linkage relationships and chromosome assignmentthe assignment of this linkage group to chromosome 3...

LINKAGE RELATIONSHIPS AND CHROMOSOME ASSIGNMENT OF FOUR ESTERASE LOCI IN THE MOSQUITO

ANOPHELES ALBlMANUS

SHARANJIT S. VEDBRAT'. AND GREGORY S. WHITT

Department of Genetics and Development, 515 Morrill Hall, Uniuersity of Illinois, Urbana, Illinois 61801

Manuscript received June 13, 1975 Revised copy received October 30, 1975

ABSTRACT

Although anopheline mosquitoes are important vectors of malaria, their genetic makeup has not yet been extensively investigated. The present studies concentrate on the genetic basis of esterases in Anopheles albimanus. Nine zones of esterase activity have been resolved by gel electrophoresis. Four of these esterases: EST-2, EST-4, EST-6, and EST-8 are present throughout all developmental stages and also possess allelic variation. Mass matings were carried out with homozygous males and females heterozygous for two or more loci. The analyses of the progeny from single egg batches revealed that the four esterase systems mentioned above are encoded in separate loci with codominant alleles. Analyses of two-point and three-point crosses have indicated the following linkage relationships: Est-8 - - 12% - - Est-4 - - 22% - - Est-2 - - 9% - - Est-6. The assignment of this linkage group to chromosome 3 has been accomplished by the use of a Y-2 chromosome translocation.

G E N E T I C analysis of Anopheles albimanus and other anopheline mosquitoes has, until relatively recently, been very difficult due to technical problems

such as: (1) lack of suitable genetic markers, (2) an inability to achieve single- pair matings in cages, or by forced copulation, and (3) the relatively long generation time of approximately thirty days as compared to two weeks for Drosophila and for Aedes aegypti. Previous information on the genetics of Anopheles albimanus has been limited to the description and mode of inheritance of certain morphological mutants such as stripe, bisignatus, and mutants resistant to dieldrin and DDT (KITZMILLER and MASON 1967). However, most of these naturally occurring mutants display continuous variation, probably due to poly- genic control and/or sensitivity to environmental factors, and thus are not very useful markers.

Earlier attempts have been made to establish linkage groups in anopheline mosquitoes for characters such as sex determination, stripe, and dieldrin resistance (FRENCH and KITZMILLER 1964; GEORGHIOU, GIDDEN and CAMERON 1967). However, within the last few years biochemical markers which are relatively

Present address Department of Cell Biology, Medical Center, University of Kentucky, Lexington, Kentucky 40506. Reprint requests should be sent care of G. S. WHITT.

Genetics 82: 451-466 March, 1976

452 S . S. VEDBRAT A N D G . S. WHITT

simple gene products have been successfully employed. Allelic isozymes have proven to be particularly useful gene markers.

The Esterases (EC 3.1.1.-) have been used to study the inheritance of simple gene-enzyme systems and to differentiate between different mosquito species (FREYVOGEL, HUNTER and SMITH 1968; TREBATOSKI and HAYNES 1969). The pattern of inheritance of two or more codominant alleles at different esterase loci has been described in Anopheles stephensi (BIANCHI 1968), A. atroparvus (BIANCHI and RINALDI 1970) , A . punctipennis (NARANG and KITZMILLER 1971a and b), Aedes aegypti (TREBATOSKI and CRAIG 1969; TOWNSON 1972), Culex pipiens ( GARNETT and FRENCH 1971 ) , and in C. tritaeniorhynchus ( IQBAL, SAKAI and BAKER 1973). More recently, a linkage relationship has been established for autosomal loci coding for an esterase, an acid phosphatase, and an alcohol dehydrogenase in A. stephensi (IQBAL et al. 1973). Autosomal acid phosphatase and alcohol dehydrogenase loci are linked with a recombination distance of 13.39% in females and 21.81% in males, and have been assigned to linkage group 11. The esterase locus, which assorts independently of sex and linkage group 11, has been assigned to linkage group 111.

The present linkage analyses of esterase loci in the mosquito Anopheles albimanus have a dual purpose: (1) to investigate the linkage relationships of homologous enzyme loci; (2) to assign the linkage group to a specific chromosome in this species. The present genetic analyses utilize the allelic variants previously described for four esterase loci (VEDBRAT and WHITT 1975). The pattern of esterase inheritance, the linkage relationships for the four esterase loci, and the chromosome correlation for the established linkage group will be presented in the present report. The significance of these results will be discussed in the context of evolutionary and developmental genetics.

MATERIALS AND METHODS

The basic techniques of rearing the A . albimanus stocks and the electrophoresis procedures for resolving and histochemically staining the esterases have been described elsewhere (VEDBRAT and WHITT 1974a, 1975).

Estublishment of a stock homozygous at each of four esterase loci

The homozygous stock was derived from a partially inbred laboratory stock of A. ulbimanus in three generations. Single-pair crosses cannot yet be carried out in this species; therefore, an indirect method had to be employed. It was assumed from indications in the literature that an egg batch, laid by a female, is a product of fertilization by sperms of a single male. Cages were spt up with ten freshly emerged males and 40-50 females from the laboratory stock. They were allowed to mature and mate f3r about a week. The females were then blood-fed and after three days, they were isolated in 3” x %‘’ glass vials. The lower one-third of the vials was lined with a strip of filter paper, and deionized water occupied approximately one-sixth of the vial. The females were isolated in such vials with the assistance of a mouth aspirator made from a glass tube with an extension of rubber tubing. The vials were covered with a cotton plug which permitted some aeration. The females laid eggs within a day or so. In this manner, individual egg batches were obtained. The female parent was frozen for a short period, then homogenized and subjected to electrophoresis to determine her esterase phenotypes. The egg batches of females which were homozygous for two or more loci were kept and raised until determinations of

ESTERASE LINKAGE IN A . albimanus 453

enzyme phenotypes could be made for individuals. If the male parent were homozygous, all the individuals from the egg batch would be homozygous for the locus of interest. Fifty percent of the individuals would be heterozygous at this locus if the male parent were heterozygous. All individuals from an egg batch would be heterozygous if the male parent was homozygous for the alternate allele. A sample of 5-6 individuals from each egg batch was analyzed at the pupal stage for esterase phenotypes. The egg batches in which all the sample individuals were homozygotes were considered to have a high probability of being a 100% homozygous egg batch and these egg batches were pooled and raised to adults. From this strain of pooled egg batches, 10 males and 40-50 females were placed in a cage and the whole selection process was repeated. Most of the females from this strain turned out to be homozygous for all the four loci. Their egg batches were raised to the pupal stage and a sample of 5-6 individuals was tested from each egg batch. Those egg batches, where all 5-6 individuals sampled were homozygous, were selected to be raised to maturity. The females which produced the eggs for the next generation were all homozygous at all esterase loci. In addition their egg batches were also tested before considering the stock to be homozygous. This stock was periodically retested to assure the maintenance of its homozygosity.

Crosses Males (40-50) from the homozygous stock and females (40-50) from the polymorphic

laboratory stock were kept together in a cage for approximately a week to ensure effective mating. The females were then blood-fed, isolated in vials after three days, and allowed to lay eggs. The egg batches of the females determined to be heterozygous for one or more loci were reared to the pupal or adult stage for the electrophoretic analyses. By employing this procedure, one could analyze crosses equivalent to single-pair matings. Pupae were used in most of the analyses because the esterases were well resolved at this stage. Pupae were frozen or allowed to develop to the adult stage if all the pupae could not be analyzed on the same day. Analysis of egg batches from females heterozygous for one locus provided inheritance data only, whereas egg batches from females heterozygous for twn or more loci were used for linkage relationships as well. Reciprocal crosses were analyzed only after the inheritance of alleles at these loci was clearly understood, i.e. when it was possible to predict the genotype of one parent if that of the other was known. At that time females from the homozygous stock were crossed with males from the polymorphic stock.

For the analyses of chromosomal correlation of the linkage group, the sex of the pupae was determined before they were used for electrophoresis. The sex was determined by examining the genitalia, already developed in the pupae, and also from the size of the individuals, the females being larger.

A translocation Y-2 chromosome stock was used to establish the chromosome correlation of the linkage group studied. The rationale behind the use of this stock is described in the RESULTS

section.

RESULTS

Electrophoretic uariants and their inheritance Nine zones of esterase activity are resolved in A. albimanus by starch gel

electrophoresis. The most anodal zone is referred to as EST-I and the most cathodal as EST-9. Allelic variants have been observed for four of the esterases: EST-2, EST-4, EST-6, and EST-8 (VEDBRAT and WHITT 1974b). EST-2 and EST-4 each has two different electrophoretic forms-EST-eA, EST-2B and EST-4A and EST-4B. In each case, the A variant is the more anodal isozyme. EST-6 and EST-8 exhibited three variants, A, B, and C according to their relative mobility on the gels. A is fast, B is intermediate and C is slow (Figure 1). The crosses were analyzed to study the inheritance of the variants of the four esterases. Each of the esterases is encoded in a separate locus with codominant alleles

454 S. S. VEDBRAT AND G. S. WHITT

ESTERASE ALLELlZ VARIANTS I & \ -

.,

c * a 0- . * - - * - * - - - - - - -. - - 1 - \ ( -1 I 70

FIGURE 1.-Allelic variants of Esi-2, Est-4, Est-6, and Est-8. The left zymogram displays the allelic isozymes encoded at the esterase loci 2,4, and 6. The right zymogram indicates the allelic isozymes of Est-8.

TABLE 1

Phenoiype frequency for EST-2, EST-4, EST-6, E S T 4 in the progeny of various crosses

Progeny phenotypes Totnl Estcrnscr Crorsrr AA An BB AC nC progeny x? Probability

EST-2 AB X BB 147 141 288 0.125 0.9-0.7

EST-4 B B X A A 48 48 -- BB x AB a5 84 169 0.004 0.95

A B X A A 100 102 202 0.019 0.9-0.7 A B X BB 23 20 43 0.210 0.7-0.5 B B X AB 20 23 43 0.210 0.7-0.5

A A X AB 135 163 298 2.63 0.2-0.1 A B X AB 16 13 11 60 1.43 0.5 [2 df]

EST-6 A C X BB 150 156 306 3.117 0.9-0.7 A C X AB 7 10 15 11 43 3.046 0.5-0.3[3 df] A B X AB 16 27 17 60 0.63 0.9-0.7[2 df] A B X BB 15 3 24 1.50 0.3-0.2 B B X AB 93 99 192 0.187 0.7-0.5

A A X BB 12 12 - BB x A A 43 43 -- BCX BB 19 29 48 2.08 0.2-0.1

EST-8 A C X A A 82 84 166 0.012 0.95-0.9 A A X AC 30 30 60 0 1

ESTERASE LINKAGE IN A. albimanus

TABLE 2

455

Test of linkage between the four esterase loci, Est-2, Est-4, Est-6 and Est-8 from two-point-cross analyses

LOCl Progeny % Recombinants A B Parentals Recombinants xz (95% confidence limit)

Est-2 Est-6 227 20 173.5 8.77 t 3.5 Est-2 Est-4 205 54 88.03 21.84 t 5.93 Est-4 Est-8 80 11 52.32 12.53 t 6.80 Est-2 Est-8 26 17 1.88 39.53 t 14.94 Est-6 Est-8 82 41 13.66 32.77 2 8.295 Est-6 Est-4 234 85 63.60 25.90 t 4.81

(Table 1 ) . The alleles appear to be segregating properly as indicated by the x2 analyses. The double-band phenotypes observed in heterozygotes suggest that these enzymes are monomers.

Linkage relationships for four esterase Loci Linkage relationships between four esterase loci have been established from

all possible types of “two-point cross” data (Table 2). x2 values for a test of inde- pendent assortment show significant deviation. There is moderately close linkage between the following pairs of loci: Est-2 - - Est-6; Est-2 - - Est-4; Est-4 -- Est-8. The recombination values for these pairs of loci always remain below 50% even after including the maximum likelihood limit of recombination (95 % confidence limits). Loose linkages between Est-6 and Est-8 as well as between Est-6 and Est-4 are also evident from the data. The summation of all these linkage data also suggests that Est-2 and Est-8 are linked even though the backcross data for these two loci alone show recombination near 50% at the maximum likelihood limit of recombination and the x2 value agrees well with the hypothesis of inde- pendent assortment. It may be assumed that the Est-2 and Est-8 loci are distant but on the same linkage group. The data from Table 2 show that the map distance between Est-2 and Est-8 and that between Est-6 and Est-8 are similar. They further suggest that Est-2 and Est-6 are closely linked. Thus the following two alternative gene orders may be constructed for all the four linked loci based on the average distance between all pairs of loci:

I Est-8 -- 12.5% - - Est-4 -- 25.9% - - Est-6 - - 8.7% -- Est-2 ___-__c-_ 4 I____- 32.7%---- ---_-- -_.... _ _ _ - _ --- _ _ _ _ _ _ - - - _ _ 39.5 yo- - - - - - - - - - - - - -- - - _ _ _

_-_-__-._- 21 go/ . o

I1 Est-8 - - 12.5 % - - Est-4 - - 21 .8% - - Est-2 - - 8.7% --Est-& --c___--- __-- ---39.5 yo- ---- --- - - _ y d J I - _ i - 4 4 d l _ _ l - -LUtdL 32.7% __-- -___-- C C r r - _ _ - -

-_LI-l-L-L 25.9%- -_- - --_ -- The map distance value between Est-2 and Est-4 does not fit well into arrange- ment I; thus gene order I1 has a higher probability of being correct. In order to

45 6 S. S. VEDBRAT AND G . S. WHITT

help establish the correct gene order for these loci, three-point backcrosses were also analyzed.

Analyses of the relative positions of Est-2, Est-4 and Est-6 from the three-point backcross data (Tables 3,4, and 5 ) reveal that Est-2 is in the middle with Est-4 and Est-6 on either side. No double recombinants were observed, probably because of the small sample size in each cross.

Data from a three-factor cross to analyze the order of Est-2, Est-4 and Est-8 indicate that the gene order is Est-8 - - Est-4 - - Est-2 (Table 6 ) .

The backcross data for Est-4, Est-6 and Est-8 reveal that the gene order for these loci is Est-8 - - Est-4 - - Est-6 (Table 7 ) .

Linkage data from the two-point crosses and three-point crosses have been summarized in Table 8, with a tentative linkage map at the end of Table 8.

Chromosomal correlation of the linkage group Anopheline species including albimanus are known to have a heteromorphic

pair of chromosomes in addition to the two pairs of autosomes (KITZMILLER 1967). The genetic proof of an XY system of sex determination has not yet been

TABLE 3

Linkage relationships between Est-2, Est-4, and Est-6 determined from the progeny of a backcross,

Est -9 Est-2’ Est-6a Est-& Est-2” Est-6” x _ _ _ _ ~ - - Est-4a Est-2b E ~ t - 6 ~ Est-4b Est-2b Est-6c

Progeny [ la-s

Parentals

Single recombinants between Est-2 and Est-4

Progeny genotypes

Est-la Est-2a Est-& Est-4a Est-2’ Est@ Est-4’ Est-2’ Est-6c Est-4” Est-2b Est-@ Est-4’ E ~ t - 2 ~ E~t-bc ______-- I Est-4a Est-2’ Est-6’ Est-4’ Est-2a Est-&


Double recombinants

Est-4a E ~ t - 2 ~ Est-6b

Est-4” E s t 3 Est-bb Est-4’ Est-Za Est-6‘ I1 --

Est-4’ Est-Zb Est-@ Est-4a E ~ t - 2 ~ Est-@ Est-4a Est-2’ Est-& Est-4a Est-2’ Est-6’ Est-4’ Est-2” Est-6c

Percent recombination

Gene order

Est-4a Est-2b Est& Est-2 - - E s t 4 15.91 Est-2 - - Est-6 6.82 Est-6 - - Est-4 22.73 Est-4 - - Est-2 - - Est-6

N

14

23

2

5

2

1

0

0

ESTERASE LINKAGE IN A. albimanus 45 7

TABLE 4


Est-+ Est-2a Est-6a -_______ x---- Est-4” E ~ t - 2 ~ Est-Gc Est-+ Est-2” E~t-6b

Est-4a Est-2b Est-6”

Progeny class Progeny genotypes N

Parentals


Est-4’ Est-2‘ Est-& Est-4’ Est-2” Est-6” Est-4b Est-2b Est-& _---____- Est-4’ Est-2” Est@ Est-4’ Est-Zb Est& Est-4a Est-2” Est-@

I

Single recombinants between Est-2 and Es t4

Double recombinants

Est-4’ Est-2” Est-6” Est-4‘ Est-2‘ Est-6‘ Est-4‘ Est-P Est-6”

I1

Est-4” Est-2” Est@ Est-4a Est-Zb Est-6” --_______ Est-4’ Est-2” Est-6’ - _ _ ~ _ _ _ Est-4a Esi-2b Est@ Est-4” Est-2’ Est-6c


Gene order

Est-4’ Est-2b Est-6” E s t 3 - - Est-4 22.97 Est-2 - - Est-6 10.80 Est-6 - - Est-4 33.77 Est-4 - - Est-2 - - E s t 4

26

23

8

9

4

4

0

0

obtained in this genus. It is not known whether A . albimanus possesses an X Y system as found in mammals and Drosophila, where most of the Y chromosome is heterochromatic and non-homologous with most of the X chromosome, or whether it possesses a sex-determining system which is intermediate between the X Y system of Drosophila and the Mm system characteristic of mosquitoes such as Aedes aegypti and Culex.

If the latter mechanism is possessed by A. albimanus, it would mean that this species has a heteromorphic pair of chromosomes (like the X Y system) which carry a sex-determining locus (or loci) on the Y chromosome, and that this chromosome also shares alleles with a number of loci on the X chromosome. Thus, it would be possible to obtain males heterozygous for enzyme loci in this kind of sex determination system in contrast to a true X Y system in which males are hemizygous.

In order to assign the linkage group containing these four esterase loci to a particular chromosome, the first step is to determine whether these loci are on a heteromorphic X Y sex chromosome pair or are autosomal. The data derived from employing heterozygous males in the present study of inheritance of the four

45 8 S. S. VEDBRAT AND G . S. WHITT

TABLE 5 Linkage relationships between Est-&, Est-4, and Est-6 determined from the progeny of a backcross,

Est-+ Est-2' E ~ t - 6 ~ Est-4" Est-2b Est-6a x-----.-.-- Est-+ Est-2b Est-6b Est-4' Est-2a Est@

~

Piogeny c1a.s Progeny genotypes N

Parentals Est-4a Est-2b Est-&



Double recombinants


Gene order

Est-4a Est-2b Est-@ Est-46 Est-% Est& Est-4a Est-2b Est-@ Est-4a Est-Za Est-66 Est-4a Est-2b Est-@ Est-4b Est-Zb Esi-6a Est-4a Est-2b Est@ Est-4a Est-2b Est-6b Est-4a Est-Zb Est& Est-4b Est-2a Est-@ Est-40 Est-Zb Est& Est-4a Est-$ Est-& Est-4a Es t -P Est-@ Est-4b Est-2b Est-6b Est-4a E ~ t - 2 ~ Est&

Est-2 - - Est-4 17.44 Est-2 - - Est-6 3.50 Est-6 - - Est-4 20.94 Est-4 - - Est-2 - - Est-6

-___-__- I

______-_

I1

_________

33

35

2

13

1

2

0

0

esterase loci (Table 1) suggest that these loci are autosomal, if the sex determination mechanism is of the true XY system type. However, these data are not able to exclude the possibility that the esterase loci are on the same chromosome containing one or more sex-determining genes.

Test of whether the four loci are autosomal or sex-linked In order to distinguish between the possibility that these loci are sex-linked in

an M m system with heteromorphic sex chromosomes and the possibility that they are on an autosome, it is necessary to construct the following testcross:

If males heterozygous for at least one of the four loci, e.g. at the Est-6 locus, are used, the variant allele in the heterozygote can be either on the X or the Y chromosome, which permits us to generate the two alternative hypotheses described below.

I. If the variant allele is carried on the Y chromosome which may also have the M allele for the sex locus, the result expected is shown in Figure 2. The majority of individuals will be homozygous females and heterozygous males, and only a few individuals, the products of recombination, will comprise heterozygous females and homozygous males for that locus.

ESTERASE LINKAGE IN A. albimanus 459 TABLE 6

Linkage relationships between Est-2, Est-4, and Est-8 determined from the progeny of a backcross, Est-8' Est-4' Est-2' Est-8' Est-4" Est-2' -.-----_x----- Est-8' Est-+ Est-2a Est-8a E s t 4 Est-2'

Progeny class

Parentals

Single recombinants between

Est-2 and Est-4

Single recombinants between

Est-4 and Est-8

Double recombinants


Gene order

Progeny genotypes

Est@ E s t 4 Est-2b Est@ Est-4b Est-2b Est-db Est-4' Est-2a

~

Est-@ E~t-4b Es t -9 Est-8' Esi-46 Est-2'

I Est-8' E s t 4 Est-Zb Est-8' Est-4' E~t-2b _ _ _ _ ~ - - Est@ Est-46 Est-2b Est-@ E s t 4 Est-2b Est-8' E s t 4 Es t -9 ___-_-- I1

Est-8' Est-4' Est-2' --_____ Est-@ Est-4b Est-2b Est-8' Est-4' Est-2b -____-- Est-8' E s t 4 Est-2b Est-@ Est-4b Est-2' Est-8a Est-4b Est-2b

Est-8 - - Est-4 20.93 Est-2 - - Est-4 27.90 Est-8 - - Est-2 48.80

______--

Est-8 - - Es t4 - - Est-2

N

14

10

4

6

3

4

2

0

Test Of Sex-Linkoge With Esterase - Loci

Parents in 6b m 6b x Q X 6'y U

m 6b M i

c Progeny

m 6b m eb

m 6b

X and 6a ] Major class

Y M

Homozygous 9 Heterozygous d

x m gb The

Homozygous 6bl d I Minor class m sb

Recombinants X

X and

6a ' M Heterozygous 0

FIGURE 2.-Test of sex linkage with esterase loci. X and Y represent the sex chromosomes. M and m represent hypothetical alleles of a sex locus. The Est-6 locus has been used as a hypothetical marker.

460 S. S. VEDBRAT A N D G . S . WHITT

TABLE 7


Est-8' Est-+ Est-6' Est-8' Est-4' Est-6'

Est-8a Est-4' Est-6'



Double recombinants

N

13

Progeny class Progeny genotypes

Parentals Est-8' Est-4a Est-6b

Est-@ Est-4a E s t 4 Est-Sh Est-4b Est-6c Est-Sa Est-4' Est-6h E ~ t - 8 ~ Est-4h Est-& _________ 1 E ~ t - 8 ~ Est-4a Est-@ Est-Rh Est-4' Est-@ -_-- 1 E ~ t - 8 ~ Est-4' Est&

16

I

Est-8' E~t-4a Est-6' 11 _--______ I1

Est-Sa Est-4a Est@ Est-86 Est-4h Est@ E ~ t - 8 ~ Est-4a Est-6h Est-8h Est-4a Est-& Est-Sa Est-4a Est& Est-8a Est-4b Est& Est@ Est-4a Est&

Percent recombination Est-8 - - Est-4 4.16 Est-4 - - Est-6 35.42 Est-8 - - Es t4 39.58 Est-8 - - Est-4 - - E s t 4

6

0

0 -_---- ___

Gene order

11. If the variant allele is carried on the X chromosome in males, the results would be opposite to those of Figure 2. The majority class ~vould be composed of heterozygous females and homozygous males, while a minor class, again the product of recombination, would be homozygous females and heterozygous males.

Table 9 gives the results of such analyses for three of the four linked loci and demonstrates that the linkage group is not associated with the heteromorphic sex chromosomes but is autosomal.

Assignment of the linkage group to one of the autosomes The linkage group for the four esterase loci is autosomal and should reside

either on chromosome 2 or 3 , named according to the salivary gland chromosome map (KEPPLER, KITZMILLER and RABBANI 1973). A stock of mosquitoes carrying the Y-2 translocation has been used to determine which one of the two chromosome pairs is correlated with this linkage group.

The use of the Y-2 translocation provides a test for pseudolinkage between the loci in question and sex. The procedure is essentially the same as that used for distinguishing whether the loci are autosomal or sex-linked. Again, testcrosses

ESTERASE LINKAGE IN A . albimanus 46 1

TABLE 8

Linkage relationships for four esterase loci: Est-2, 4, 6, and 8

Two-paint Number of progeny Gene sequence and map distances crosses per cross 8 4 2 6

I I I I , ~ - _ _ ~ - - - - - - I

Est-2 & 6 Est-4 & 6 Est-4 & 2 Est-8 & 6 Est4 & 6 __ - Three-point crosses

Est-8,4 & 6

Est-8,4 & 2

Est-4,2 & 6

Est-4,2 & 6

E ~ t - 4 , 2 & 6

43 43 12 46 29

48

43

74

86

43

I I_______ 39.5 --___-- I--- 4.2 - - I - - - - 35.4 _____ I

1--20.9--1--27.9--- I______ 48.8 ----I

I - - - - 33.7 -___ I

[----21-___- I

)----- 22.7 I

1- 23 --I- 10.8 -1

I--1 7.4----~--3.5-~

I--15.9---1---6.8---~

I Est-6

I I I Est-8 Est-4 E s t 2

were analyzed by using males carrying the translocation and heterozygous for at least one of the four loci, e.g. at the locus Est-6. These males were crossed with the homozygous females. It was not possible to determine the genotype of the male parent of a particular egg batch. Therefore, a sample of individuals was tested from each egg batch. The egg batch which showed some heterozygotes in the sample indicates the genotype of the male parent because the females used were homozygous. Such egg batches were used for the rest of the progeny analyses.

If the esterase loci were on chromosome two, the progeny would show the expected pseudolinkage as indicated in Figure 3. (Only chromosomes 2 and Y are shown in Figure 3 . ) Again, two alternatives are expected depending on the location of the variant allele Est-6". If the variant allele is on the translocated chromosome two, the main class of individuals will be homozygous females and heterozygous males. If the variant allele is carried on the nontranslocated homolog of 2 as opposed to the translocated chromosome 2 described above, homozygous males and heterozygous females are expected as a major class. The loci in question should be on chromosome 3 if all types of individuals are present in a 1 : 1 : 1 : 1 ratio. The data in Table 10 reveal that the linkage group is associated

462 S. S. VEDBRAT AND G . S. WHITT

TABLE 9

Test to determine whether there is sex linkage for three of the esterase loci

Progeny Female Male - Probability

Locus Cross no. Homo Hetero Homo Hetero XZ (P) 3 df.

Obs. Exp.

Obs. Exp.

Obs.

Exp.

2

3

4

Obs. Exp.

5

Obs. Exp.

Est-2 1

Obs. Exp.

Obs. Exp.

2

3

Obs. Exp.

Est-6 1

Obs.

Exp. 0 1

10 23 15.5 15.5 16 22

19.25 19.25 15 13 11 11 17 27

21.5 21.5 6 9 7.25 7.25

11 17

11 11

13 11 9.75 9.75

20 24

21.5 21.5

23 21 21.5 21.5

26 36 29 29

10 19 15.5 15.5 21 18 19.25 19.25

9 7 11 11 21 21 21.5 21.5

7 7 7.25 7.25

8 8 11 11

7 8 9.75 9.75

8.32

1.18

3.636

2.372

0.655

4.91

2.33

26 16 21.5 21.5

16 26 21.5 21.5

28 26

29 29

2.744

2.465

2.345

0.05-0.01

0.90-0.70

0.50-0.30

0.53

0.90-0.70

0.25-0.10

0.70-0.50

0.50-0.30

0.53-0.30

0.70-0.50

TABLE 10

Tests to determine whether the linkage group of four esterase loci is on chromosome 2 or 3 by utilizing a translocation Y-2 stock

Progeny Female Male - Probability

Locus Cross no. Homo IIetero Homo Hetero XZ (P) 3 df.

Obs. 11 9 12 12 Exp. 11 11 11 11

Obs. 9 11 13 11

Exp. 11 11 11 11

Obs. 14 13 14 11 Exp. 13 13 13 13

Obs. 6 8 10 4

Exp. 7 7 7 7

Est-6 1 0.545 .95-.90

Est-4 1 0.727 .90-.70

2 0.461 .95-.90

3 2.857 .50-.30

ESTERASE LINKAGE IN A. albimanus 463

Parents

Progeny

&2)

X U gb Q 6" (2-Yl,

gb gb

4 gb Major class

gb 6b

and

Q Homozygous d Heterozygous

Minor class

Recombinants IThe 6 O gb (2-& and

gb 6b, Heterozygous (Y-2) Homozygous

U FIGURE 3.-Use of the Y-2 translocation for chromosomal correlation of the esterase linkage

group. Only chromosomes 2 and Y have been shown in this figure. The Est-6 locus has been used as a hypothetical marker.

with chromosome 3 because of the 1 : 1 : 1 :I ratio observed in the progeny (the x2 values and their probability values agree with this hypothesis). It has been possible to reach this conclusion on the basis of studying only two of the four loci. Although the numbers are low, they are sufficient to exclude the possibility of pseudolinkage which would be expressed if the esterase loci were on chromosome 2. Because the other two loci, Est-2 and Est-8, are linked to these two loci (Est-6 and Est-4) , they are therefore presumed also to reside on chromosome 3 .

DISCUSSION

The genetic variation observed in the laboratory stocks of Anopheles albimanus for esterases is consistent with the variation reported for this group of enzymes in a wide variety of olrganisms (ALLEN 1960; RUDDLE and RODERICK 1966; JOHN- SON, RICHARDSON and KAMBYSELLIS 1968; JOHNSON and BURNS 1966; LEWONTIN 1973). The esterases have proved to be one of the most useful markers to differentiate between the neutral and the balanced natural selection hypothesis for the maintenance of definite polymorphic levels in populations and/or sibling species of Drosophila (AYALA et al. 1972; AYALA, POWELL and TRACEY 1972; RICHMOND 1972), vertebrates (SELANDER and JOHNSON 1973) and man (HARRIS and HOP- KINSON 1972). Such analyses can now be carried out with A. albimanus, which has a wide distribution and a moderate level of esterase polymorphism in its natural populations. The existing data indicate inversion polymorphism for some of the species of the subgenus Nyssorhynchus (KITZMILLER, KREU~ZER and RABBANI 1974) and A. albimanus is a member of this subgenus. The polymorphism in polytene chromosome structure can be supplemented with analysis of allele frequencies in these species to facilitate their biosystematics. Future investigations will also allow us to determine whether there is any correlation of a particular allele with a particular inversion prevalent in a population, as has been demonstrated in DrosophiZa robusta (PRAKASH and LEVITAN 1973).

464 S. S. VEDBRAT AND G . S. WHITT

The four esterase loci, for which linkage has been established, are expressed at the same time during development (VEDBRAT and WHITT 1975). This linkage of simultaneously expressed loci may be just a chance event, and this possibility cannot be excluded with confidence because of the low number of chromosomes in this species. However, the linkage of homologous esterase loci has been reported in a number of different dipteran species. In Drosophila montana, four esterase loci have been shown to be so tightly linked (ROBERTS and BAKER 1973) that the authors suggest they have all arisen from a single locus which has undergone repeated tandem duplications to produce four loci. In Drosophila melanogaster and D. simulans Est-& and Est-C loci are linked and are within a distance of 15 CM on chromosome 3 (TRIANTAPHYLLIDIS and CHRISTODOULOU 1973). In Drosophila virilis, Est-2 and Est-4 are linked and are on the second chromosome. In A. albimanus four linked loci are spread over a 50 map unit distance. But this distribution could conceivably have arisen from initially closely linked loci (originally products of tandem duplication events) by a series of inversions. Many inversions have been reported to occur in natural populations of related anopheline species (KITZMILLER, KREUTZER and RABBANI 1974). This postulate will be experimentally tested once a correlation of a particular enzyme locus to a particular band on the polytene chromosome is established.

It has been shown that three linked loci, Dieldrin resistance, black larvae and white stripe are also on chromosome 3 (M. G. RABBANI, personal communication). Chromosomally rearranged stocks will be used to associate these loci and the esterase loci with particular bands on the salivary gland chromosome. Thus genetic studies of these markers and other biochemical markers can be easily combined with the cytological analysis of salivary gland chromosomes to establish cytogenetic maps. These investigations will also greatly facilitate the study of cytogenetic problems of sex determination, somatic crossing over, dosage com- pensation, differences in crossing over frequencies between the two sexes, etc., in anopheline mosquitoes. The present study indicates that crossing over takes place in both sexes, whereas in some other dipterans, Drosophila males and Culex tritaeniorhynchus females, recombination does not occur in one sex between genes on the same linkage group.

Some translocations and inversions have been produced and studied in order to be able to employ them for genetic control (RABBANI and KITZMILLER 1972; and personal communication with KITZMILLER and RABBANI) . These studies are based on salivary gland chromosome analyses, hatching data and their relative compatibility as compared to normals. A better understanding of these chromosome anomalies could be obtained in terms of their effect on gene expression if genetic and cytological analyses are combined. For example, the effect of gene expression due to the proximity of a heterochromatic part of X , Y chromosome or chromocenter to any of these esterase loci (due to chromosomal rearrange- ments) could be determined from the zymogram analyses of the esterases. Thus, the knowledge of chromosome correlation to the linkage group of four esterase loci can help us to choose the type of translocations or inversions needed to study such a phenomenon.

ESTERASE LINKAGE IN A. albimanus 465

This research was supported by NSF grants GB-161.25 and GB-43995 to G.S.W. and PHS grant AI-03486 to J. B. KITZMILLER. We thank DR. J. B. KIZMILLER for the translocation stocks, and DRS. J. B. KITZMILLER and D. L. NANNEY for their helpful suggestions concerning the manuscript. Portions of this research were submitted in partial fulfillment of the degree of Doctor of Philosophy in the Cell Biology Program at the University of Illinois at Urbana.

L I T E R A T U R E C I T E D

ALLEN, S. L., 1960

ASLAMKHAN, M., M. AAQIL and M. HAFEEZ, 1972

Inherited variation in the esterases of Tetrahymena. Genetics 45: 1051-

Genetical and morphological variations in a natural population of the malaria mosquito, Anopheles stephensi, from Karachi, Pakistan. WHO/VBC/72.337, WHO/MAL/72.762 pp. 13.

Enzyme variability in Drosophila willistoni group. V. Genic variation in natural populations of Drosophila equinoxialis. Genet. Res. 20:

AYALA, F. J., J. R. POWELL, M. L. TRACEY, C. A. MOURAO and S. PEREZ-SALAS, 1972 Enzyme variability in the Drosophila willistoni group. Genic variation in natural populations of Drosophila willistoni. Genetics 70 : 113-139.

Genetica formale di una proteina dotata di attivita catalitica esterasica in Anopheles stephensi. Acc. Naz. Lincei. Rend. Sc. Fis. Mat. e Nat. 45: 3-4.

New gene-enzyme system in Anopheles atroparuus: occur- rence and frequencies of four alleles a t the Est-6 locus. Can. J. Genet. Cytol. 12: 325-330.

Linkage groups in Anopheles quadrimaculatus. Mosquito News 24: 32-39.

Nonspecific esterases in mosquitoes. J. Histochem. Cytochem. 16: 765-790.

A genetic study of an esterase in Culex pipiens, quinque- fascialus. Mosquito News 31 : 379-386.

A stripe character in Anopheles albimanus (Diptera: culicidae) and its linkage relationship to sex and dieldrin resistance. Ent. Soc. Am. Ann. 60: 323-328.

HARRIS, H. and D. A. HOPKINSON, 1972 Average heterozygosity per locus in man: an estimate based on the incidence of enzyme polymorphisms. Ann. Hum. Genet. 36: 9-20.

&?BAL, M. P., R. K. SAKAI and R. H. BAKER, 1973 The genetics of an esterase in Culex tritaeniorhynchus. Mosquito News 33 : 72-75.

IQBAL, M. P., M. K. TAHIR, R. K. SAKAI and R. H. BAKER, 1973 Linkage group and recombination in the malaria mosquito Anopheles slephensi. J. Heredity 6p: 133-136.

Electrophoretic variation in esterases of Colias eurytheme (Pieridae). J. Lepidopterists Soc. 20: 207-211.

Isozyme variability in species of the genus Drosophila. 111. Quantitative comparison of the esterases of D . aldrichi and D. mulleri. Biochem. Genet 1: 239-247.

KEPPLER, W. J., J. B. KITZMILLER and M. G. RABBANI, 1973 The salivary gland chromosomes of Anopheles albimanus. Mosquito News 33 : 42-49.

KITZMILLER, J. B., 1967 Mosquito cytogenetics. pp. 133-150. In: Genet'cs of Insect Vectors of Disease. Edited for WHO by J. W. WRIGHT and R. PAL. Elsevier Publishing Co., Amsterdam, London, New York.

1070.

AYALA. F. J., J. R. POWELL and M. L. TRACEY, 1972

19-42.

BIANCHI, U., 1968

BIANCHI, U. and A. RINALDI, 1970

FRENCH, W. L. and J. B. KITZMILLER, 1964

FKEYVOGEL, T. A., R. L. HUNTER and E. M. SMITH, 1968

GARNETT, P. and W. L. FRENCH, 1971

GEORGHIOU, G. P., F. E. GIDDEN and J. W. CAMERON, 1967

JOHNSON, F. M. and J. M. BURNS, 1966

JOHNSON, F. M., R. H. RICHARDSON and M. P. KAMBYSELLIS, 1968

466 S . S. VEDBRAT A N D G. S. WHITT

KITZMILLER, J. B. and G. F. MASON, 1967 Formal genetics of Anophelines. pp. 3-15. In: Genetics of Insect Vectors of Disease. Edited for WHO by J. W. WRIGHT and R. PAL. Elsevier Publishing Co., Amsterdam, London, New York.

Chromosomal polymorphism in populations of Anopheles albitarsis. Genetics (Suppl.) 77: S35.

KITZMILLER, J. B., R. D. KREUTZER and M. G. RABBANI, 1974

LEWONTIN, R. C., 1973 NARANG, S. and J. B. KITZMILLER, 1971a

Population genetics. Ann. Rev. Genet. 7: 1-17. Esterase polymorphism in a natural population of

Anopheles punclipennis. I. Genetic analysis of the esterase A-B system. J. Heredity 4 2 : 259-264. -, 1971b Esterase polymorphism in a natural population of Anopheles punctipennis. 11. Analysis 3f the esterase C system. Can. J. Genet. Cytol. 13: 771-776.

Associations of alleles of the esterase-1 locus with gene arrangements of the left arm of the second chromosome in Drosophila robusta. Genetics 75: 371-379.

RABBANI, M. G. and J. B. KITZMILLER, 1972 Chromosomal translocations in Anopheles albimanus. Mosquito News 32: 4'21432.

RICHMOND, R. C., 1972 Enzyme variability in the Drosophila willistoni group 111. Amounts of variability in the superspecies, D. paulistorum. Genetics 70: 87-112.

ROBERTS, R. M. and W. K. BAKER, 1973 Frequency distribution and linkage disequilibrium of active and null esterase isozymes in natural populations of Drosophila montana. Am. naturalist 107: 709-726.

RUDDLE, R. H. and T. H. RODERICK, 1966 Genetic control of two types of esterases in inbred strains of the mouse. Genetics 54: 191-202.

SELANDER, R. K. and W. E. JOHNSON, 1973 Genetic variation among vertebrate species. Ann. Rev. Ecol. Syst. 4: 75-91.

TOWNSON, H., 1972 Polymorphism in Aedes aegypti: the genetics and Km values of electro- phoretically heterogeneous forms. Ann. Trop. Med. Parasit. 66 : 255-266.

TREBATOSKI, A. M. and G. B. CRAIG, 1969 Genetics of an esterase in Aedes aegypti. Biochem. Genet. 3: 383-392.

TREBATOSKI, A. M. and J. F. HAYNES, 1369 Comparison of enzymes of twelve species of mosquitoes. Ann. Entomol. Soc. Am. 6 2 : 327-335.

TRIANTAPHYLLIDIS, C. D. and C. CHRISTODOULOU, 1973 Studies of homologous gene enzyme system esterase C in Drosophila melanogaster and D. simulans. Biochem. Genet. 8 : 383-390.

VEDBRAT, S. S. and G. S. WHITT, 1974a Lactate dehydrogenase and glycerol-3-phosphate dehydrogenase gene expression during ontogeny of the mosquito (Anopheles albimanus) . J. Exptl. Zool. 187: 135-140. -- , 1974b Linkage relationships of four esterase loci in Anopheles albimanus. Genetics (Suppl.) 77: S67. - , 1975 Isozyme ontogeny during development of the mosquito, Anopheles albimanus. Pp. 131-143. In: Isozymes. Vol. 111. Developmental Biology. Edited by C. L. MARKERT. Academic Press, New York.

PRAKASH, S. and M. LEVITAN, 1973

Corresponding editor: F. H. RUDDLE

linkage relationships and chromosome assignmentthe assignment of this linkage group to chromosome 3...

Documents