thus, in relation to the translocation

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PRONOTAL-WING TRAIT OF THE COCKROACH

- 12.8

No. 9=23.:"r TI= 17.5

7.3

st- e - 1 .5

Fig. 3. Diagramatic representation of cockroach chrommomes. a. T(9,10) Pw showing whoIe chromosome and s v e n t measurements. The arrows indicate hypothetical cenuorneres. b and c. Cliromosome KO. 9 shawing two possible alignments of the deficiency trait in relarion to the tsms2ocarion. a, b and c are drawn to scale. d-h. Hypo- thetical gene arrangemenn for linkage group VnI on chromosome No. 9. Numbers in parenthesis in 11 are ~ s u m e d . See tcm for. details.

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figure two possible arrangements exist as shown in Figs. 3b and c. The dcci- S ~ O R bctween these two alternatives is aided by two lines of evidence. First, thc break points in chromosome 9 produce similar phenotypic effects in both thc rranslocation and the deficiency. It seems logical that such break points wnutd occur at closely similar posirions on thc chromosome. Secondly, from the linkage data presenccd here and by Ross and Cochran (1%8b), it is clear that both T(9,lQ) Pw and Df(9) Pw arc closely linked with the group VIII traits m and st. To accommodate rhese factors the most plausible configuration is that shown in Fig. 3b. For this arrangement to be correct, one has to assume only that the locus for the deficiency Pw is at or near the broken end. In the Fig. 3c arrangement both loci would still have to be near each other, because of the linkage data, but one or both would be removed from their most likely position on the chromosome.

Crosses Between T(9,lO) Pw and Df (9) Pw

A finaI resolution of the above problem wouId be possible if a double heterozygote couId be studied cytologically. Againsr this possibility, reciprocal crosses were made betwecn Df ( 9 ) Pw and 'f (9,lO) Pw individuals. In the first set of crosses the cytological examinations were not successful for reasons which are unknown. However, it was determined that the per cent hatch from these crosses was 35.8% and a 2: 1 ratio of offspring was obtained (146 Pw: 75+; X' = 0.08; P > 0.70). The expected hatch here is 43% assuming the double heterozygote to be lethal. - - -

In view of the above results, a second set of crosses was undertaken. Thirreen successful matings occurred in this series. The per cent hatch was 36.0% with no difference existing between the reciprocaI croses. The pheno- rypic results were 136 Pw: 7 6 1 (1' = 0.53; P > 0.40), an excellent fir: to a 2 : 1 mdo. Cyrologically, 36 male nymphs were Df (9) Pzs; 35 were T(9,lO) Pw, and 3 had both traits. Theoredcally, one would expect equal numbers of each of these three types. Obviously, the double heterozygote is lethal in a high percentage of cases.

Cytological examination of the three double heterozygotes was highly successful, The presence of horh traits is signsled by the tinding of a high percentage of meiotic ceIIs with an open chain-type of translocation figure. PseviousIy obtained data on the T(9,lO) PC s310wed that about 1% of cells in diplotene-diakinesis have a chain-of-four rather than a ring-of-fafour (Coch- ran, unpubhhed). The percentage of open chain-of-four configurations in these three individuals varied, but ran as high as 40%. A number of ce1Is had adequare displays of chsomosornes (Fig. 4 a ) and were used for detailed study and measurement (Fig. 4b). Translating these resuIts into the stylized form nf Fig. Sa, and realizing that the numbers represent one cell only, it is clear that the proposed arrangement shown in Fig. 3h is the correct configuration (Fig. 4c). For Fig. 3c to be correct, the chromosome arm lacking a chiasma, and thus being open, would be that shown by the arrows in Figs. 4b and c.

With the availability of these data one other comparison can now be completed. Previously i t was reported that crosses betwecn T(810) Pa individuals give 29.2% hatch (Ross and Cochran, 1965). The expecred value is 37%. It was shown earlier in this paper that if Df(9) Pzlr x Df(9) Pw yields 58.2% hatch, while 67% is expected. Crosses between the nvo traits

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Fig. 4. a. Phoropaph showing the chmmosomal configur*tion of the double hecero- zygotc T(9,10) Pw, Df(9) Pw. I>. Drawing of same showing chrommomc measure- mmts. c. Styli7xd drawing of m e ro correspond with Figure 3a. Arrnws in b and c indicate the chromasomc arm where the deficiency would exist if the Figure 3c arrangement were correct. d. Enlaqed drawing of the T/9.10) P a cenrer sllowing the proposed location of specific loci. C

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532 MARY H. ROSS AND DONALD G . COCHRAN

gave an average of 37% hatch. Allowing for the slight viability of the double heterozygote about 47% is expected. Thus, in all three possible combina- tions from 8-10% fewer nymphs hatch than anticipated. In the latter case the reduction is clearly not similar to that associated with hybrid male Df(9) PW (Table 111), since no difference in the reciprocal crosses, reported in this section, occurred. While the reasons for these hatch reductions are unknown, it seems that they result in a slight reduction in viability of all surviving geno- types as reflected by the good fits to expected phenotypic ratios.

Discussion

From the data presented above a number of points concerning chromosome pairs 9 and 10 can be clarified with a reasonable degree of confidence. One such question is the position of the break points in the translocation. Evi- dence from cytological examination of both the translocation and the deficiency traits indicates that the break point for chromosome 9 is at approximately 15 scale units (6 !i) from the left side of the chromosome as shown in Fig. 3a. Presumably this also corresponds to the locus of the Pw traits, although it is not clear whether this is a point locus or a small region of the chromosome. In a corresponding manner, the break point for chromosome 101 appears to be located at about 16.5 scale units (6.5ci) from the top of the chromosome as shown in Fig. 3a.

This tendency of chromosomes 9 and 10 to break repeatedly at closely similar, if not identical points, is one of the more intriguing aspects of the prowing traits. Localization of breaks in heterochromatic regions has been shown in other organisms (Natarajan and Ahnstrom, 1969) but as yet we have no proof of the existence of such regions in the autosomes of B. gemmica . In fact, no pronounced chromocentres have been found in interphase cells except that attributable to the X chromosome. On the other hand, if breaks are localized in euchromatic regions, possibly these are areas of relatively high DNA content which occur in the interior of chromosomes 9 and 10. These DNA--high areas could be particularly subject to breakage (Lefevre, 1969). I t is not clear why chromosomes 9 and 10, in particular, should tend to exchange pieces if, indeed, that is the case. Perhaps there is some attraction related to the evolution of these two chromosomes.

The ability to fix the breakpoints of chromosomes 9 and 10; even approximately, allows some additional deductions about these chromosomes. From the deficiency it is clear that the centromere of chromosome 9 is not located on the lost arm. I t is not likely t o be situated on that portion of the chromosome where pairing is imperfect. Thus, it seems reasonably certain that the centromere of chromosome 9 is on the intact arm, and occupies a metacentric position, since the majority of the chromosomes of this species are metacentric (Suomalainen, 1946; Cohen and Roth, 1970). The hypothetical position of this centromere is shown by the arrow in Fig. 3a. In a corresponding manner, i t would be expected that the centromere of chromosome 10 is located on the arm of the translocation figure opposite to that proposed for No. 9 (right hand arm of Fig. 3a indicated by the arrow). This means that chromoson~e pair 10 is one of the few submetacentric or acrocentric chromosomes of this species.

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Assuming the position of these centrorneres to he at least approximately correct, the composition of the translocation chromosomes T, and Ti (Fig. 3a) can he described. Chromosome TI consists of rhe short m and centromere of Na. 10, a small portion of the Iong arm of No. 10 and most of one arm of No. 9. I t is evidenc rhat the sum of lcngrhs of these sections readily accounts for she total length af T,. Similarly, chromosome TP is composed of one complete arm and the centrornere of chrnmosorne 9, a small pom'on of the other arm of 9, and the major portion of the Iong arm of Na. 10. Again the sum of the individual parts easilv accounts for ~I le total Iength of T,.

It is unforrunare that cenkomeres of the meiotic chrnmosomes of this species have not yet been located cytologically. ,4 positive landmark would allow a better correlation of physicn1 structure and genetic constitution. However, even without this information some progress is possible. On chromosome 9 the evidence presented malies it clear that the Group VIII loci, linked with T(9,iO) P v and with Df / 9) Pw, lie close to the breakpoint. Figs. 36 and e show possible sequences of traits as indicated by the individual sets of data (Ross and Cochran, 196%; this paper). In attcrnpting to combine these results, some difficulties arise. Fig. 3f shows one possible combination. The main objection to this sequence is that ir places st benveen the two Pw traits which arc assumed to be cither a point locus ar a small region on the chmmosome, Also this arrangement does not produce 3 good accummodation of the linkage distances, although the mi-st' linkagc (5.7 2.88) is based nn F1 repulsion data which are subject to error. Another possible dignmcnt is shown in Fig, 3gb Here the t w o Pm traits are close together, bur other problem arise. Ammg them the most scrious is the relatively large distance hetween T Pw and m. The linkage data showed no cmaing over benveen these two traits (Ross and Cochran, 1967) . Two hypotheses might be offered in expIanacion: ( 1 ) that the seernjngtv complete linkage was due to suppression of crossing aver in a particular arc; of the translocation, or ( 2 ) that lack of crosqing over w a s due to some characteristic inherent to the mt/m sock, since it must he noted that crossovers were also ahsenr in the Df Pw backcrosses of hybrid males to ru. The alignment proposed in Fig. 3g is based on thc assumption rhac the Df Pw linkagc data give a more accurate picture of the sequence of loci than do those of T Pw, hut the fit is still not precise.

T h e arrangement depicted in Fig. 3h represents what ii probably the best integration nf all existing data. The map distances are quite consistent, tbc two Pw traits are dose together, and this arrangement allows a sadsfacrory explanation of two perplexing facets of P w linkage data as will be explained in the nest paragrilph. T h e principle objection to this scheme is that one of the two traits m or st must lie distal of the Pur locus. This means that the mit in quesrion requires both alleles to express itself. We have chosen to show st in this position for two reasons. First, eye color mutants are often the result of the absence of an enzyme necessarv to produce the eye pigment (Zeig- ler, 196 1). Either one or two doses of the allele should be adequatc to resuit in the mutant eye, bur F, individuaIs from the cross Df ( 9 ) Pw x nc were wild t-vpe. Thus ~ l k does not seem ro fit here. Second, the st trait is very complex with six or seven separate effects havjng been noted (Ross and Cochran, 1965; Ross I966a, l966b). Because of this complexity i t is a reasonable possibility rhat both alleles are required for the trait to be expressed. If this is correc'E, thcn a position for st distal of Pw presents no particular problem. Accord-

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ingly, we are tentarivelv assigning the traits sf, Df(9) Pw, T(9,IO) Pw and ric, respectively, to the reiion of chromosome 9 extending from just beyond the breakpoint (IS units) toward the hypothetical centromere (Fig, 4d). In a similar manner, but with Iess precision, the group I11 traits ru and r are ten- tarivelp being assigned to chromosome to in the region 14,418.3 units from the top of Fig. 3a (Fig. 4). These assiprnents represent the first such attempts for this species.

This locus arrangement for Group t71T traits (Fig. 3h) also has the unify- ing attrihute of aIlowing a satisfactory explanarion for certain facets of the linkage data as nrensioned above. First, Fig. 3h indicates that T(9,lO) PW and nr are not coincident even though no crossing over occurred between them (Ross and Cnchran, 1967). This can be explained on che basis that both loci we in that porrion of the translocation figure where no crossing over is possible due to nonalignment (Fig. 4df. Thus, the absence of crossover classes here does not necessarily rncan a coincident location. Second, the linkage data for T(9,lO) Pw and st were disturbing hecanse one crossover class (Pw, AT) was missing (Ross and Cochran, 1967). This roo can be explained using Fig. 4d as a model. Mere st is on an arm which does not contain a ccntro- mere, and u~here alignment does occur since the crossover class Pw*, st* was found. The ahscncc of thc crossover class Pw, st is prohahIy attributable to the reduced viabilitv associated with each of these traits which in combination could result in suppression of the P?a+ fi type (Rnss and Cochran, 1965; this paper). In this connection it should also be noted that one crossover class was missing in linkage studies involving +o with T (9,10) PW (ROSS and Coch- ran, 1967) and R-cyclo with T(2,11$ Czc (,VlcDonald et al., 1969). Decailed analvses of thesc cases have not pet heen accomplished. Their study may pro;jdc additional evidence on thc validity of the above hypothesis. Finally, we must consider the linkage data for Df (9) PC* and st reported herein. in this case both crassover classes occurred. This could happcn provided the exchange took place without disrurbing the DfPw locus in either the deficient or the wild-type chrornosnme, Perhaps the easiest way of visualizing this is for a smalI piecc of chromosome tn exist on the deficiency chromosome beyond the DfPw locus. In char wav the exchange could take place as described.

Although thc nature of the prowing locus remains to be elucidated, some comments appear in order. Previously we diought it might be due to a position eflect thar brings together genes of chromrlsomes 9 and 10 which are separated in the normaI karyotype. J t now appears the principle locus is on chromosome 9, aIthough full: expression of thc trait may invoIve genes on chromosome 10 as well,

The ability to form a prothoracic wing is shared by other insects (Soko- loff et al., 1967). However, the prowing trait of cockroaches is most nearly identical to that of the paranotal lobes of ancestral insects (Ross, 1964). A deveIopmental pathway which, withnut one of the normal alleles at the prowing locus, would produce wing-like expansions on all thoracic segments, may still exist in the coclrroach. Conceivably, loss, alteration, or inactivation of one such allele by cliromnsnma! breakage would permit the expression of a third wing-like expansion (prowing). Hcre it is of interest that another cockroach wing mutant, balloon-wing (bn), affects all thoracic segments, pro-

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ducing bubble-like swellings on the lateral areas of the pronota of nymphs and, later, in the meso- and metathoracic wings of adults. In Ephestia, the fore- and hind wings have identical prepatterns (Kroeger, 1959). In B. germmica this may be true for the wing-forming portions of all thoracic segments.

Acknowledgemente

This work was partially supported by grants from the National Com- municable Disease Center, Department of HEW, Atlanta, Georgia (No. CC-00264) and the National Science Foundation (No. GB 28954).

References Cochran, D. G., and Ross, M. H. 1969. Chromosonle identification in the German cock-

roach. Wild type and mutant stocks. J. Hered. 60: 87-92. Cohen, S., and Roth, L. M. 1970. Chromosome numbers of the Blattaria. Ann. Entomol.

Soc. Am. 63: 1520-1547. Kroeger, H. 1959. Determinationmosaike aus kombiniert implantierten Tmaginalscheiben

von Ephestia Auh?iiella Zeller. Arch. Entwicklungsmech. Organ. 151: 113-135. Lefevre, G., Jr. 1969. The eccentricity of vern~illion deficiencies in Drosophila melano-

gaster. Genetics 63: 589-600. McDonald, I. C., Ross, M. H., and Cochran, D. G. 1969. Genetics and linkage of aldrin

resistance in the German cockroacl~. Bull. World Health Organ. 40: 745-752. Natarajan, A. T., and Ahnstrom, G. 1969. Heterochromatin and chromosome aberra-

tions. Chromosoma 28: 48-61. Ross, M. H. 1964. Pronotal wings in Blattella genizanica and their possible evolutionary

significance. Am. Midland Naturalist 71 : 161-180. Ross. M. H. 1966a. Notched sternite: A mutant of Blattella gennmzica with possible

implications for the homology and evolution of ventral abdominal structures. Ann. Entomol. Soc. Am. 59: 473-484.

Ross, M. H. 1966b. Embryonic appendages of the notched sternite mutant. Ann. Ento- mol. Soc. Am. 59: 1160-1162.

Ross, M. H. 1971. Genetic variability in the German cockroach. VII. Studies of pale- body and bulge-eye. J. Hered. (in press).

Ross, M. H., and Cochran, D. G. 1965. A preliminary report on genetic variability in the German cockroach, Blattella germanica. Ann. Entomol. Soc. Am. 58: 368-375.

Ross, M. H., and Cochran, D. G. 1966. Genetic variability in the German cockroach. I. Additional genetic data and the establishment of tentative linkage groups. J. Hered. 57: 221-226.

Ross, M. H., and Cochran, D. G. 1967. Genetic variability in the German cockroach.. 11. A description of new mutants and linkage tests. J. Hered. 58: 274-278.

Ross, M. H., and Cochran, D. G. 1968a. Genetic variability in the German cockroach. 111. Linkage relationships of the curly, hooded and pallid mutants. J. Hered. 59: 105-110.

Ross, M. H., and Cochran, D. G. 1968b. Genetic variability in the German cockroach. IV, Linkage studies with markers for groups 111 and VIII. J. Hered. 59: 318-320.

Ross, M. H., and Cochran, D. G. 1969. Red-rose linkage in the German cockroach. Ann. Entomol. Soc. Am. 62: 665-666.

Ross, M. H., and Cochran, D. G. 1970. Genetic variability- in the German cockroach. VI. Studies of fused-antennae, crossveinless and downturned-wing. J. Hered. 61: 123-128.

Sokoloff, A., Ackermann, M., and Overton, L. F. 1967. Linkage studies in Tribolium confusum Duval. 11. The map position of three homeotic mutants. Can. J. Genet. Cytol. 10: 490-502.

Suomalainen, E. 1946. Die Chromosomenverhaltnisse in der Spermatogenese einiger Blat- tarien. Ann. Acad. Sci. Fenn. 4(10): 1-60.

Sybenga, J. 1970. Simultaneous negative and positive chiasma interference across the break point in interchange heterozygotes. Genetica 41 : 209-230.

Zeigler, I. 1961. Genetic aspects of ommochrome and pterin pigments. Adv. Genet. 10: 349-403.

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CYTOLOGY AND GENETICS OF A PRONOTAL-WING TRAIT IN THE GERMAN COCKROACH