Louis M. Roth and Samuel H. Cohen.
Chromosomes of the Pycnoscelus indicus and P. surinamensis Complex (Blattaria: Blaberidae: Pycnoscelinae).
Psyche 75:53-76, 1968.

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CHROMOSOMES OF
THE PYCNOSCELUS INDICUS AND
P. SURINAMENSIS COMPLEX
(BLATTARIA: BLABERIDAE : PYCNOSCELINAE)* Pioneering Research Laboratory
U. S. Army Natick Laboratories
Natick, Massachusetts 01 760
The chromosome numbers of both arth he no genetic and bisexual forms of Pycnoscelus surinamensis (L.) have been reported to be 38 (2) and 37 ( ) by Matthey ( 1945, specimens from the botanical garden and crocodile house in Berlin; 1948, specimens from Kuala Lumpur, Malaya), and 36 (9 parthenogenetic) by Suomalainen (1945, specimens from the botanical garden and crocodile house in Berlin). Reproductive behavior (Roth and Willis, 1968), and cross- ing experiments (Roth, 1967) have shown that the parthenogenetic and bisexual "strains" are physiologically sexually isolated from one another and Roth (1967) applied the name Pycnoscelus indicus (I?.) to the bisexual taxon from which the parthenogenetic form (P. sur- inamensis) apparently arose. The specimens used in the 1967 study originated from Florida, Hawaii, Australia, and Indonesia. In this paper we report the results of a study of the numbers and morphology of the chromosomes of P. indicus, and
clones of P. surinamensis
from different geographical areas. Pycnoscelus surinamensis is cos- mopolitan, whereas P. indicus appears to be restricted mainly to the Indo-Malayan region.
MATERIALS AND METHODS
The Pycnoscelus which were cultured in the laboratory originated from Florida, Jamaica, Panama, Brazil, Hawaii, Australia, Indo- nesia, and Thailand (see acknowledgements). It is possible that certain cytogenetic forms have been eliminated in those cultures of Pycnoscelus (e.g., from Florida and Australia) which have been kept for years in the laboratory. However, the specimens from Jamaica, Panama, Brazil, and Thailand were for the most part Fi or Fa offspring from the original material collected. The methods used in preparing slides of mitotic figures were modifications of Tjio and Whang (1962) and Wolstenholme *,Manuscript received by the editor April 8, 1968 5 3
Pu&e 7553-76 (1968). hup Ytpsychu einclub orgi75175-051 html



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54 Psyche [March
( 1966). Last instar nymphs were injected with twenty microliters of colcemid (in two instances adult parthenogenetic males were used), 16 to 24 hours later their gonads were dissected in an insect Ringer's solution (Bodenstein, 1946) and transferred to 1% sodium citrate solution for 5 to 7 minutes. The gonads were placed in acetic alcohol (I part glacial acetic acid to 3 parts absolute alcohol) for about I minute, then in a drop of 45% acetic acid, on separate albuminized glass slides, until they became clear. Coverslips were placed over the gonads, squashed between 2 pieces of highly ab- sorbent paper, and the slides were immediately placed on dry ice. After 5 to 10 minutes, the coverslips were flipped off with a single- edged razor blade, and the slides were immediately placed in acetic acid-absolute alcohol (I :I) for TO minutes. The slides were quickly rinsed in absolute alcohol, placed in 2 changes of 95% alcohol for 5 minutes each, removed, and allowed to dry. A drop of acetic-
orcein was placed on the dried tissue, covered with a coverslip, and after about I minute, the excess stain was removed with absorbent paper. The slides were placed in 95% alcohol, the coverslip was removed, the specimen rinsed in 95% alcohol, then in 2 changes of Table 1. Chromosom
Nymph
Number
Source^
Hawaii0
Hawaiid
Hawaii0
Hawaiid
numbers of Pycnoscelus indicus malesa.
^he chromosomes in a maximum of 20 cells per individual were counted. If this number could not be found, as many cells as possible were examined. ^The original source from which laboratory colonies were started. ("From a culture of Hawaiian $ 9 X Hawaiian 8 8. Wrom a culture of Bogor 9 Q X Hawaiian $ 8. fiNymphs reddish.
Cells
Examined
Number of cells with
the following chromosome counts :
35 3 6 3 7 3 8




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19681 Roth å£ Cohen - Chromosomes of Pycnoscelus 5 5 100% alcohol for I minute each, and finally in xylene for about 5 minutes. Slides were made permanent by using a. mounting medium such as Permount or Permaslip.
The photographs of the chromosomes (mitotic metaphase) were taken with Kodak Contrast Process Pan 4 X 5 sheet film. Develop- ment time was 3.5 min
at 70å¡ in Kodak D-I I. Enlargements
were made up to 26ooX on Kodak Polycontrast paper. For karyotyping, the photographs of the chromosomes were cut out and roughly arranged by type and size. Centromeric indices
were then calculated and the values were used to place the chroma- somes in their respective classes (Levan et al., 1965). No attempt
was made to pair the chromosome,s and they are simply arranged by size, in each class.
RESULTS AND DISCUSSION
Pycnoscelus indicus. -The numbers of chromosomes found in P. indicus are summarized in Tables I and 2. Most of the specimens examined from our laboratory cultures have one of two modal diploid numbers of chromosomes: 2n ( ) = 35 or 37 (Table I ; Figs. I, 2) and 2n (9)
= 36 or 38 (Table 2; Figs. 3, 4). Of the 21 female nymphs examined, only I had 37 chromosomes (Table 2, nymph no. 18). It is possible that this female resulted from a cross between a male with 2n = 35 and a female with 2n = 38; a successful cross between these two fo'rms should give females with 3 7 chromosomes.
Whether these 2 chromosomal forms exist in nature is unknown. The original P. indicus culture was started from several females origi- nating in Hawaii in 1954, and additional specimens from the same island were added in 1958 (Roth, 1967). It is possible that both forms were originally established in the laboratory; but it is also conceivable that one of the chromosomal forms arose during the 14 years that this culture has been maintained (approximately 25 gen- erations - thousands of individuals were produced during this period). The scarcity of individuals with intermediate numbers of chromosomes, i.e., males with 36 (none were found among the 14 nymphs examined) and females with 37 chromosomes (only one found), suggests that the 2 chromosomal forms are not interbreeding successfully. There is apparently no way of distinguishing the 2 forms other than by chromosome count. Reddish or black nymphs may have either numbers of chromosomes (Tables I, 2, footnotes e and f). We are attempting to establish separate cultures of the



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56 Psyche
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two chromosomal types (by rearing offspring from isolated females) for further study.
Pycnoscelus surinamensis. - Although Ma,tthey ( 1945, I 948) found that parthenogenetic P. surinamensis has the same number of chromosomes as the bisexual species, it is evident that the number of chromosomes in this complex is highly variable. The modal diploid numbers of chromosomes for the various clones of P. surinamensis (Table 3)
are 34 (Brazil, Fig. 9; Australia,, Fig. 5 ; Thailand, Fig. 8), 35 (Thailand, Fig. 10) 37 (Indonesia, Fig. I I), 53 (US., Fig. 13; Brazil, Fig. 15), and 54 (Panama, Fig. 16; Jamaica, Fig. 17).
Of 650 cells examined from one specimen from Bogor, Indonesia (normally with 37 chromosomes), 38 cells ha,d 74 chromosomes, and Table 2.
Chromosome numbers of Pycnoscelus indicus femalesa. Sourceb
Hawaii0
Indonesiad
Indonesiad
Hawaii0
Indonesiad
Nymph
Number
Cells
Examined
2 0
2 0
20
2 0
2 0
2 0
Number of cells with
the following chromosome counts :
31 33 34 35 36 37 38
aThe chromosomes in a maximum of 20 cells per individual were counted. If this number could not be found, as many cells as ~ossible were examined. ^The original source from which laboratory colonies were started. CFrom a culture of Hawaiian $ $ X Hawaiian 8 8. dFrom a culture of Bogor $ 9 X Hawaiian $ 8. eNymphs reddish.
fNymphs black.




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Table 3. Chromosome numbers of the Pycnoscelus surinamensis complex. Number Examined 1 Number of Cells with the Following Chromosome Counts: Nymphs Cells
120 26 27 28 29 30 31 32 33 34 35 36 37 38 45 47 48 49 50 51 52 53 54 55 56 58 62 izil (Serra Tamendaui) Q
stralia (Fraser Island, off
eensland coast near
ryborough) - Q
- 8b
ailand (P,ak Thong Chai
mty, Sakaerat District) $
ailand
stchbouri County) Q
ailand
ak Thong Chai County) Q
lonesia (Bogor) 9
rida (Clearwater) Q
narna (Changuinola) $
maica (Mount Plenty 3
les from Goshan) 9
'he original source from which laboratory colonies were started. arthenogenetic males are periodically found in this culture from Australia; adult testes were examined.



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58 Psyche [March
612 had the normal 37. In 63 cells of a female from Florida (normally 53 chromosomes), 40 were normal and 23 cells had 106 chromosomes. These 2 individuals which had some cells with double their normal number of chromosomes may possibly have resulted from the colcemid treatment.
None of our clones of P. surinamensis had modal numbers of 36 or 38 as reported by Suomalainen (1945) and Matthey (1945, 1948). These writers did not indicate the number of cells counted, so it is unknown if the numbers given by them are the usual numbers present. Considering the variation in the clones we examined, it is quite possible that the numbers they give predominated in the in- dividuals of their colonies.
The types of chromosomes found in Pycnoscelus (Figs. 18-26) from different localities are summarized in Table 4. There are 3 classes of chromosomes: median, submedian, and subterminal. Me- dian chromosomes predominate and usually there appear to be only I or 2 subterminals. The males of P. indicus have one less sub- terminal chromosome than their respective females. This is also true of the Thailand parthenogenetic male and suggests that the subterminal chromosomes may be the X chromosomes. However, arguing against this is the fact that the Bogor female appears to lack subterminals and the parthenogenetic females from Florida and Belkm (53 chromosomes) lack one subterminal (Table 4), which should have made them males, if the subterminals were sex chromosomes and if these females have only 2 X chromosomes. In addition, if the Panama and Jamaica clones are triploids, the females should have 3 X chromosomes, and they apparently have only 2 subterminals (Table 4). It should be pointed out that several sources of error may affect the categorizing of some of the chromosomes. Not only
may colcemid treatment affect the relative lengths of the arms be- cause of contraction (Sasaki, 1961 ), but some of the chromosomes are so small that errors could be made in determining the location of the centromeres, and also in measuring the lengths of the arms. Most of the median chromoso~nes are readily classified, but there are some chromosomes which are on the borderline of the 3 categories. In P. indicus some individuals have 2 less chromosomes than others. There is no way of telling whether the parthenogenetic forms arose from bisexual stocks with 2n 36 or 38 chromosomes (Table 5). For example, the Brazilian and Australian forms (34 chromosomes) may have lost 2 submedians if they came from a 2n = 36 Q or, they may have lost 2 medians and 2 submedians if they came from a



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19681 Roth &? Cohen - Chromosomes of Pycnoscelus 59 Table 4.
Classification of the chromosomes of the Pycnoscelus complex. Source
Modal
Numbers
of
Chromo-
somes
P. indicus (Hawaii)
8
?
8
0
P. surinamensis
Thailand (Pak Thong Chai
County, Sakaerat District) $
Brazil (Serra Tamendaui) Q
Australia 9
Thailand (Pak Thong Chai
County) Q
Indonesia (Bogor) Q
Florida 9
Brazil (Belgm) 9
Panama (Changuinola) Q
Jamaica 9
3 7
I?,
3 5
3 6
3 3
3 4
3 4
3 5
37
5 3
5 3
54
5 4
Number a of:
1 Sub-
Median
Sub-
median
--
terminal 1
Figures
terminology after Levan et al. (1965). Other nomenclature systems refer to the 3 classes of chromosomes as metacentric (= median), submetacen- tric (= submedian), and acrocentric (= subterminal j. 2n = 38 stock. It is possible that the missing chromosomes may have been incorporated into other chromosome sets by translocation. The 2 fo,rms with 53 and 54 chromosomes are of interest because they ap- parently differ markedly in their chromosomal morphology. It is likely that these arose from parthenogenetic females rather than a diploid bisexual stock. Females with 53 chromosomes (Florida and Belgm) apparently have one less subterminal than those with 54 chromosomes (Panama and Jamaica). However, they differ marked- ly in that the Floridian and Brazilian females have 8 more medians and 8 less submedians than the Panamanian and Jamaican forms. This suggests that the 2 clones probably arose independently from different stocks; i.e., clones with 53 chromosomes did not necessarily arise from a form with 54 chromosomes like those found in the Panama and Jamaica clones, simply by a loss of one subterminal. The results confirm White's suggestion (in Roth 1967) that there could be several thelytokous biotypes of P. surinamensis related to the bisexual taxon of P. indicus. P. surinamensis is polymorphic, c~togenetically diverse, and probably arose polyphyletically by ~oly-



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60 Psyche [March
Table 5. Expected and observed diploid and triploid numbers of chromo- somes in the P. surinamensis complex.
n complement of
Expected diploid and polyploid numbers
the bisexual
of chromosomes in P. surinamens^s
taxon (P. indicus) 3 n
^The 2 numbers in parentheses indicate the deviation from the expected number if n = 18, or 19 in the bisexual taxon. 18
19
ploidy and/or aneuploidy. The 54 chromosome form could be con- sidered to be a triploid of the bisexual 36 chromosome species (n = 18). The aneuploid forms may have resulted from a loss of chi-omo- somes or from Robertsonian changes ; unfortunately, some of the chromosomes are so small that it was impossible to determine if Robertsonian changes were involved.
3 6 5 4
3 8 57
Modal number of chromosomes observed in
parthenogenetic clones
34 (-2 or 4 ) a
53 (-1 or -3)
35 (-1 or -3)
54 (å± or -3)
The color of the adults and nymphs may differ considerably. Nymphs may vary from red to bla,ck and adults vary from blackish 1 37 (+I or -1)
brown to almost solid black. The adults in clones with 34 chromo- somes [Australia, Petchburi (Thailand), and Serra Tamendaui (Brazil)] are the darker more blackish form. The relatively lighter colored adults from BeGm (Brazil) (53), Florida (53), Jamaica and Panama (54) have high chromosome numbers. However, this correlation is not constant since the Bogor (Indonesia) nymphs are reddish and adults are reddish brown, but they have 37 chromosomes. It is interesting that both the Serra Tamendaui (34), and Bel6m (53) forms are found in Brazil (the 2 areas are more than a thou- sand miles apart) and the adults of these 2 clones differ in color, the latter being more lightly colored; the parthenogenetic forms with the low chromosome numbers are not restricted to the Indo- Malayan region.
Roth (1967) found I I parthenogenetic males in his colony of P. surinamensis from Australia,. We have since noted additional males periodically produced in this laboratory culture and it seems that males are produced more frequently, probably by non-disjunction of



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1 9 6 Roth ^S Cohen-Chromosomesof Pycnoscelus 6 I
Figs. 1-4. Pycnoscelus indicus. Chromosomes of males and females from Hawaii (X 1300). 1. 8 2n = 35. 2. 8 2n = 37. 3. 9 2n = 36. 4. ? 2n = 38.




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62 Psyche
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the x chromosome, by the Australian females than by Floridian fe- males. Two of these Australian males had 33 chromosomes, as com- pared with 34 for the parthenogenetic female (Table I). No males have ever been observed in the Floridian culture in the more than 15 years that this colony has been maintained. Seiler ( 1967) stated that in the moth Solenobia triquetrella F.R., the older and more stabilized the parthenogenesis becomes, the rarer are the males which are produced. Whether the occasional production of parthenogenetic males in the Australian colony indicates a more recent development (in nature) of parthenogenesis than the Floridian clone is unknown. Dr. Narbel-Hofstetter (personal communication) has suggested that if non-disjunction and therefore loss of an x chromosome occurred in the triploid form, the result would be ^/aaa and not an ^/aa like that formed in the diploid clone. We have never seen intersexes in the triploid clones.
One male nymph from Thailand (Sakaerat District) had 33 chromosomes (Fig. 7). This was collected in the field and is pre- sumed to be the parthenogenetic species whose female chromosome number is 34 (Table I, Fig. 8) or 35 (Table I, Fig. 10) in Thailand.
The parthenogenetic male from Australia (33 chromosomes ; Fig. 6) and the Hawaiian males of P. indicus (2n = 35 or 37 ; Figs. I, 2) are non-functional when crossed with Floridian (53 chromo- somes; Fig. 13) or Australian females (34 chromosomes; Fig. 5) (Roth, 1967). Thus, males of P. indicus or the occasionally pro- duced males of P. surinamensis cannot introduce genes into the parthenogenetic population even though they mate with females; every parthenogenetic female is genetically isolated. This incom- patability can be accounted for in P. surinamensis by the fact that its parthenogenesis is apomictic; although the parthenogenetic male undergoes meiosis, the diploid number is maintained in the female as a result of the absence of chromosome reduction (Matthey 1948), and amphimixis cannot occur (Seiler, 1967). The lack of success in crossing the parthenogenetic male with P. indicus may be explained by the difference in chromosome numbers between these two species. The thelytokous parthenogenesis exhibited by P. surinamensis is not unique ; White ( I 954) and Narbel-Hofstetter ( I 964) have listed several cytologically similar (polyploids and aneuploids) par- thenogenetic species. Parthenogenesis in P. surinamensis conforms well with White's (1954, p. 341) statement that since no pairing of chromosomes takes place during maturation of the eggs in an apomic-



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19681 Roth &? Cohen - Chromosomes of Pycnoscelus 63 Figs. 5-7. Chromosomes of Pycnoscelus surin,amensis (X 1300). 5. Fe- male from Australia, 34 chromosomes. 6. Male from Australia, 33 chromo- somes. 7. Male from Pak Thong Chai County, Sakaerat District, Thailand, 33 chromosomes.




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64 Psyche [March
tic species, ". . . there is no 'mechanical' barrier to the establishment of any type of polyploidy in such forms and various forms of aneu- ploidy, due to irregular reduplication of some chromosome elements, must be expected to occur."
ADDENDUM
Since this paper went to press we have obtained the following ad- ditional information concerning Pycnosceius. Dr. Barbara Stay gave us several specimens of P. surinamensis which she collected in Queen Elizabeth Park, Lake Edward, Uganda, Africa. A colony has been established and there are 54 chromosomes in this clone (20 cells in 2 nymphs were examined). The number of chromosomes is the same as that found in the females from Jamaica and Panama. The African females also resemble the Jamaican and Panamanian forms in size, coloration, and wing length.
A colony of P. surinamensis was established from 2 adult females collected in Puraquequara, Rio Negro, Amazonas, Brazil. This clone has 39 chromo,somes (40 cells from 4 female nymphs were examined).
We also crossed 16 P. surinamensis females from Bogor, Indo- nesia (37 chromosomes) with males of P. indicus from Hawaii (35 or 37 chromosomes). All the females had sperm in their sperma- thecae when they were examined after producing young. Two hun-