SEGREGATION O F CYTOPLASMIC INCOMPATIBILITY
PROPERTIES IN CULEX PIPIENS FATIGANS
SARALA K. SUBBARA0,I B. S. KRISHNAMURTHY,z C. F. CURTIS,3
T. ADAK1 AND R. K. CHANDRAHAS4
WHO/ICMR Research Unit on Genetic Control of Mosquitos, New Delhi, India
Manuscript received April 12, 1976
Revised copy received April 1, 1977
ABSTRACT
Maternally inherited variants, which arose within a laboratory colony of
Culex pipiens fatigans, have been studied by rearing cultures from single egg
rafts. Segregation, i.e., variation of cytoplasmic incompatibility properties between the male progeny of individual females, was demonstrated. Also, from
the daughters of individual females, sub-lines were derived within which all
the males showed the same incompatibility or compatibility properties. Among
the descendants of tetracycline-treated individuals were lines which superficially simulated these phenomena, but these lines ultimately reverted to the
cytoplasmic compatibility type of the strain which was submitted to the treatment. The types of variations in cytoplasmic incompatibility properties that
have been studied are discussed.
CYTOPLASMIC incompatibility is a naturally occurring phenomenon in the
Culex pipiens complex, whereby crosses between many strains of different
geographical origin are sterile. It has been shown by backcrossing that the
crossing type is a maternally inherited character without any involvement of
chromosomal genes (LAVEN1957). Field release experiments (e.g., LAVEN
1967) suggest that Cytoplasmic incompatibility could be used in the control
of the main mosquito vector of bancroftian filariasis. The Paris strain was
reported to be bidirectionally incompatible with the Delhi strain (KRISHNAMWRTHY and LAVEN
1972). However, SUBBARAO
et al. (1974, 1977) found
polymorphism in cytoplasmic types in Culex pipiens fatigans populations from
various parts of the Indian subcontinent. Males of the minority type (De 19)
were found to be compatible with all the cytoplasmic types tested. The majority
type (De 2), whose males are incompatible with Paris females, could be separated from the De 19 type by rearing single egg raft cultures. All the males
from any one of the cultures showed the same compatibility properties.
In addition to the polymorphism observed in natural populations, SUBBARAO
et al. (1977) reported the variants ISB 20 and ISB 49, which arose in a laboratory colony of the IS31B strain that has Paris cytoplasm (KRISHNAMURTHY
and
Piesent
Present
Present
Present
address
address
address
address
Malaria Research Unit (ICMR), 22 Sham Nath Marg, Delhl 110054, India
N.M E P ,569 5th Block, 11th Main Rd , Jayanagar, Bangalore, India.
London School of Hygiene and Tropical Mrdicine, London WClE 7HT, UK.
Vector Control Research Centre, 5/38 Lenin St , Kasapalyam, Pondichemy, India
c/o
Genetics 8 7 : 381-390 October, 1977.
382
s. K. SUBBARAO et al.
LAVEN1976). This paper reports studies on ISB 20 and ISB 49, which revealed
the occurrence of variation among the progeny of individual females. A similar
phenomenon has been briefly reported by FRENCH
(1970).
YEN and BARR(1973) treated larvae with tetracycline and derived a line
whose males showed universal compatibility and whose females showed sterility
in matings with all untreated mosquitos. It is not known to what extent the
mechanism of the sterility in these matings is similar to the incompatibility
between different wild-type strains. Electron microscopy revealed that the treated
mosquitoes were free from rickettsiae, while the untreated mosquitoes contained
large numbers of rickettsiae (YEN and BARR1973, 1974), and the progeny of
treated female mosquitoes were referred to as aposymbiotic lines. The authors
proposed that these rickettsial organisms are responsible for the cytoplasmic
incompatibility phenomenon. The present paper also reports studies on tetracycline treatment of both larval and adults mosquitos.
MATERIALS A N D METHODS
Stocks
The following stocks were used in the experiments:
De
: a laboratory colonized strain of Delhi origin ( SINGHet al. 1975).
De 2 : Single egg raft isolate of the majority type from De.
De 19 : Single egg raft isolate of the minority type from De.
: Paris cytoplasm with Delhi genome (KRISHNAMURTHY
and LAVEN1976).
Pa
IS31B : Paris cytoplasm with Delhi genome, including a male-linked translocation (KRISHNAMURTHY and LAVEN
1976).
Ha
: Hamburg cytoplasm with Delhi genome (KRISHNAMURTHY
and LAVEN1976).
Ba
: Bangkok cytoplasm (type A) with Delhi genome (KRISHNAMURTHY
and LAVEN1976).
Pr
: Prague cytoplasm with Delhi genome (SUBBARAO
e t al. 1977).
Experimental procedure
To avoid the effects of aging i n causing partial compatibility (SINGH,CURTISand KRISHNA1976), all mating were carried out with two- t o three-day-old males. Chicks were provided as a blood source on the fourth day after mating, and egg rafts (which are clumps of about
150 eggs laid by individual females) were collected on the ninth day. The hatchability of the
egg rafts was scored after two days. In the great majority of cases incompatible and compatible
egg rafts were qualitatively distinct with no egg hatch in the former and a high percentage
of egg hatch in the latter. However, in a few cases rafts gave a hatch of 1-5 larvae and these
were classified as incompatible, since a small percentage of eggs are known to hatch after incompatible matings as a result of parthenogenetic development (LAVEN1957). From compatible
matings, in which the male parent had inherited the male-linked translocation of the IS31B
stock, the egg hatch was reduced to about 50%. This nuclear genetic marker provided a check
against contamination of the experimental stocks by untranslocated material maintained in the
same laboratory o r from the local wild papulation, and it did not reduce the egg hatchability
into the range where it could have been confused with that of incompatible egg rafts.
MURTHY
Single raft cultures
In order to study samples of progeny from individual females, egg rafts were reared in
individual bowls. Some of the males and females from each culture were inbred and the remainder were test-crossed with various cytoplasmic types.
383
SEGREGATION O F CYTOPLASMIC GENES
Tetracycline treatment
(a) Larval treatment: larvae of the Pa strain were reared in concentrations of 0.025 and
0.05 mg/ml tetracycline, the solution being changed on alternate days. Treated males and
females were inbred and males were testcrossed with Delhi females. Progeny of treated inbred
lines were testcrossed with all the available cytoplasmic types.
(b) Adult treatment: in the normal rearing procedure, cotton pads with 1% glucose solution
are provided for feeding adults, and for experimental purposes 0.1 or 1 mg/ml tetracycline was
added to the solution for the first four days of adult life.
RESULTS
Variation in IS31B colony
As reported by SUBBARAO
et al. (1977), the males from 50 single egg raft cultures from the IS31B colony were testcrossed with Delhi females to determine
their crossing type. Males from two of the rafts, designated ISB 20 and ISB 49,
showed compatibility, while all males from the other 48 cultures showed incompatibility with Delhi females (Figure 1 ) .
All males of ISB 49 showed compatibility with females of all the available
cytoplasmic types, but females of ISB 49 retained the Paris compatibility and
incompatibility properties (Figure 1) .
ISB 20 females, like ISB 49 females, retained Paris compatibility/incompatibility properties, but among males of ISB 20, unlike ISB 49, some individuals
showed compatible and others incompatible properties with respect to De 2,
De 19, Ba and Ha females (Figure 2). We refer to such variation among the
progeny of a single female as “segregation,” as was done by FRENCH
(1970).
SINGLE R A F T CULTURES FROM I S 31 B ( P a
CYTOPLASM)
I
ISB 49
I S B 1-19,21-48
AND 5 0
IS6 20
5
-De2??
19
V
+
+
SEE FIGURE 2
[
I
IS 31 B COLONY
HAMBURG
Ba
Pr
TT
&
--y
X
Y
PARIS
BANGKOK
PRAGUE
TETRACYCLINE T R E A T E D
COMPATIBLE
INCOMPATIBLE
NO. OF COMPATIBLE R A F T S
NO. OF INCOMPATIBLE R A F T S
FIGURE
1.-Effect of isolation of single egg rafts from the IS 31B colony.
384
s. K.
SUBBARAO
et al.
IS B 20
Ba 66
Pr
66
SINGLE
~
I
I
I
I
(6) ( e ) W l
(1) (2) ( 5 )
(POOLED DATA FOR
PURE COMP. LINES)
-
??A
??
R A F T CULTURES
I
I
I
I
(13)k(l4)
(POOLEO DATA FOR
PURE INCOMP. L I N E S )
Den??
+
?:9:-3~-De2??
b ~ f ~ D e 2 ? ???
bb-,;.-Da2??
I
I
I
I
I
I
(3) (4) (7)(9) (IO) (11)6(121
(POOLED D A T A F O R
SEGREGATING L I N E S )
,uI-1
??y&cf-%-DE2?
?
S E E FIGURE 3
FIGURE
2.-The behavior of the ISB 20 line continued from Figure 1 . Segregating lines are
defined as descendants of individual females among which some males show compatibility and
others incompatibility with De 2 females. Pure lines are defined as those i n which all the males
tested were either compatible or incompatible with De 2. Strain designations are as defined in
Figure 1 .
Fifteen single-raft cultures were reared from ISB 20 to investigate the genetic
constitution of the ISB 20 line. These were designated ISB 20( 1)-( 15). Females
and males were mated and males were also testcrossed with De 2 females (Figure
2). Six lines (with a total of 111 rafts) gave males, all of which were compatible,
two lines (with a total of 30 rafts) gave all incompatible males, while the remaining seven lines showed a segregation among the males similar to the original
ISB 20 lines.
Two of these segregating sublines ISB 20 (7) and ISB 20 (9) were tested further
by rearing single raft cultures, ISB 20 (7) a-m and ISB 20 (9) a-o, and test-crossing
the males (Figure 3 ) . I n sublines ISB 20 (7) g and ISB 20 (9) f, it was found
that male progeny were, respectively, all compatible and all incompatible with
De 2 females but, in the other sublines, segregation continued.
To test whether the properties of ISB 20 and ISB 49 were cytoplasmically or
chromosomally inherited, males and females of each type were outcrossed to
the normal Pa stock. When the males of the ISB 20 and ISB 49 stocks were outcrossed, the male progeny showed the properties of the Pa stock, i.e.,bidirectional
incompatibility with De 2. Conversely, when females of ISB 49 and of several of
385
SEGREGATION O F CYTOPLASMIC GENES
I
I
IS0 20(9)
IS0 20(7)
??
'-dd=$r=De2??
ISB20(7lo-m
S I N G L E R A F T CULTURES
b
o - f ' a h-m
(POOLED DATA FOR A L L
SEGREGATING L I N E S )
(PURE COMP.
LINE)
SlNGL:E R A F T CULTURES
is6 2 0 (9)a-o
I
I
a - e AND g - 0
(POOLED DATA F O R A L L
SEGREGATING L I N E S )
??
: -i-
; __
1
COMOATIBLE
INCOMPATIBLE
NO O c COMPATIBLE R A F T S
NO O F INCOMPATIBLE R A F T S
FIGURE
3.-Further
f
( P U R E INCOMP.
??
]
data on two sublines of the ISB 20 stock continued from Figure 2.
the sublines of ISB 20 were outcrossed, the male progeny retained the properties
of the line to which their maternal parent belonged. This remained true when
four successive backcrosses of females to Pa males were carried out.
Studies with tetracycline
(a) Larual treatment 01 the Pa strain: There was extension of larval development time and larval and adult mortality following tetracycline treatment, and
from the 0.05 mg/ml treatment the few rafts obtained from the surviving adults
failed to hatch.
In the matings between males and females treated with 0.025 mg/ml there
were abnormally small rafts but a small number of larvae were obtained.
Treated males were also testcrossed with De females, and the rafts showed a
mean hatch of 20% (range 0.7-67%), i.e., the incompatibility barrier had been
partially broken. The F, male progeny from treated females x treated males were
compatible with both De and Pa females (Figure 4). The F, female progeny
were incompatible with De 2 males, but a majority of females were compatible
with Pa males. Thus, the majority of individuals appeared to belong to the cytoplasmic type that has been designated ISB 49, while the minority resembled
YEN and BARR'Saposymbiotic lines in that the females gave sterile rafts when
mated with the Pa type, i.e., the strain from which they were derived.
In the F, generation, males varied in their compatibility with De, and females
again showed either fertility o r sterility when mated with Paris males (Figure
4). To study this variation further, 15 single raft cultures, TT1-15, from the F,
generation were reared. and the males and females from each were testcrossed
with De females and Pa males, respectively (Figure 4). Twelve cultures were
386
s. K.
SUBBARAO
P a L A R V A E T R E A T E D WITH
TETRACYCLINE
T T 1-15
SINGLE R A F T {ULTURES
n
P a 66
-?P
i
et al.
,
I
1
TT5 T T 7
I
I
, ,
I
T19,
66%,
De??
---- COMPATIBLE
INCOMPATIBLE
- k $ f PARTIALLY COMPATIBLE
--
X
Y
SEGREGATION O F
COMP. AND INCOMP.
INDIVIDUALS
NO. OF COMP. RAFTS
NO. OF INCOMP. RAFTS
d
SINGLE R A F T CULTURES
a-g. i-m, o, q-1
cL4
FIGURE
4.-The effects of treating larvae of the Pa strain with 0.025 mg/ml tetracycline.
Designations are as in Figure 1, except that the symbol - - - is defined as “incompatible/sterile”
since it is not known whether the mechanism of sterility is the same in matings of the female
progeny of tetracycline-treated material as it is in matings between different wild-type strains.
found to regain Paris cytoplasmic characters, i.e., the males were incompatible
with De and the females were compatible with Pa. In two lines, TT5 and TT7,
males were compatible with De females, but females produced sterile rafts when
mated with Pa males. At the next generation, males and females from these two
lines were testcrossed with females and males of all the available cytoplasmic
types (Figure 4). Males from both lines were found to be compatible with all
cytoplasmic types, but females produced sterile rafts when mated with the Pa
type (from which they were derived) and with several other types. However,
the T T 5 and TT7 females were partially compatible with De 19 males: the
mean hatchability was 8.4% with a range from 0.0-25.3% hatch. Eggs from
SEGREGATION O F CYTOPLASMIC GENES
387
unhatched and partially hatched egg raFts from these crosses were predominantly
unembryonated. The fertility in matings within the TT5 and T T 7 lines was
almost normal (mean egg hatchability of 88.8%).
As shown in Figure 4, the single-raft culture TT9 showed a mixture of female
types, and further single-raft isolations from it produced lines that had reverted
to Paris properties, and other lines that showed segregation among males similar
to that of the ISB 20 line described above. However, subsequent results (not
shown in Figure 4) indicated complete reversion of the T T 9 line to the Pa type.
(b) Adult treatment of Pa strain: Following 1 mg/ml tetracycline treatment,
only one of the rafts laid by the treated females hatched, but following 0.1
mg/ml tetracycline treatment, all the rafts hatched.
Progeny from both the treatments were reared, and the males and females
were then testcrossed with De females and Pa males, respectively. Males derived
from parents treated with 1 mg/ml showed compatibility with De females and
the females were compatible with Pa males. These properties resemble the ISB 49
isolate described above. Some of the males from the 0.1 mg/ml treatment showed
compatibility with De females, while others showed incompatibility, but all
females were compatible with Pa males, i.e., the results resembled those with
ISB 20. However, after two generations of breeding (without any further treatment) of both the 1.0 mg/ml and 0.1 mg/ml treated lines, it was found that the
compatibility of the males with De females had reverted to the Pa cytoplasmic
type, i.e., to the properties of the stock from which they were derived.
DISCUSSION
SUBBARO
et al. (1977) showed that the origin of the ISB 20 and ISB 49 types
was a mutation-like process from a previously cytoplasmically invariable colony.
The present paper demonstrates that the properties of the two new variants ISB
20 and ISB 49 were maternally inherited. ISB 49 provides a n interesting analogy
with the naturally occurring De 19 variant of Indian populations (SUBBARAO
et al. 1974, 1977). The males of both of these types were compatible with all the
strains available €or testing, and the females show typical compatibility/incompatibility properties. In the case of ISB 49 the properties of the females resemble
those of Pa, and in the case of De 19 they resemble the majority Delhi type,
De 2.
In the ISB 20 line there was variation in the compatibility properties of sons
of an individual mother. In addition, the line gave rise to pure sublines, within
which all males showed the same properties with respect to females of several
other cytoplasmic types. It seems probable that the segregations between the
sons of individual females and between the lines derived from the daughters of
individual females are related phenomena. We suggest that, between the oocytes
produced by individual ISB 20 females, there is an unequal assortment of the
cytoplasmic determinants of incompatibility. It might be that, in females of the
ISB 20 line, two different types of rickettsia co-exist, and that some of the progeny
inherit rickettsiae all or predominantly of one type. Alternatively, one might
388
s. K.
SUBBARAO
et al.
postulate only one type of rickettsia and that some individuals inherit reduced
numbers of them. However, the numbers of rickettsiae in normal C. pipiens
oocytes are very large (YEN and BARR1974), and the segregations we found
were clear-cut, and individual males with an intermediate level of compatibility
were not found. It appears, therefore, that either of the above two hypotheses
would require the additional postulate of threshold densities of rickettsiae, above
which a male produces all incompatible sperms and below which it produces all
compatible ones. The existence of such sharp thresholds seems to us rather difficult to imagine, and we suggest as another alternative hypothesis that the process
of unequal assortment may not act at the level of the rickettsiae themselves, but
rather may involve other cytoplasmic factors that control the rickettsiae. It is
suggested that these controlling particles may be present in small numbers SO
that they could be entirely absent from some oocytes due to “sampling error.”
SAGER
and RAMANIS
(1968, 1970) have mathematically analyzed their data on
cytoplasmic variants in Chlamydomonas. Though there is some quantitative
information in our Culex p . fatiguns data (Figures 1, 2 and 3 ) , we consider it
premature to attempt to frame any detailed hypothesis on the segregation process.
The occurrence of several sublines of ISB 20 all of whose males were compatible with De 2 females suggests that the ISB 49 variant is probably a secondary derivative of the original ISB 20 “mutation”.
As shown in Figure 4, from treatment of larvae with 0.025 mg/ml tetracycline,
two lines (TT5 and TT7) were derived whose properties resembled those of the
aposymbiotic lines of YEN and BARR (1973), i.e., the males showed no incompatibilities and the females were compatible with males of their own lines, but
produced sterile rafts when mated with males of the Pa strain from which they
were derived. Electron microscope studies by C. M. S. DASS,University of Delhi,
India (personal communication) revealed no rickettsiae in oocytes of the TT5
and TT7 lines, whereas rickettsiae were readily demonstrable in the De 2, De 19,
IS31B, ISB 20 and ISB 49 strains. Thus, we regard 73’5 and 1 T 7 as aposymbiotic. It was found that these females were partially compatible with the
naturally occurring De 19 type, and there therefore appears to be a limited
degree of resemblance between the De 19 type and the aposymbiotic type. It
should be recalled, however, that De 19 females show conventional compatibility/incompatibility properties, unlike aposymbiotic females. The partial
resemblance of De 19 males to the aposymbiotic type suggests that it may not
be possible to find a cytoplasmic type whose females are incompatible with it.
This would have important practical consequences for the application of cytoplasmic incompatibility for genetic control because it would allow “recombination” of ti released cytoplasm with indigenous Indian genome (CURTIS1976).
As noted in Figure 4, ISB 49 differed from De 19 in being incompatible with
aposymbiotic females. The results reported suggest a gradation of properties in
males as follows: (a) De 2 and Pa; (b) ISB 20; (c) ISB 49; (d) De 19; and (e)
aposymbiotic. Whether this gradation reflects an underlying gradation in density
of rickettsiae, or of some other particle, remains to be determined.
SEGREGATION O F CYTOPLASMIC GENES
389
The effects of tetracycline treatment of larvae were not uniform; in addition
to the TT5 and T I ” 7 (aposymbiotic) derivatives of the treatment, other types
were found (Figure 4) some of which superficially resembled ISB 20. Unlike the
latter stock, these tetracycline-derived segregating stocks all ultimately reverted
to the parental Pa type. By contrast, from ISB 20, in addition to the Pa-type
revertants [ISB 20 (13), ISB 20 (14) and ISB 20 (9) f in Figures 2 and 31, there
were ISB 49-type sublines [ISB 10(1), ( 2 ) , (5), (B), and (15) in Figure 2 and
1SB 20 (7) g in Figure 31 and lines where segregation of male types continued for
further generations [ISB 20(7)a-f and h-m, and ISB 20(9)a-e and g-o in
Figure 31.
Adult treatments with tetracycline at different concentrations gave lines which
initially simulated ISB 49 and ISB 20. However, once again the tetracycline lines
reverted to Pa type after a few generations, unlike ISB 49 and ISB 20. The
eventual reversion to parental type of lines derived from tetracycline treatment
of larvae or adults suggests that in many cases rickettsiae are incompletely
cleared by tetracycline (as also found by PORTARO
and BARR1975) and that
normal concentrations can be restored after several generations.
The effect of tetracycline on adults could be of importance in a genetic control
program because the chickens kept for blood ieeding of mosquitoes in a massrearing colony (SINGHet aZ. 1975) have been dosed with antibiotics to protect
them from intestinal infections. Studies are required of the effects on incompatibility of feeding mosquitoes on chickens dosed with various concentrations of
tetracycline.
This paper and other recent publications have shown that the earlier “all-ornone” picture of cytoplasmic incompatibility is complicated by the following
phenomena:
(a) Polymorphism of cytoplasmic types in wild populations, e.g., De 19 and
et al. 1974 and 1977) and a similar
De 2 in Indian populations (SUBBARAO
phenomenon in Bangkok (KRISHNAMURTHY
and LAVEN1976).
(b) Segregation between the sons of a single female in their compatibility
properties, e.g., ISB 20 (Figure 1).
(c) Segregation of sublines from the daughters of one female, the males of any
(1970) and ISB 20 (7) g
one subline showing invariable properties, e.g.,FRENCH
and ISB 20 (9) f in Figure 3.
(d) Partial compatibility of certain wild-type males leading to partial egg
hatch of individual rafts, e.g., Hanford females x Scauri males (BARR1970).
(e) Partial compatibility of aged Pa males with De females (SINGH,CURTIS
and KRISHNAMURTHY
1976),
(f) Partial compatibility of aposymbiotic females x De 19 males (Figure 4 ) .
(g) Partial compatibility of newly tetracycline-treated males (YEN and BARR
1974and 1975, andFigure4).
We are grateful to R. C. JUYAL,
D. C. JOSHIand SATISHKUMARfor their technical assistance.
for his encouragement throughout this work and DR. K. R. P.
We thank DR G. D. BROOKS
SINCHfor his comments on the manuscript.
390
s. K.
SUBBARAO
et al.
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Corresponding editor: N. W. GILLHAM