MUTANTS OF THE PARASITIC WASP DAHLROlMINUS FUSCZPENNIS (ZETT.)

(HYMENOPTERA : EULOPHIDAE) W. F. BALD~VIN, E. SHAVER AND A. WILI~FS

Atonaic Etaergy of Cnnndn Lhtrited, Cbnlk River mad Entm~zology Rcsenrch Institzrte, Cn~indn Depnrtnrent of Agriczdture, Ottnwn, Ontnrio

In the wasp Dahlbo7?zi1zzrs fzrscipe~z~zis (Zett.), an insect which produces haploid males from unfcrtilized eggs and diploid fernales from fertilized eggs, many individuals that are strikingly different from the wild type have appeared in recent breeding work. A number of these individuals have been found t o

V

be true mutants and, although some have already been re fe l~cd to in the litera- ture, none has previously been tested genetically or described. It is the purpose of this report to describe and assign names and symbols to those proven so far to be true mutants. T h e s~ec ies will be dcscribcd brieflv and an account given of its lifc historv and 1ab;ratorv rearin; methods. r he illore detailed V i i u

data on biology of the species is intended to provide a source of basic informa- tion and general introduction to further genctic rcsearch with this insect.

Although two of the mutations tested to date involve pupal and body colours and a number the development of the appendages, many recessive eye- colour mutants have appeared ranging from black, approaching the dominant wild-tvue blacltish brotvn, throuph claret, carmine. chestnut and russet. Some

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of the eye-colour mutants occur at the saine locus, but most of thosc referred to in the present report are scattered, segregating independently and, as far as is known at present, ~v i t h no linltage. Allost of the nlutations hivc arisen from irradiation studics of Baldwin (1962).

MATERIALS AND METHODS Life History md Descriptio~z

Dilhlbo~?zinz~s fz~scipe1~17is is a sillall ectoparasite placed by most authors in the hymenopterous fanlily Eulophidac. Although first reported by Zctterstedt (183g) under the name Entedon fZ~scipe?mis (Zett.) as a parasite of lepid- opterous pupae, it is 110117 recognized as Dahlbo1?7i~7z~s fZ1scjpe7l1zis (Zett.) and almost exclusively as a parasitc of diprionid sawflies. For details of its syn- onyinv and bibliography to 1959 sce Peclc (1963).

- This parasite has a rather wide distribution in Europc and Scandinavia where it reachcs its maximum abundance in the warmer, more southerly climates; the temperature and rainfall in thesc countries appear to favour its development. Until its introduction into Canada in 1934 by the Canada Department of Agriculture as an agent of control of the spruce sawfly, Diprio~z he7.cy1ziae (Htg.) there were no records of the occurrence of this species in North America. T h e only papers dealing with its lifc history are those of h!Iorris and Cameron (19j5) and Finlayson and Reelts (1936). Thesc are somewhat i~icornpletc and the general features of thc lifc history, supplemented by more current observations, arc thereforc outlined here.

Adults enierge from host cocoons at any tiille during the 24 hours. Usually only onc emergence hole is made through thc exposed surface of the

Issued as AECL-2105. Manuscript rcceivcd Aug. 3, 1964. Can. J. Genet. Cytol. 6 : 453-466. 1964.

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454 W. F. EALD\\'IX, E. SHAVER AND A. \VILI<ES

cocoon. In cocoons containing both lllales and females, the hole is gnan-cd by the female. Her emergence is followed immediately by that of a male; the remaining adults continue to pass through, using the edge of the hole to assist in the removal of thc pupal integument. Where cocoons contain only males, thc emergence hole can be cut by a male but only when the cocoon is moist and pliable.

i\/Iating takes place immediately after emergence. Following a prelinlinary courtship of less than a minute, during \vhich the nlale n ~ o ~ l n t s the female and stroltes her antennae, the female raises her abdoinen and copulation is cffectcd in a fen, seconds. T h e male inlmediately thereafter goes in search of another female. Fclnales will inate rnorc than once and sometimes after intervals of sevcral days, but usually they resist any attempts by thc males to copulate more than oncc. iblales arc capablc of mating successfullv with at least 25 females.

Oviposition may ta le place immediately aftcr mating. Virgin fcmales are at first disinclined to oviposit, but after a short delay they will lay their eggs as freely as do mated individuals and with no significant difference in thc number of eggs deposited. Before oviposition begins the female picrces the cocoon several times \vith her ovipositor, indicating that paralysis of thc host is not always cffccted by the initial puncture. Following paralysis of thc host, the eggs are deposited hosely in clusters of about 20 on the integument of the host larva. Fronl laboratory stock the average number of progeny per female is 90; the maximum obtained was 21 5 from one individual.

The tiine required for hatching of thc egg and subsequent developmcnt of the immature stagcs is determined largelv by the tcmpcrature to nrhich they are exposed, within the range 12" to 3 5 " ~ . T h e eggs are l;illed by 1 8 hours esposurc to 35°C and the larvae and pupae by 96 hours esposurc (Willtes, 1959). At 23"C, the period from oviposition to adult emergence is about 18 days. The time required for developnicnt of the different stagcs at 23OC and at 80% relative humidity is sho\vn in Tablc I. Fro111 19" to 26°C there is a decrease or increase of approri~nately onc day in total development t i ~nc for each degrec of tcnlperaturc. T h e larvac of both sexes are sterilized at tcm- peratures above 26°C. T h e threshold for development is 12°C.

There arc five larval instars, differing mainly in size, shapc of the mandibles, tracheal systenl and the number and sizc of the papillae. A detailed description of the stadia is given by Morris and Cameron (1935). Changes in the pupal stage are described briefly in Table I. T h e cggs, larvac and pupae are shown in Fig. I , as sccn through an opening made by removing part of the wall of the host cocoon.

T h e carliest stam at which the sexes can be identified is the fourth instar, 0

when the width of the head capsule of the fcinalc is slightly greater than that of the male (Table I). T h e diffcrencc is nlucll greater (0.031 in11>.) in the fifth instar. Thc scses can be more readily separated, howevcr, in the pupal stage and with less danger of affecting their survival. Although male pupae are usuallv smaller than those of females. the lnost reliable criterion is the

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difference in thc relative lengths of the antennae and prothoracic legs (Fig. 2C and D). Both of these appendages of the nlale extend the same distancc posteriorly with their distal tips reaching the same point on the ventral thorax, whereas the antennae of the female are shorter and never extend as far distally

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Time rcqi~irctl for tlevelopme~~t of each irn111~iti11c scagc of D J I I S C I ~ P I L I I I J

from egg to a d u l ~ a t 23'C ant1 80% R.H. - - --

I I

r\ge ill days Stagc Description of stadia --

Oviposition 1 2 3 4 5

7 ' 5th i~ l s t ,~ r 8 i ~ h (prep~~p<le) 9 5th (prcpi~pac)

10 1 j ~ h (prcpupac) 11 I'iipae 12 13 I'iipac 14 15 16 Pupac 17 Pupac 18 i .\clults c~ucrgc

-- -- -

1 \\'bite, smooth, sub-reniform, 0.32 X O . l l mln I

1 Hcad capsulc, \viclth, 0.093 mln IIcacl capsule, \vicitli, 0.135 nim Hcad capsule, \\.itltli, 0.183 mill

8 3 0.218 Ililn I-lead capsule, \\lidtli, 0.225 Il lr l l

1 End of fccdiilg; bod! or;~llge-1-elloii

Ilcfaecation; bod!. \\.hi tc \\'hi tisli-ycllo\v Rody yel\o\\~ish-brown Rocly light bro\vll; eves pilrl;

' Botly light brown; cycs rctl

I Botly light- bro\\rn; eyes <lark rcti Body dark bro\\rn, dorsal sclericcs black Body black throughout

as do the prothoracic Icgs. T h c adults are striltingl!r dimorphic, although somewhat minute in sizc (Fig. 2A and B). T h e body of both sexes is unl- forinlv black, thc illale bcing usually smallcr (2.0 mm.) than thc female (2.8 inn.). T h c niorc strilting secondary sexual characters, and thc niost useful in separating the seses of adults, are thosc of the appendages. T h c antennae of the males arc black throughout and noticeably branched \\:it11 branchcs

Fig. 1. Stages in the life history of D. fz~scipe?z~~is . Eggs on host larvac iri cocoon. B, Early fifth instar larvae. C, Fourtecn-day old pupae. D, Seventeen-day old pupae.

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456 m. F. BALDWIN, E. SHAVER AND A. IVILICES

Fig. 2. D. filscipennis showing differences between adult male (A) and female (B) and pupal male (D) and female (C). Arrows indicate apices of pupal antennae and pro- thoracic legs in female and male.

arising fro111 thc lst, 2nd and 3rd funiculars. T h e female antennae are regularly club-shaped and blaclc with the esceptivn of the scapc which is white. T h e cosae and trochanters of the male arc blaclc xvhercas in the feii~ale they are white. T h e fore wings of the male are inembranous and clear while those of thc female are fumous but tvithout a pattern.

T h e !ength of life of adults in the laboratory avcragcs f rom 5 to 15 days. A t 23°C and 80% R.H. and without food, males live an average of 5 days, virgin females 7.5 days and mated females 9 days (Willtes, 1917). A t lo.icer tc~nperatures adults -1ivc much longer. Reela (1937) reports thc average longevity of field populations during the summer in Quebec as 31 days. A t 35°C most adults live less than three da~7s and those that live longer are rendered almost co~npletely sterilc.

Wild t y p e stocks There are two wild type stoclcs in current use, both of u ~ h i c h originated

fro111 parasitized sawfly cocoons collected jo Central Europe and imported to Canada in 1934. From the original stock some 500 million were rcared b y mass mating at the Enton~ological Laboratory, Belleville, Ontario for libcra- tion as biological control agents in the forests of eastern Canacla and United Statcs of America during tlle following 10 years. Sincc then this stoclc has

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hlUT.4NTS OF THE WASP DAHLBOR~IINUS 457

been continuously inbred through hundreds of generations largely by sib- mating and pedigree lines and is now known as the Belleville or B stoclc. A second stoclc, nanlcd Wisconsin, with the symbol W , was established from a collection made in the forest of northern Wisconsin, U.S.A. in 1959. This stoclc has been carried by mass mating. Altl~ough few exact comparisons have hcen made, the biology, structure and behavior of both stoclcs appear to be alilcc.

D. fz~sc j$e~~nis can be reared with comparative ease on diapause larvac in cocoons of nos t diprionid sawflies. In the present work Neohiprio7z leco7ztei (Fitch) was used. After mating, one or more females u:erc placed in a shell vial(10 x 60 mm) containing two or threc cocoons and the vial was stoppered with loose cotton wool. T o prevent damage to the parasites by n~ovc- mcnt of the host larvae and to avoid the enlergence of unparasitized sawflies, the cocoons were previously immersed for two nlinutes in water at 60°C. 'This produced an artificial paralysis. T o avoid breakdown of the host larvae, exposure to this treatment should not escced two minutes. A t 23°C and 75 to 80% R.H. oviposition began soon after the females were placed with hosts. About 40% of the total coniplement of eggs were laid in the first day, about 25% during thc second day and 17% during the third day. Oviposition was usually completed by the sevcnth day (Willtes, 1963). After 1 2 davs at 23"C, the parasite pupae were removed froill thc host by opening the end of the cocoon with a scalpel and allowing thcm to fall freely into a watch glass. The pupae were thcn scgrcgated bv sex and phenotypc at room temperature, without affecting survival, and returned to the vials to complete their devel- opmcnt. At this stage pupal mctabolisn~ could be safely reduced and adult emergence rctarded for months by placing the vials in a refrigerator at 10" to 12°C. Adost breeding stoclts at the n~hitish-yellow pupal stage could be held for ovcr a year at 2" to 5°C wit11 less than 30% mortality. A t rooin telnperature indi;idual pairs lnated readily in the vials used for rearing; mating rarely occnrred below 20" and abovc 3obC. Both pupae and adults could bc handled without injury by the usc of a "sucl;er" madc by fittino the glass tube

3. of a medicine droppcr u:ith a loosc cotton plug and a short piece of rubbcr tubing.

cblethods of testbzg the 7nzttntio7ls

In I). jctscipe1~7lis, mutations arc idcntificd w~ith uilusual ease, sincc everv male is haploid. Visible mutations, xvhcthcr dominant or rcccssive, appear at once in thc malc if the genc were present in the cgg from which the nlalc developed. In this inscct, diploid males do not occur as a result of close inbreeding as they do in Hnhrobmco71 jz~gln.lldis (\Vhiting, 1961).

Thc majority of thc mutations described in the present report originated as nlalcs froill irradiated females or from inbrcd lines of irradiated stocks. Variant malcs lvcre mated to homozygotis wild fcmales. The F1 heterozygous females from this cross were separated from the malcs at the pupal stage and propagated as unmated females. If the 1<1 femalcs carried cithcr a dominant or reccssivc mutant genc u~it11 a normal gene, half the male progeny would cxhibit the mutant phcnotype.

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458 IV. F. BALDT'I'IN, F. SHAVER AXD A. 'I'I'II.I<ES

All pigmcnt mutiltioiis urcre classified according to the Yillalobos- Dominguez Colour Atlas (\'illalobus, 1947). T h c colours were identified b y comparison to colours in the atlas by thc use of a dissecting microscope undcr a uniforllily controlled incandescent light source.

RESULTS In the prcsent report 11- visible mutations are described and grouped

accordiilg to those affccting pigmentation, morphology and sex. Eight muta- tions affect eyc and body colour, thrce affect changes in thc antcnnae and \vino.s, and one thc sex ratio. Soillc behavioral differences have beell observed, ? particularly in males during mating, but tests have not been llladc to checlc the accilracv o r possible inheritancc of the differences.

.\,Ianv mosaics for mutant traits have appeared among offspring of feinalcs irradiated during the larval and adult stages of development. Tliesc somatic variants talcc inany forms, iilcludi~lg males with a small area of one eye display- ing a "bright" colour, males with deformed eyes, and sterile gynandromorphs. ,\~Iosaics are rarely discovered in uilirradiatcd control groups, and usually take the form of gyandromorphic males. Also, clusters of mutations arc icldorn found as either spontaneous or radiation-induced variants. A description of the mosaics discovered in D ~ ~ ~ ~ O O ~ ? Z ~ I ~ Z ~ S \vith data collcernillg thcir frequcncy following acute and chronic irradiation is in preparation.

iMutations Affecting Pigmentation

Eye Colozlr i44zltntio.l~~ Fivc rccessive eve-colour mutations have been identified LID to the vresent

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time b y cross-testing. T h e colours range f rom a blaclc u:hich is darlcer than the blaclcish brown wild typc, through a gradient scrics consisting of claret, carmine, chestnut, to a russet colour. In appearance, the claret, carminc, chestnut, and russet arc distinctive colours '~vhich can be readily distinguished from the wild-type, and verv liltcly correspond to thc "bright" colours des- cribed in iUor7nonielln ( ~ a y and Whiting (1950)). These four distiilctive eyc-colours have been utilized in studies of the effects of radiation 011 mutation fi-equency in Dnklhomh71,s by Bald~vin (1964), and it has been detel-mined that irradiation of female L)ahlbo7/zi~7zu as larvae has increased the frequency of "bripht" eve mutants bv a factor of 40 over the ratc in unirradiatcd stock.

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Spontaneous mutations are comparativelv rarc, occurring at a frequency of 2.3 X 10." ( 1 1 mutations among 459,482 ~ilales).

Wild type: T h e wild type eye colour, identified in the colour catalogue as S.3.6", is blaclcish b r o ~ v n 2nd is casily distingx~ishable from the mutant eye colours. In the pupal stagc the eyes-change f rom whitish-yellow in carly pupac, through pink, red and finally dark red prior to emergence. In voung adults thc eyes are blaclcish brown but in older insects the eyes darlccn and thev becorn; similar in'appearance t o the blaclc mutant described below.

Blnck ( 0 ) : T h e black-eyed rnutant, only rccently discovcrcd in exper- iments with irradiated female larvae, seems t o occur less frequently than the "bright" mutants. T h e eye pigmentation (Villalobos classification R-5-2") is slightly darlcer than that of the wild type and in older adults the two can be separated only with difficulty. T h e male progeny of hybrid females (b+/++) segreg-ate 1: 1 wild type and black (Table 11). T h e phenotype of the

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.\IC'Si\STS Oli THE XV.4S1' DAHLBOAIINUS

'I'ABLE I1 Phenotype frequencies of F:! male progeny from mutants crossed

with wild Bclleville stoclc of D. fi4scipennis.

Mutation \Vilcl type 1 Mutant x 2 / P*

E y e Black ( b ) Claret ( c t ) Carmine ( c ) Chestnut ( c s ) Russet ( r z ~ ) Bodv sep<a ( s e ) LVhite legs ( w ) Brown pupa ( b p )

*Significant a t the 5 per cent level.

Morplzology Withered wings ( w d ) 1 902 Stubby wings ( s t ) 23 1 Club antennae ( c l ) 1 596

hybrid female is indistinguishable froin homozygous wild type. Development of the eye colour in the "black" mutant pupa .follows the same course as wild type, with each stage being slightly darlter than the previous stage.

Clnl-et ( c t ) : This "bright" mutant can be easily separated f rom wild type and the other mutants by its characteristic claret brown colour. I t occurs frequently in radiation studies. Classified as S-4-8" (Villalobos, 1947), this eye colour mutation sho~vs some variation in intensity, whereas thc intensity of all others does not vary. Wi ld type and ct males from unmated h e t e r o ~ g o u s females ( c t+ /++) segregate i: 1 (Table 11). T h e claret cyc colour mutation has previously been referred to in worlc on radiation-induced mutations (Baldwin, 1962), as claret-brown. T o avoid the possibility of confusing this mutant with others of the "bright" series the name and symbol have been changed.

Canvisze ( c ) : This is the most brilliant of all the eye-colour mutants and was the first mutation discovered in Dnh1bon;rinzis. I t was originally produced by x-irradiation of pupae in 1939 by LVilltes (1940) and referred to as "red" ( i ) (Willtes ( 1 940, 1963, 1964), but was subsequently lost. Thus , the original strain cannot be co~iipared t o the present carmine. Following the discovery of the other "bright" c o l o ~ ~ r s (especially claret) it has been necessary t o reclassifv the "red" mutation, and it is now renamed "carmine" ( c ) . i'his mutant hppears in irradiated stocla in numbers cornparablc to the claret muta- tion, and at frequencies much greater tl-lan chestnut and russet. T h e mutant colour has been matched t o that found under the formula RS-7-11" in the Villalobos atlas. T h e hybrid females ( c+ /++) produce males which segregate 1: 1 (Table 11). T h e colour of the eyes of hybrid females is the same as the wild type.

Chesnzzit (cs) : This mutation, classified as SSO-5-7", does not appear as frequently as carmine and claret in irradiated populations but is clearly dis- tinguishable by its brownish chestnut colour. A noticeable characteristic of this mutant is a tendency of the eye t o deflate o r collapse a day o r t w o after

880 247 597

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460 XV. F. BALDTITIN, E. SHAVER AND A. WILI~ES

emergence. I\/lalcs froill unfertilized heterozygous cs+/++ females segre- gate equally into cl~estnut and wild type (Table TI). T h e eye colour of the heterozygous females is the same as that of the wild type.

Rzrsset (rzs): This mutant, characterized by a lack of pigment, appears as a tan colour and liltely corresponds to "oyster" described in Mor7izo1ziellr (Ray and Whiting, 1950). Inbred lines of this mutation have been difficult to maintain in the laboratory because inutant males seem to have difficultv in locating females during courtship. Survival of this mutant is also lower than others at low temperatures; very few adults emerge froin pupae stored for inore than 3 months below 3°C. A tendency towards "dishing" of the eyes, found in the chestnut, is also evident in this niutant, altl~ough it occurs i ~ & c l ~ less frequently. T h e russet colour has been identified as SO-5-9" in the Villalo1)os atlas. As shown in Table IT, heterozygous females produced an almost 1: 1 ratio of russet to wild type illale progeny. T h e eye colour of heterozygous fenlales is the same as that of wild type.

T o determine if the four "brigl~t" eye colour nlutants originated froin separate loci, and to test for linltage between the loci, test crosses were made between claret, carmine, chestnut and russet. I11 Table 111, the data indicate that crosses 1-5 resulted in independent segregation of the eye-colours. In cross 3, pl~enotypically wild-type hybrid females from original crosses of c x cs produced carmine, chestnut, wild-type and "tan" sons on a 1: 1:l: 1 ratio, the "tan" colour resulting from the recombination of genes c and cs. In crosses 1, 2, 4 and 5 the ratio proved to be 1:2: 1, with only 3 instead of 4 phenotypes appearing in the F2 sons. This was a result of russet recombining with chestnut, carnine and claret in crosses 1, 2 and 5 respectively and mask- ing thein while chestnut maslted claret in cross 4 when cs ct recombined. Independent segregation did not occur in cross 6, ct and c being linlted with a cross-over value of 22.5%.

Body Colour Mzrtatio7zs Sepia (se): The sepia mutant, one of the less frequently occurring

variants, is characterized by a body colour which is in distinct contrast to the black, mild type. Although the general body colour of the mutant is sepia, a range of intensity can be found in different parts of the body. T h e head, thorax and the coxal and fe~noral segments of the metathoracic legs appear darltcr thnn all other areas. In contrast, the antennae, abdomen, tibiae and tarsi and all legs are a light shade of sepia. The femoral segments of the meta-

TABLE I11 Frequencies of eye colour mutations in males produced by unfertilized hybrid females.

Exptl. no. of crosses

F1 mothers FZ sons

cs, 184:ru, 388:+, 187 c, 320:rz~, 643:+, 308 c, 225:cs, 257:+, 266:tan, 264 cl, 236:cs, 447:+, 223 ct, 149:ru, 264:+, 157 ct, 153:c, 157:+, 42:tan, 40.

*Significant a t the 5 per cent level.

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>IUT'\Y1'S 01; THE \TrASI' I).4HLBO,\lIKUS 46 1

thoracic legs arc uniformly darkcr than the mcso- and prothoracic lcgs, whereas the trochantcrs of all the lcgs are light.

Thc colour standard of 00s-5-4" established for this mutant \vas based on thc colour of thc abdomcn. The abdominal wall is translucent. rcvcalinrr

0

somc of thc intcrnal organs. Thc eves of the scpia mutant, althougl~ identical to those of thc wild type, appear illnost blacli against thc sepia bacliground colour of thc hcad capsulc.

Unmatcd hcterozygous fc~nalcs (se+/++) xvhich arc phenotypically identical to thc wild type, produce sc and + males which scgrcgate in- dependcntlv 1: 1 (Tablc 11). The sepia mutants are the most activc of all

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mutant adults, and unlilic the eye colour mutation russct, successful matings occur quite rcadily. Thus purc lines can bc maintaincd indefinitely without difficulty.

While legs ( w ) : Thc xvllitc Icg mutation, found rcccntly among male progeny of irradiated mothers, appears to be a deviate close in colour to that of the wild-tvue but differentiated from it bv a reduction of melanin in thc cuticle. T I I ~ Striliinp featurc of the whitc l e mutant is the xvhitencss of thc " " coxae and fcmor of the prothoracic legs. In thc wild typc malc, these leg seg- ments are black (Fig. 3A) xvllereas in the female the coxae are white and the femora black. In the mutants of both sexes the femur is white Fig. 3B). The antennae of this mutant are noticeablv liphter in colour than those of thc wild

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type, but they do not approach the very light tan of sepia. Independent segregation is found among male progeny of 'i~-+/++ females, the ratio being 1 : 1 (Table 11).

Fig. 3 . Con~parison of the white leg mutant and the wild typc. A, prothoracic leg of wild type and B, prothoracic leg of the white mutant.

Pupal Mutation Brourz pupn (bp): In contrast to the previous mutations, which arose in

the first generation of males from irradiated females, the brown pupa mutant was first discovered among the males of the F j generation of an inbred, sib- mated, stock of carmine. When the two bp males discovered were mated to their sisters two thirds of the sisters produced all wild type pupae; the other third produced pupae of which half were wild and half similar in colour to the male parent. Segregation of the bp factor in brecding tests with wild Belleville stock and their hybrids clearly indicates that the brown condition of the pupae is produced by an autosoma1 recessive gene (Table 11).

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462 IT'. P. BALDII'IN, E. SHAVER ASD A. IVILICES

There is considerable difference in survival between certain crosses with wild stoclt. Although the lethals associated with this mutation have not been studied in detail, it is clear that the survival of some genotypes involving l ~ p is much lcss than that of wild stoclt. In a study of 220 offspring f rom a ++ Y bp+/+Dp cross, the mean number of progeny nras 20.0 per female, of 2278 f rom ++ X ++l+bp 59.9, of 3103 f rom +bp X ++/+bp 57.4 and fro111 130 offspring of +bp X +bpl+bp it uras 26.0. Thus, crosses involving homozygous, +bp/+bp, females produce only about half as many offspring as heterozygous o r wild females. Among the mutant offspring that reach maturity, structural abnormalities are unusually comnlon. Many of the offspring die at emergence, the adults are nluch smaller than in the wild type (males 17 and females 29% smaller) and development from egg to adult taltes 2 days longer at 23°C. Although mating is norillal and the sex ratio is not affected, many hon~ozygous feinales fail t o produce offspring when placed with host cocoons. I t has been in~possible to inbreed pure recessive lines of this mutant stoclt bevond the 5th generation.

T h e primary phenotypic effect of the bp gene is t o alter the colour of the pupae f rom light yellow to olive brown (Villalobos 0-5-7"). T h e first detect- able difference in colour is evident in mature larvae. which in wild stoclt are white and in the nlutant light brown. In wild a n k heterozygous pupae, the entire bodv is initially whitish-yellow, becoming dark brown and finally blacli (Table I): In the mutant pupae the body is olive brown throughout pupal development and remains dark until ecdysis when the integument is shed and the adult emerges as a black, phenotypically normal individual. T h e cast exuvia of the pupa is cl~aracteristically dark brown as coinpared to the golden yellow of the wild and heterozygous individuals. This must be ltept in mind when classifying mutant brown-bodied pupae for eye colour.

Mutations Affecting Morphology

Wiug il/lutnnts Withered (wd) : T h e first wing iuutation discovered anlong male

progeny of irradiated L). fz~scipemzis females was an insect with crunlpled and shrivelled wings. In coinparison to the eye-colour mutants, very f ew w d individuals have been found in our experiments t o date. In the pupal stage, the wing buds of the nlutant are large, oval shaped lateral protuberances, differing marltedly with the inconspicuous s\vellings of the wing buds on the mesothorax of the wild type pupa (Fig. 4D, E). T h e lnesothoracic wings of the newly emerged nlutant adult appear first as swollen distended sacs; the wings of the metathorax are small and inconspicuous. A few hours after emergence, the fore wings collapse into ventrally concave, spoon-shaped paddles extending t o a polnt mid w a y along the abdomen; the distal tip of each n-ing is crumpled. In a few nlutant specimens the mesotl~oracic wings do not seem to be affected; in others, they are atrophied and shrunlten beyond recognition. T h e nlutant differs from the wild t i p e only in wing forkat ion (Fig. 4B). -

Crosses of the mutant males with hon~ozygous wild females results in hybrid females phenotypically identical to the wild type form. T h e wild and niutant males from the unmated hybrid females segregate independently (Table 11). I t is quite evident that w d is an autosoma1 recessive gene.

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Fig. 1. Comparison of thc \\,ing mutations and tlic \\.ild typc. A, s t u l ~ b y \ving of thc adult ant1 B, \vitlicrcd \\ling of tlic adult; C, s tubby \\.ing of thc pupa, D, \\~ild typc pupa a n d L.:, \\.ithcrcd \\ring of thc pupae.

StrlOOy ( s t ) : T h e second \ving a n o i ~ ~ a l v to appear in sons of irradiatcd mothers \\;as an ;~dul t n ~ i t h short, dorsally con;cx lobes extending laterally from the wing bases (Fig. +A). O n pupae, thc \ \~ ing buds of stubby arc small phcr ica l s\vcllings cstending laterally from the upper margins of the meso- thoracic segments (Fig. 4C). T h e ne\vly emerged adult wings arc swollen bulbs which soon collapse to thc form described. In this mutant, the wings never extend beyond the seco~ld abdo~lli~lal segnlent. Although precisc qumtitativc determinations of bodv sizc havc not bcen ~nadc, st appears to be smaller than the wild type.

Brecding tests have sho\vn that 11lale progeny from unmated heterozygous females (phenotypically i~~distinguishable from I I O I I I ~ Z ~ ~ O U S wild) scparate

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464 W. F. BALDWIN, E. SHAVER AND A. WILICES

equally into + and st inales (Table 11). T h e stubby wing mutation segregates independently of the wild type and is recessive.

Since the effects on wing formation of the withcred and stubby mutations were somewhat similar, further crosscs were nlade behveen them to determine whether or not they were indeed at different loci. Homozj~gous + wd/+wd females were crosscd with st + males. Twenty-five unmated hybrid females resulting from this cross (+wd/st+, pheno~ypically wild) produced 755 wd+, 851 st+ and 34 ++ males. Thus, rod and st arc clcarly mutations. Although apparently soine linltage is involved, the amount has not as yet been adequately determined.

Mz~tations of the antennae Club (el): T h e antenna of wild U . fuscipesznis, beginning at the basc,

consists of a scapc, a pcdiccl, a very sinall rina segment, 4 funicular and 2 club s segments. T h e male antenna is plun~ose beanng a branch on each of the first three funiculars (Fig. 5A). On the female, the segments arc thiclter and the branches are absent (Fig. 5D). In the mutant the abnormality in both male and female is confined to the funicular and club segments. T h e segments of the female are fused into a characteristic, triangular-shaped club (Fig. 5E, F) . T h e illale mutant also displays a son~ewhat elongated club which varies in shape and form. In many individuals, seginentation of the club may be absent; in others there may be an indication of one or more divisions (Fig. 5B, C). The branches on the first threc funiculars of the wild male antennae are, in most cases, absent in the mutant. Occasionally there may be one small attenuated branch (Fig. 5B).

In test crosses, hybrid females (+el/++) identical to wild type in appearance, produced an equal number of el and + males (Table 11).

mutations ajfecting sex Sex ratio (SR): This mutation causes a reversal of the "nonnal" sex ratio

of progeny froin inseminated females. It was first discovered during the selective inbreeding of Belleville stock in an attenlpt to establish a line of females producing a high proportion of males. A description and preliminary account of its inheritance has been reported by Wilkes (1964).

T h e frequcncy distribution of the sex ratio of progeny from inbred wild females producing offspring of both sexes (mated females), although slightly slewed negatively, is remarkably regular with a inode at 92% females and a mean of 89%. In laboratory populations of wild stocks, the ratio of the sexes is quite constant, with little variation except when environnlental conditions are extremely adverse. When the SR mutant is present, the sex ratio of the progeny of any one female rarely goes above 15% and the population mean is near 5 % (\Vilkes, 1964).

Test crosses between wild and SR stoclcs have shown that the low percentage of females in the SR stock is sex-limited, being transmitted by females through their sons. In crosses of SR males with +/+ females, the percentage of females in the progeny was very low. Reciprocal crosses using wild males and both heterozygous and l~omozygous (+SR/SR+) females produced progenies of high or "normal" percentages of females. T h e number of progeny per female parent (in all crosses) was comparable.

T h e abnormal ratio of the sexes produced by the SR mutation is not due to cytoplasmic factors transmitted through the egg. Progeny from normal

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Fig. 5. Comparison of club antennae mutant and the wild typc. -4, wild typc male antenna; B and C, two mutant males; D, wild typc female antenna; E and F, two mutant females.

females mated succcssively to marlted wild and SR males showed that the eggs were fertilized by both types of sperm, and the ratio of fertilized to unfertilized eggs was the same as in feinalcs that rcceived one typc of sperm only. The sex ratio among 19,000 offspring from females inseminated by both types of males was, by wild sperm 89.1% females, and by SR spenn 6.3%. Froin thc records of adult emergence, the utilization of sperm in fertilization appears to have becn random. The tcstes and the number, morphology and motility of thc spermatozoa of wild and SR males are alilte.

Although the inodc of action of this male-producing mutation is unltnown, it is clear that its effects are not due to selective mortality, nor is it transinittcd by the egg alone. Its prescncc xvould appear to hinder successful fertilization of thc egg, either through failure of a much greater proportion of SR sperm to enter the cgg or through the inability of the SR sperm nilcleus to unite

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466 \\T. F. BALD\\'I~-, I.:. SHAVER AND A. XVILICES

xvith the fenlalc pronucleus after it has entered the egg. T h e action of thc nlutation in D. fzlscipennis is clearly independcnt of the factors considered t o be responsible for causing variations in the sex ratio of most arrhcnotoltous animals. I t is thought that the mutation, if it occurs in nature, \\~ould account for the variability of the sex ratio of this spccies in natural populations.

SUMMARY T h e main phe~lotypic effects of 12 visible mutants arc reportcd in

the arrhcnotoltous \Imp, Ilfihll~o71r'i7~1~~ fllxcipe7?1?is (Zett.) ( H y ~ ~ ~ c i ~ o p t c r a : Eulophidae). All hut t w o of the mutations occurrcd among haploid ~nalcs follo\ving irradiation of wild females, the others during pedigree breeding of irradiated stock. Eight of the mutations affcctcd pigmentation of the cvcs o r body, thrce produced morphological abnormalities in the \\7ings and antcnnac and one rcvcrsed the sex ratio. All but the last one \vere controlled by recessive factors, each of which has bccn assigned a name and symbol. T h e sex revcrsing factor \!>as dominant and scs-limited, being transmitted by fcmalc:; to their sons. Alt11oua.h tests for linltagcs bctwecn the mutations .have not . P been completed, in initial crosscs betucen four of the "bright" cyc-colours and the wing mutations segregation uras independent indicating that thesc arosc as separate mntations.

An account is givcn of the life history of thc spccics and a description of the adult and immature stages of the wjld stock as a basis for comparison with thc mutant lincs. A siinple method is givcn for rearing largc numl)ers in the laboratory.

REFERENCES Bald\\~in, \A'. 1:. 1962. T h c cffcct of radiation dosc ratc o n thc production of cyc colour

mutations in the chalcid L)nhlDo.ttzi./nrs. Radiation Rcscarch 1 7 : 127-132. 13ald\vin, W. 1;. 1961. Visiblc miltation frcqucncics in I l n h l b o ? ~ ~ i ~ ~ ~ l s oogonia produccd

by acute X-rays and chronic gamma radiation. S:Iutation Rcscarch (In prcss). ITinIayson, L. R., and Rcclts, 11'. A. 1936. Notes on the introduction of Di/?riou parasites

to Canada. Can. Entomol. 68: 160-166. 3/lorris, I<. R . S., and Camcron, 1.:. 193.:. T h c biology of ibficroplectro71 firscipelnris

Zctt. (chalcid), a parasitc of thc pine sandy ( D i p r i o ~ ~ seltifer, Gcoff.). Bull. lint. Iics. 26: 107-419.

Pcclt, Os\vald. 1963. A catalogue of thc Ncarctic Chalcidoidca (Insccta: I-Iy~~~cnoptera) . Can. Entorn. Suppl. 30: pp. 108-113.

Ray, D. T., and Whiting, P. W. 1954. An X-ray dosc-action cur\.c for cyc-colour rnuta- tions in i\lontzo~~ielln. Biol. I3ull. 106: 100-106.

Rcelts, W. A. 1937. Notes on the biology of ~Microplectro71 l:lrscipelmis Zctt., as a cocoon parasite of Dipriorl polyt07tzzr1tz Hartig. Can. Entomol. 69 : 185-187.

Villalobos, C. y J. 1917. Atlas de 10s colorcs. El Atenco, Buenos Aircs, Argentina. Whiting, Anna R . 1961. Genetics of Hnbrobrncmz. Advances in Gcnct. 1 0 : 295-381. Willtes, A. 1910. T h c sex ratio, its variations and control in a chalcid parasite, Alicvo-

plectron fztscipew~zis Zctt. Ph.D. thesis, University of Toronto, Toronto. pp. 172. Willtes, A . 1917. T h e effects of selecti\re breeding on thc laboratory propagation of insect

parasitcs. Proc. Roy. Soc. B., Lond. 134.: 227-215. Wilkes, A. 19j9. Effects of high temperatures during postembryonic dc\:clopmcnt on thc

sex ratio of an arrhcnotoltous inscct, Dnl?lbm)til?zrs fllligil~osrls (Nces) (Hymcnoptera: Eulophidae). Can. J. Gcnct. Cytol. 1: 102-109.

\Villccs, A. 1963. Environmental causes of variation in the sex ratio of an arrhenotoltous inscct, D n I ~ l b o ~ ~ z i ~ ~ l l s fz~ligi lzos~~s (Nces) (Hymenoptera: Eulophidae) . Can. Entomol. 95: 183-202.

Willtes, A. 1961. An inherited male-producing factor in an insect that produces its malcs from unfertilized cggs. Science, 144 : 305-307.

Zetterstedt, J. W. 1838. Insecta Lapponica, pp. 427-428.

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