An liirroducfiofi lo The Genefics Ol Habrobracon Juqiandis Ashmecid ALBERT MARTIN, JPt a An Introduction To The Genetics Of Habrobracon Juglandis Ashmead By ALBERT MARTIN, JR. Assistant Professor of Biology Mount Mercy College Lecturer in Genetics University of Pittsburgh * * Jf * .i\u\ * • ^ • THE HOBSON BOOK PRESS 52 VANDERBILT AVENUE NEW YORK, N. Y. 1947 Copyright 1947 By ALBERT MARTIN, JR. Manufactured by the Hobson Book Press, Cynthiana, Ky., U.S.A. TO MY WIFE FOREWORD This summary is designed for students of Habrobraconology. Although written primarily for use in course work, it is hoped that it may serve as a reference for those engaged in ex- perimental genetics. The aim of the work has been to present a clear and readable account of the results of investigations made thus far in the genetics of Habrobracon juglandis (Ashmead). The insect is described, and its method of culture as well as that of its host is outlined in detail. A complete list of extant and dis- carded mutant types is included. The statisti- cal formulas utilized in determining crossover values and other linkage problems are pre- sented. Tables in the appendix summarize pres- ent linkage information and effects of X-radia- tion. A glossary and a complete bibliography of Habrobraconology accompany the summary. I am deeply indebted to Professor P. W. Whiting, of the University of Pennsylvania, for reading the manuscript and for making valuable suggestions, and to Dr. G. M. McKinley, of the University of Pittsburgh, for his encourage- ment. Special thanks are given to my wife, Dr. Phyllis C. Martin, of the Pennsylvania College VII for Women, who read the text at several stages in its preparation, and assisted in the clarii- fication of the language. Albert Martin Jr. University of Pittsburgh May, 1946 VIII TABLE OF CONTENTS FOREWORD VII I INTRODUCTION 1 II DESCRIPTION 6 III CULTURE 12 IV GENE MUTATIONS 22 V RECORDING OF DATA 69 VI SEX CONDITIONS 74 VII SEX DETERMINATION 91 VIII LINKAGE 103 IX ENVIRONMENTAL EFFECTS 121 X CONCLUSION 139 APPENDIX 143 BIBLIOGRAPHY 149 GLOSSARY 181 INDEX 193 ' 160 LIST OF ILLUSTRATIONS 1. Hahrohracon Female 7 2. Habrobracon Male 8 3. A Variegated Eye 26 4. Mutant Eye Fonns 32 5. Wild-type Primary Wing 40 6. Wing Types 52 7. Wing Types 53 8. Antennae Types 61 9. Leg Types 64 10. Individual Fi Female Data Chart 71 11. Master Sheet of Fi Female Progeny 72 12. Chromosomes of Habrobracon 78 13. Compound Eye of Male Mosaic 86 14. Genitalia of a Gynander 88 15. Male Mosaic 89 16. Crosses Involving Sex-determination 99 17. Habrobracon Linkage Groups 105 18. Diagram of Enzyme Color Determination 125 19. A Single Habrobracon Ovariole 131 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD Chapter I INTRODUCTION For the purpose of studying inheritance, the Insects have been of primary importance, chief- ly because their life cycle is short, and be- cause they produce large numbers of progeny. The fruit-fly, Drosophila melanogaster, and the bee, Apis mellifica, have been studied inten- sively by a number of workers; but the parasit- ic wasp, Habrobracon juglandis (Ashmead), has been given special study by P. W. V/hiting and his co-workers during the past thirty years. Habrobracon is a parasitic hymenopteran of the superfamily Ichneumonoidea, family Braconi- dae, subfamily Vipiinae, genus Microbracon, species hebetor (Say) (Muesebeck, 1925). The species M. hebetor, was first described in 1836 by Say who, however, called it Bracon hebetor. Later that same year he described another spe- cies, which he called B. dorsator. The spe- cies, B. hebetor and B. dorsator, are now known to be the same. Many other names have been ap- plied to this insect since the original ones proposed by Say: B. brevicornis (Kirby, 1884; Marshall, 1885), B. juglandis (Ashmead, 1889), Habrobracon hebetor (Johnson, 1895), Bracon (Habrobracon) honestor (Riley, 1895; Howard, 1895), Habrobracon benef icientior (Yiereck, 1911), H. brevicornis (Cushman, 1914; P. W. THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD Whiting, 1918, 1921e), and H. juglandis (Cush- man, 1922 )■. The type specimens of Bracon hebetor (Say) and of B. dorsator (Say) have been lo^t, but those of B. juglandis (Ashmead), and Habrobra- con benef icientior (Viereck) are in the United States National Museum, Washington, D. C, the former bearing type catalogue number 2913, the latter, number 13494. The species, Microbracon hebetor (Say), is) exceedingly close to M. brevicornis (Wesmael), and the tv/o have been much confused in litera- ture. In early papers on the genetics: of Habrobracon juglandis, the name, H. brevicornisi (Wesmael) was used, Cushman (1922) cleared up thisi matter, calling attention to th'e differ- ence in habit between the two species, and pointing out some morphological differences-. Habrobracon brevicornis is known to parasitize the European corn-borer, Pyrausta nubilalis, while Habrobracon juglandis parasitizes the Mediterranean flour moth, Ephestia kuehniella (Zeller). The females of Microbracon hebetor are readily distinguished from those of M. brevicornis by the antennae, which consist of 13-15 segments in the former, and 17-19 seg- ments in the latter. At this time, however, Cushman did not regard Bracon juglandis (Ash- mead) as identical with B. hebetor (Say). It appears, after a careful consideration of Say's description of B. hebetor, that there can be nO' reasonable doubt that Say and Ashmead were dealing with the same species (Muesebeck, 1925)t. The name Hadrcbracon brevicornis (Wesmael) waS' used by Doten (1911) in describing the relation of food to reproduction and longevity in cer- INTRODUCTION tain hymenopteroLis parasites, and by Cushman (1914), and P. W. Whiting (1918, 1921a) in their work with Habrobracon juglandis. This use of Hadrobracon is clearly a misspelling since no such generic name is recognized in scientific literature (Neave, 1939). There have been some genetic studies reported on Microbracon brevi- cornis (Wesraael) (Speicher and Speicher, 1940) and M. pectinophorae (Watanabe) (Inaba, 1939, 1940) under the generic name, Habrobracon. The present account, however, deals mainly with the work done on the genetics of Microbracon hebe- tor (Say), but since the name Habrobracon juglandis (Ashmead) has been used as a synonym f-or Microbracon hebetor (Say) and is so well entrenched in genetic literature, it is prefer- able to use it in a work of this kind. At present five wild-type stocks of Habro- .Ibracon juglandis are being reared by Whiting for genetic studies. These stocks have been named for the geographic locality in which they were found, and have been arbitrarily numbered «toi differentiate one from the other in genetic 'crosses. Thus' wild-type stock number one (1) lis) from Lancaster, Penhsylvania, stock number eleven (11) from Iowa City, Iowa, stocks number 'thirty-two (32) and thirty-three (33) from Cal- ifornia, and stock number twenty-five (25) from New York City, New York. Habrobracon juglandis is ecto-parasitic on various cereal-infecting caterpillars. Refer- ences in literature to Bracon or Habrobracon brevicornis, Microbracon hebetor, or Habrobra- con juglandis as parasites of the Mediterranean flour moth, Ephestia kuehniella (Zeller) (Wash- burn, 1904), of the meal moth, Plodia inter- THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD punctella (Grandi, 1931), the fig moth, Ephes- 'tia cautella (Walker) (Simmons, and Reed, 1929) and of the bee moth, Galleria mellonella, con- cern this species. Habrobracon is- distributed throughout the v;orld wherever its hosts, par- ticularly the flour and meal moths, are presents (Fahringer, 1928; Goidanich, 1934; Muesebeck, 1925; Watanabe, 1935). It does especially v/ell in laboratory cultures if raised on the cater- pillars (larvae) of Ephestia kuehniella. The original Lancaster (1) stock was derived from seven females bred from a single infested Ephestia caterpillar and mated to their sons in June, 1919. Genetic research with Habrobracon was begun at the University of Pennsylvania in the fall of 1916 by P. W. Whiting. The first mutant type, a recessive orange eye color, appeared March 27, 1920 (P. W. Whiting, 1921c). Tests) made with this first mutant indicated that three types of individuals occur regularly in the species where the parents^ are related. Males arise from unfertilized eggs, and are, therefore, haploid and display only maternal traits; females develop from fertilized eggs, and, therefore, may show both maternal and pa- ternal characters. In a few cases diploid males) have appeared (P. W. Whiting, 1921e). These may be recognized because they show paternal characteristics. Habrobracon is, therefore, typical of the order, in that it reproduces) both gamicly and parthenogenetically. Habrobracon illustrates complete metamorpho- sis and furnishes rich material for experimen- tation because of the striking effect of temper- ature on its body color, its habit of parasit- INTRODUCTION ism, its parthenogenetic method of producing haploid males, the occurrence of gynanders and other genetic mosaics, its impaternate or par- thenogenetically produced females, and its^ in- heritance of easily distinguished mutant traits. Chapter II DESCRIPTION Habrobracon juglandis in the adult stage is approximately three millimeters in length. Var- iations in size are directly related to the quantity of food available to the larvae. Gen- eral body coloration varies from honey-yellow to jet black, depending largely upon the tem- perature during development. Females are, in general, less deeply pigmented than males in any given strain. Body colors are similar on dorsal and ventral sides of head and thorax. The distribution and quantity of integumental pigmentation are exceedingly variable, but nor- mally produced individuals are bilaterally sym- metrical. The color pattern of the praescutum, scutum, mesoscutellum, and the dorsal plate of the mesophragma is used in classifying degrees^ of pigmentation since it is consistently re- lated to muscle areas and is not complicated by other factors. There is a high degree of asym- metry of pigmentation in specimens mosaic for mutant factors and for sex. As compared with females (Fig. 1), males (Fig. 2) have longer antennae. The antennae consist of a scape, and a pedicel, in the males a flagellum of 20 to 22 segments, and in the females a flagellum of 13 to 15 segments. The males have larger ocelli, smaller wings and legs, and larger compound Fig. !• Dorsal view of Habrobracon female. X 18. Fig. 2. Dorsal view of Habrobracon male. X 18. DESCPJPTION eyes, although the facets of the ommatidia closely approach those of the females. The wing size of a normal specimen varies' consider- ably along with differences in general body size. Measurements from tip of sclerite at base of costa to end of radius vein on the cos- tal margin vary from 2.04 to 2,35 millimeters* for female wings and from 1.89 to 2.30 for male v;ings. These figures make it clear that, while males have slightly smaller wings than females, variations which normally occur within each sex are such that measurements are ordinarily of no. avail for sex identification. The microchaetae, however, may be used to differentiate diploid males from haploid males and diploid females, since each bristle on the wing corresponds to and gives indication of the presence of a wing surface cell which is larger in diploid males than in haploid males or diploid females (Spei- cher, 1935; Grosch, 1945). Males also have smaller abdomens with abdominal sternites small- er and thinner than those of females. Abdomi- nal tergites, especially the first and second, are thinner in the males. A transverse sub- basal depression is evident especially on the fourth and fifth tergites of the males. In all types the abdomen is covered, ventrally and laterally, by a cuticle which is thin and trans- parent except for small sclerotized regions at the lateral ends of the abdominal sternites. Female genitalia consist of a pair of elon- gate sensory gonapophyses blackened distally, a brownish sting, and a vagina anterior and ven- tral to the gonapophyses. The paired gonads of Habrobracon females each consist of two ovari- oles morphologically composed of three regions: THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD (1) dorsal, apical end-chamber with' oogonia, followed by (2) masses of nurse cells alternat- ing with (3) developing oocytes each of which is surrounded by follicle cells. In mature fe- males which have fed upon host caterpillars the lower end of each ovariole is expanded into a storage chamber (uterus) in which there are from two to five eggs with chorion and yolk fully developed and with follicle cells still present but with nurse cells gone. In females about to emerge from the cocoon the uteri are small and empty. The first maturation division is initiated before oviposition. Speicher (1936) showed that when the egg reaches the uterus, acquires a chorion, and loses its nurse cells, its nucleus increases in size, and the chro- matin elements appear. They quickly shorten and with the disintegration of the nuclear mem- brane move to the center of the nuclear area and become arranged on a flat metaphase plate. A spindle forms at the same time. The chromo- somes continue to late metaphase stage, and here maturation is stopped until after oviposi- tion. Oviposition will not occur unless the female has fed upon the host caterpillar. With- in the nuclei of the younger oocytes the ear- lier processes of the meiotic pronhase are go- ing forward. The genitalia of the male consist of an out- er and an inner pair of claspers and a penis. In normal haploid males the paired, rounded, yellowish testes (about one-fourth millimeter in diameter) are fused and lie dorsally to the intestine in the posterior part of the body. Each gives off a lateral duct which passes around the intcctine and unites with the acces- 10 DESCRIPTION sory gland to form a common duct which connects with the penis. Internally the testes are sub- divided into a number of cysts. Scattered throughout the testes of immature individuals are spermatogonia! cells many of which are in compact rosettes. Some are in larger, looser rosettes. In the gonads of late pupae matura- tion division figures and even some spermatids may be observed, but gonads of freshly eclosed males show the highest percentage of cells in active division and have, therefore, been stud- ied most intensively. Definite types of reactions characteristic of sex are found in Habrobracon. The females sting the host caterpillars on which they sub- sequently feed and lay their eggs. The males are entirely indifferent to caterpillars, but they show characteristic reactions towards fe- male wasps, such as flipping wings, mounting, and beating with wings and antennae in the proc- ess of mating. 11 Chapter III CULTURE Habrobracon juglandis has proved to be ex- cellent material for laboratory study and re- search investigations because of the ease with which it can be handled, the low cost of feed- ing the stocks, and its brief ten-day genera- tion period. These factors together with its fecundity make possible the accumulation of large numbers of progeny in a short time. Habrobracon is cultured in shell vials, twen- ty millimeters by seventy millimeters, plugged with cotton covered with fine mesh cheese-cloth through which neither caterpillars nor wasps will burrov/. When the wasps are cultured in this manner it is possible to observe the mat- ing procedure and the complete life cycle. Af- ter mating, the female is given one caterpillar of the host, Ephestia kuehniella, and incubated at 30° C. After stinging the caterpillar the female sucks juice from the puncture but depos- its no eggs until her victim has become torpid. Young females are given only one caterpillar at first because they do not always sting immedi- ately, and the caterpillars, if not stung, start to spin webs upon exposure to incubator temper- atures. The caterpillars may in this way es- cape being stung by the wasp, or the wasp may become trapped in the web and be killed by the 12 CULTURE caterpillar. After a v/asp has once stung cat- erpillars and oviposited, she usaally stings as soon as she is confronted with fresh caterpil- lars, and as many as five can be given at once. Female wasps feed on the juice which is essen- tial to reproduction. This is borne out by the fact that females will not lay eggs until they have fed upon a stung caterpillar, although they mav be kept alive for v/eeks on diluted honey. On the other hand, Habrobracon males subsist on a diluted honey diet entirely and will not feed on caterpillar juice even when it is available. Both the males and the females may be kept alive in shell vials for extended periods by feeding on a mixture of honey and water. It is convenient to keep the honey and water in separate small containers, for if the two are mixed, fermentation occurs. A metal rod such as a dissecting needle or a dental probe is dipped into the honey, then into the "water and touched to the inside of the vial where the drop will adhere. At ordinary room temperature it is necessary to feed the wasps every other day, but if it is desired to keep any individuals isolated for an extended period it is better to feed them once and set them in the ice chest at approximately 10° C. Follov/ing the stinging of the caterpillar, the female, if mated, deposits both fertilized and unfertilized eggs on top of or on the under side of the caterpillar; if a virgin, only un- fertilized eggs will be laid. These eggs may be easily observed. If it is desired to study the ovipositing wasps or to count their eggs the paralysed caterpillars are placed upon a piece of glass which is set about three-fourths 13 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD of an inch above a mirror. Then by focusing either upon the insect directly or' upon the image, upper or lower sides may be observed. There is no danger of contamination from the food even if wasps have infested the caterpil- lar culture for eggs are never laid except upon torpid and flaccid hosts. Such should, of course, be discarded in selecting caterpillars) for the culture vials. An occasional female lays many sterile ova. Data on eggs of such exceptional females are not included in general summaries. There is' likewise a tendency on the part of many females* to lay "shells," apparently filled only with fluid. This soon evaporates, and the egg be- comes brittle. In one series (A. R. Whiting, 1940a) the percentage of such "shells" was 2.26' for the first ten days of life of the females^ and 12.06 for the second ten days. Some females^ produced no "shells," although all were from the same inbred line. Those "shells" are like- wise omitted in computing hatchability ratios. It has been thought that the "shells" might be the result of the females stinging their eggs. It has been concluded that females sting eggst very rarely if at all, and that stung eggs can continue develooment after such injury. In or- der to determine the time of death of eggsi which, for various reasons, do not hatch, the eggs may be collected at intervals and placed in the mineral oil, "Nujol," where development proceeds normally and conditions can be clearly observed. The developmental limits for the egg have been found to be 12-38° C. at a relative humid- ity of 76-98 per cent (Maercks, 1933). The 14 CULTURE life cycle is completed in the shortest time at 33.5° C. and a relative humidity of 32-98 per cent. The optimum temperature is, however, 29-30° C. with a relative humidity of 80 per cent. The temperature optimum is considered to be the temperature at which there is 0 per cent\ mortality and the smallest variation in the ef- fect of various percentages of relative humid- ity. The relative humidity optimum is found on the humidity scale at the point where there is the greatest range of temperature with the same mortality. The duration of development is) influenced especially by temperature and only slightly by relative humidity. At the tempera- ture optimum the mortality is not influenced by slight deviations from optimum humidity, but at^ non-optimal temperatures, either high or low, there is an increase in mortality the farther the temperature and humidity are removed from the optimum. The mortality at non-optimal tem- peratures is influenced considerably by humid- ity; however, the duration of development of the survivors is less affected. At optimum or near optimum conditions, the deposited eggs hatch into larvae in two or three days. The larvae feed on the juice of the host caterpillar and grow to the size of IS THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD tend to cling to the sides of the vial and must be brushed off into the nev; vial. Transfer at three day intervals is continued until the fe- male dies. The larval stage lasts about two days, at the end of which time the larvae spin cocoons* with silk from the salivary glands. The cocoon entirely surrounds the larva, but only a very thin layer of threads is laid down where it ad- heres to the inner surface of the vial; thus it^ is possible to watch the development of the pupa from this side. The caterpillars by this* time are reduced to shrivelled remnants which may be removed from the vials. In the higher hymenopterans the end of the stomach is closed during larval life. The fecal matter is, therefore, retained in the stomach until pupation and then discharged into the end of the pupal case. This discharge appears as a dark brovm mass and marks the posterior end of the developing pupa. After two days, the pupa changes externally *from the worm form of the larva into approxi- mately adult insect form. Legs and wings are not fully formed until the third day of pupal existence when pigmentation and sclerotization take place rapidly. Compound eyes are formed externally by the end of the second day, but the internal tissues are not completely devel- oped until the third day, and facets are not apparent until the wasp is almost ready to^ hatch. Near the end of pupal life the thorax and antennae become deeply pigmented. The time of pupal life varies somewhat, but most wasps average about four days. In three day pupae, the ventral side lies^ 16 CULTURE squarely under the thin attached layer of the cocoon. Antennae are folded down the ventral side, and in females they reach to about the posterior end of the thorax; in males, they ex- tend to about the second sternite of the abdo- men. The sexes may be readily separated by observing the long antennae of the males and the prominent sting and sensory gonapophyses of the females. At eclosion both males and females are ei- ther mature sexually or almost so; consequently females are certainly virgin only if males are not present. In obtaining virgin females , pupae should be isolated, or great care should be taken to see that no mature wasps are present in the culture. These insects have the habit of crawling back into the cocoons after eclo- sion. For this reason it is important to, see that cocoons are either empty or intact. Since virgin females are often necessary in experimental work, methods have been devised for obtaining them in numbers and with a mini- mum of attention. The method that has proved, most successful is also the simplest. Cater- pillars are stung by females of any kind. The females are removed before any eggs are laid. Then about five stung caterpillars are spread out along one side of a shell vial, and the mated female that is to be the mother of the desired virgins is placed in the vial with them. The vial is laid on its side and kept in that position. The female then lays her eggs on the caterpillars, the larvae hatch from them and feed, and when they are ready to pupate, they crawl off the host caterpillars and spin their cocoons on the side of the vial instead of pil- n THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD ing up on each other as they would if the vial v/ere kept upright. As they develop into mature wasps, they can be observed through the vial by means of a hand lens or a binocular dissecting microscope, and as the sex becomes evident, the males or any questionable pupae may be killed with a needle, thereby insuring the eclosion of virgin females only. If it is necessary to rear all the progeny but still to retain the females as virgins, another method is effective. This consists of placing the caterpillars and wasp larvae on cellophane discs placed in petri dishes. When the larvae have pupated, the sex of the pupae may be determined by viewing them from the un- der side of the cellophane by means of a hand lens or a microscope. The sexes may be sepa- rated by cutting the cellophane surrounding them, and the males and females may be placed in separate vials to await eclosion. Various non-genetic influences affect the ratio of offspring. Sperm supply in the semi- jial receptacles influences the female ratio. Production of females tends to be somewhat re- duced in later culture vials preceding their total absence after exhaustion of sperm supply. Matings with old males result in lower female ratios than matings v;ith young males, probably l)ecause of lowered sperm supply. In much of the earlier work, entire fraternities were sum- marized including the haploid males produced after depletion of sperm. Ratios thus obtained are subject to great variation. They depend in large measure on the length of life of the mothers, who may die before they have exhausted 18 CULTURE their supply of sperm received from the initial mating, or who live for many days afterward. The average size of fraternities varies ac- cording to length of life of the mother and the technique of the investigator, being reduced by poor cultural conditions such as small size, scarcity, or disease of the host caterpillars, overcrowding and presence of recessive lethals or semilethals, as well as by crossing between related stocks (close-crossing). Average off- spring per day or per vial may be determined for individual females or for groups of females. The average number of fertilized eggs and hence females per vial or per fraternity may be twice as high in crosses between unrelated stocks' (outcrosses) as in close-crosses. The number of eggs remaining unfertilized and hence the average number of haploid males will be unaf- fected, except for crowding in the larger, out- cross fraternities. To facilitate the counting of Habrobracon progeny, it is necessary to etherize and to ex- amine the anaesthetized individuals under a binocular dissecting microscope at about twenty diameters- magnification. " Since Habrobracon is much more resistant to ether than is Drosophila, it is usually safe to allow one group of waspsi to remain in the etherizing bottle while another group is being counted. For anaesthesia two wide mouthed bottles are used. A cork with cotton suspended on a wire and saturated v;ith ether may be transferred from one bottle to the other. One group of wasps is placed in one of these wide mouthed bottles and etherized while those from the other bottle are being counted and examined. After examination the wasps may 19 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD be returned to their shell vial, destroyed by dropping them into a jar of mineral oil, or preserved for future use. Preserving fluid is nine parts of 95 per cent alcohol to one part of glycerine. The glycerine prevents drying out. In this medium the colors are retained, and the specimens re- main opaque as is desired for observation by reflected light. A binocular dissecting micro- scope is best for observing preserved material. The insects, well-covered with fluid, should be placed in a Syracuse dish and are best seen against a v;hite background. For detailed study low magnification with a compound microscope id desirable (A. R. V/hiting, 1933a). A few speci- mens may be placed in a drop of glycerine on a slide covered with a small cover glass. Al- though the glycerine clears eventually, opacity will be retained long enough for careful study and drawing during a laboratory exercise. Re- immersion in alcohol will restore opacity, and the same specimens may be used several times. A pipette is useful for returning the alcohol to the vials, and the insects may be picked up with a camel's hair brush. The rearing of Habrobracon as an experimen- tal animal is dependent upon many factors, the most important of which is the culturing of itsi host, Ephestia kuhniella. These insects show a complete metamorphosis, and it is the larval stage that is utilized in rearing Habrobracon. The Ephestia larvae are about five-eighths of an inch in length when full grown. Success in rearing the moths is attained with rolled wheat (Pettijohn's Breakfast Food) or untreated yel- low corn meal. These cereals appear to furnish ao CULTURE the optimum stimulation for oviposition, and the larvae feed well upon them and grow large and fat if ample floor space is provided in breeding boxes. The moths scatter their eggs over the cereal and these hatch in approximate- ly a week, the time varying according to the temperature. The young larvae spin webs attach- ing particles of cereal together. After two or three weeks, an inspection should reveal webbi- ness of the cereal, denoting successful pairing and fertility. If temperature is high the lar- vae will attain full size in four weeks from time of isolating the parental moths. A warm temperature 27-30° C. gives best results, but humidity must be fairly high, 80-90 per cent, or the young larvae will not develop. Pupae are formed in silken cocoons. The entire peri- od from egg to eclosion may be reduced to five weeks, but usually six weeks are required for a generation in summer weather. Eclosion beginsi at the end of this time, and moths will contin- ue to emerge for three or four weeks or longer. Moths rest on the cover, sides of box, or on the cereal, and may be conveniently collected in a shell vial. A vial "is placed over each one, and at the same time the culture box or cover is tipped in such a way that the insect, will fall down into the vial when touched by it. If many moths have emerged and are inconve- niently active, the box may be cooled slightly to quiet them. Several individuals, approxi- mately tv/enty, are collected in the vial which is then inverted into nev/ culture medium. The development of Ephestia larvae m.ay be arrested for prolonged use by placing the culture in a cold place. 21 Chapter IV GENE MUTATIONS Habrobracon juglandis has proved to be ex- cellent material for the study of gene muta- tions which produce visible effects, since the haploid nature of the males prevents recessives from being carried over more than one genera- tion before the mutant individuals appear. Most, mutations occurring in the germ tract of a nor- mal wild-type female will appear in her mutant, sons if her eggs are not fertilized. Should her mutant eggs be fertilized, her daughters that develop from these eggs will produce sons, fifty per cent of which will show the new trait. Recessive mutations occurring in the germ tract of a normal wild-type male will become evident in certain of his grandsons; that is, in half the sons of those daughters which developed from eggs fertilized by sperm bearing the mu- tant gene. Of course, this ratio may be dis- turbed by the lowered viability of the mutant. Several mutations have occurred spontaneously in wild-type stocks. These have involved eye color, shape, and size; body color, shape, and size; wing pattern, shape, size, venation, and position; antennal length, structure, and posi- tion; leg length and structure; foot structure; varying degrees of lethality; and sex, Muta- 22 GENE MUTATIONS tions have also been induced through X-radia- tion and temperature variation. Variations in eye color were among the first mutations to be observed. A quadruple allelic series of factors affecting eye color was re- ported (A. R. Whiting and Burton, 1926). ORANGE, 0 (eyes). The normal wild-type, jet black eyes and dark brov/n ocelli, mutated to orange, in which the ocelli have but a slight trace of pigment while the eyes are orange varying to pink and red. The first appearance of orange (P. W. Whiting, 1921c) was in a sin- gle male found March 27, 1920. Crosses between orange and wild-type gave the first definite .genetic check on the supposition that males, being haploid and from unfertilized eggs, in- herit only from the mother. They also gave the first indication that related parents occasion- ally produce a few sons from fertilized eggs which are diploid and like their sisters in ap- pearance. IVORY, oi (eyes). On June 24, 1924, there ap- peared in a fraternity of orange stock, four ivory-eyed males. Ivory has colorless ocelli and compound eyes of a greenish v;hite v/ith oc- casionally a trace of pink. This mutation has proved to be recessive to both wild-type and to orange, forming the third in an allelic series. LIGHT-OCELLI, ol (eyes). In January, 1925, there appeared from a cross of ivory female by wild-type male a single female with light ocelli but with the compound eyes black as in wild- type. This mutant proved to be an allele of 23 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD ivory, and hence the mutation took, place in spermatogenesis involving at least one sperm. Light-ocelli has black eyes but ocelli are very light in compound v/ith ivory or with orange. In pure light stock the ocelli may be charac- terized as light brown, somewhat darker in the males. It was found to be recessive to type and, as regards compound eyes, dominant to orange and to ivory. In combination with orange or ivory the ocelli were lighter than in the homozygous condition. Light-ocelli was lost before the appearance of a fifth allele, dahlia, and, therefore, was never tested v/ith that or v;ith any eye colors found later. DAHLIA, 0^ (eyes). The fifth allele in the orange locus appeared December 1, 1929, from a cross of an ivory female by a wild-type male. A single female with light ocelli and eyes very dark reddish-brown appeared. This mutant, called dahlia, proved to be dominant to orange and to ivory although somewhat lightened in combina- tion with either. In view of decreasing domi- nance associated with decreasing pigmentation in this series, it is probable that light-ocelli vmuld have come between wild-type and dahlia, could it have been tested. The locus, orange, appears to be especially unstable, but there is certainly no striking increase of mutability correlated with X-ray treatment (P. W. Whiting, 1932a). P. W. Whiting (1928b) reported a very high correlation be- tween mosaicism and apparent mutation for this locus. The orange series is female fertile with good viability of both males and females. These stocks, except for light-ocelli, are be- 24 GENE MUTATIONS ing carried in the homozygous state in the lab- oratory for genetic studies. CANTALOUP, c (eyes). The first eye mutation not in the orange locus was cantaloup, reces- sive to wild-type. A female from orange stock was treated by P. W. V/hiting in March, 1928, with X-rays (dosage about 1100 R units). One male with eyes resembling ivory was produced, and subsequent tests proved it to be a new mu- tation. At the time of emergence cantaloup eyes appear white with a slight greenish tinge. They quickly change to a light pink and fre- quently darken so as to resemble orange or car- rot, which will be described later. After death they usually become black, differing in this respect from orange and carrot, which show no corresponding change in color. Ocelli are col- orless. No other allele has been found at this locus. In a mixed culture it may be confused with ivory or with orange. Both males and fe- males are fertile, and this type may be kept in a homozygous condition. MAROON, ma (eyes). Another locus has become marked by maroon, a form resembling dahlia. This mutant was found in January, 1931. Maroon has light ocelli with the compound eyes a very dark red, so dark that it is often necessary to use the light ocelli as an aid in separating it from wild-type. This stock is male and female fertile and can be kept homozygous. WHITE, wh (eyes). Another allelic series in- volves white and carrot. White first appeared in June, 1931, and resembles ivory in color 25 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD with colorless ocelli. V/hite is' of normal viability and fertility. In combination with shot-veins, to be described later, white con- sistently shows small red flecks scattered in the posterior and ventral part of the eyes. White shot-veins is, therefore, known as varie- gated (Fig. 3). CARROT, wh^ (eyes). In March, 1932, mutant males with carrot eyes appeared. This eye color closely resembles orange. White is almost dom- inant to carrot. The white-carrot heterozy- gote has a yellowish cast by which it can be accurately separated from white. Both of these alleles are of normal viability and fertility. SPECKLED, Sk (eyes). This factor is due to a dominant gene and results in numerous small, bright red flecks of pigment in a white eye. Preliminary work indicates that the specks are not on the cornea but lie in the soft tissue beneath. They do not appear in the pupal eye until about the third day of pupal life. Speck- led varies in its expression in the two sexes) and is sensitive to temperature. At 30° C, a few specks are visible in the eyes of the fe- males, which vary among themselves in the de- gree of speckling. At room temperature, specksi are usually lacking in female eyes. In males- at 30° C, the degree of speckling is very pro- nounced both as to depth of coloration and num- ber of specks. Although specks are scattered at random throughout the compound eye, there is a denser speckling in the ventral half of the eye. Males, too, vary among themselves in the intensity and the number of specks. At room 26 Fig. 3. Right compound eye showing effect of the mutant, shot-veins, in a white eye. The darkened areas are red in the living animal. X 160 GENE MUTATIONS temperature, if the lighting is good, a few specks can usually be distinguished in males. So far, white is the only underlying eye color that will differentiate speckled. This mutant is of normal viability and fertility. RED, rd (eyes). Red is a recessive factor iqr eye color which is not allelic to orange. It varies considerably in intensity, depending on the temperature, from a light red to a dark red almost black. The ocelli are a light pink and can be used as an aid for distinguishing the very dark red eyes from wild-type. This mutant is female fertile, and the males are of normal viability. PELLUCID, pi (eyes). Pellucid is an eye mutant in which the compound eyes are semitransparent. It is female fertile, and the males are of nor- mal viability. PINK, pk (eyes). This is an eye mutation in which the compound eyes are pink. It appeared in the progeny of an X-rayed female in the sum- mer of 1945. It is of normal viability and fertility. (Discarded) TINTED, tn (eyes). This mutation can best be observed in combination with white. In this combination the compound eyes are not opaque but have a grayish cast with a semitransparent background. It is female fertile, and the males are of normal viability. MOTTLED, mo (eyes). This is an eye mutation in which the compound eyes are not uniformly black 27 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD but have purple splotches in the ventral por- tion, and the ocelli are light. It is female fertile, and the males are of normal viability. GLASS, gl (eyes, antennae, and tarsi). In Oc- tober, 1931, mutants having smaller eyes and slenderer antennae than normal were found in a fraternity from a female heterozygous for orange and maroon. Glass eyes lack facet outlines and resemble the shiny, immature eyes of developing pupae. Sections of glass eyes show incomplete tissue differentiation; hence, this mutation may result in arrested development of the eye. Ocelli apparently are not affected. Antennae are very slender and of uniform diameter. The tibial spurs of the prothoracic legs are small while those of the mesothoracic and raetathoracic legs are lacking. The foot is much malformed and reduced in size with claws very small or lacking. Glass females are weak and of de- creased fertility. Glass males are of good viability. GLAZE, gz (eyes). This mutant appeared in the summer of 1934 in the offspring of an X-rayed female (dosage about 4000 R units). Glaze re- sembles glass, but antennae and tarsi are not. affected. It is female fertile with males of normal viability. SHINY, gz^ (eyes). Shiny is an eye mutation where the compound eyes are of the v/ild-type black but show a very enamel-like gloss on their surface. In various tests it has been found to be allelic to glaze. Both sexes are fertile with normal viability. (Discarded) 28 GENE MUTATIONS TRANSPARENT, tp (eyes). This is an eye muta- tion in which the compound eyes have a trans- parent appearance. It shows up only with light eye colors. It is female fertile, and the males are of normal viability. Several recurrences of this mutation occurred in the summer of 1945. LIGHT-OCELLI, lo (eyes). This mutant appeared in the summer of 1934 in the offspring of an X-rayed female (dosage about 5000 R units). Here normal wild-type black compound eyes are accompanied by light ocelli. This mutant was not allelic with orange, and both sexes were found to be fertile. (Discarded) PORT, pt (eyes). This mutant appeared in the summer of 1934 in the offspring of an X-rayed female (dosage about 4500 R units). Here com- pound eyes are of a dark red color with light ocelli. In this mutant the males appeared to be sterile, and it was lost immediately after appearance. The several cases of eye-color "mimics" are of some interest. White and ivory are indis- tinguishable except under a high magnification, when it can be observed that white is more translucent than ivory. Both have a slight greenish tinge. Carrot is somewhat more pink than orange although very similar to it, while dahlia and maroon resemble each other v/ith dah- lia tending on the average to be lighter. Any two of these "mimics" crossed together recon- stitute the dominant in the Fl females. Multiple recessives are as light as or lighter than any single mutant type entering into their genetic constitution. In the major- THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD ity of cases they lack pigment. Dahlia and ma- roon form a double recessive which is but slightly different from either single type. The lightening influence of one mutant on another is not necessarily correlated with in- tensity of its color. For example, maroon is darker than dahlia, but when combined with car- rot it gives white, whereas dahlia-carrot is a somewhat lightened carrot ( A. R. V/hiting, 1934). Mutations for eye size and shape have not been as numerous as have those for eye color. KIDNEY, k (eyes). A mutation to kidney eye shape was found on August 18, 1930, and was de- scribed by P. W. Whiting (1932a). The mutant appeared in a male with compound eyes lacking with the exception of a very minute one on the right side. Ocelli were of approximately nor- mal size. The mother had been X-rayed with a dosage of 2600 R units. When the wasps are bred at 30° C, compound eyes and ocelli are reduced in size and elongated dorso-ventrally , some of them kidney shaped. The majority of specimens are inviable, many dying in cocoons as elongate pupae often with small heads. At lower temperature, 25° C, for example, they are of excellent viability and both sexes are fertile. W. F. Dunning (1931) obtained two mutant types, "small" and its allele "extreme small." These have proved allelic with kidney. SMALL-EYES, k^ (eyes), and EXTREME-SMALL, k® (eyes). These alleles are of normal viability and fertility. A study has been made of domi- nance in the kidney-extreme-small compound fe- 50 GENE MUTATIONS males as affected by temperat'ire differences (Speicher, 1932b, lQ33a,b, 1934a, b). The eye size of mutant-type wasps showing this charac- ter is extremely variable, ranging from a total lack of eyes to those which, though approaching, never reach the normal size (F'ig. 4). The in- dividual facets, when present, are of normal shape and size, the variation occurring as a decrease in the total number. The sizes of right and left eyes vary somewhat independently of each other. The ocelli are likewise affected and are also extremely variable, ranging from none at all to those apparently normal in size. Although no actual measurements have been made, it has been noted that the ocelli of any one individual tend to be of the same size. Aside from the modification of compound and simple eyes no other external effect is manifest, (Discarded) SMALL, sm (eyes). This mutant appeared in the summer of 1934 in the offspring of an X-rayed female (dosage about 4500 R units). This mu- tant, in v;hich the compound eyes are much re- duced in size, closely resembles small-eyes. It was discarded soon after its appearance. EYELESS, el (eyes). W. F. Dunning (1931) re- ported a mutation which she called eyeless, a recessive mutant type in which the heads are much malformed when the wasps are bred at 30° C. with large lobes on either side. At lovirer tem- peratures these lobes fail to appear. Rudi- ments of compound eyes may be present, and the wings tend to be somewhat wrinkled. Both sexes are fertile, but females are somewhat weak. 31 Pig* ^* Eyes of wild- type (A) and mutant type (B - P) wasps. From a study of the kidney-extreme -small alleles. B. R. Spelcher, 1932b* GENE MUTATIONS Viability of males is about fifty per cent that of their wild-type sibs. (Discarded.) BAR-EYES, be (eyes). W. F. Dunning (1931) re- ported on another eye mutation, which she called bar-eyes, a recessive mutant type. Males are fertile, but 121 tested females proved sterile. (Discarded) CRESCENT, cr (eyes). In August, 1931, an eye mutation affecting both ocelli and compound eyes appeared in a fraternity of 35 males. The compound eyes are slightly smaller than normal, and the ocelli are crescent-shaped. Crescent is of normal viability and fertility in the male, but fertility seems slightly reduced in females. PEBBLED, pb (eyes). In March, 1932, five males^ of a fraternity had a new eye character, peb- bled, with facets irregularly arranged and eyes^ somewhat smaller than normal. Pebbled males' are rather small and they mate with difficulty. The females are weak or sterile. Viability of males is normal. (Discarded) BULGE, bu (eyes). In this mutant type the com- pound eyes are abnormally bulged transversely. Both sexes are fully fertile with normal via- bility. Mutations for body color have not been aS' numerous as have those for eye color. The wild-type individuals vary in color from honey- yellow to almost black, depending upon the tem- perature at which they are reared; the higher the temperature, the lighter the color. During 95 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD the time in which Habrobracon has 'been reared in the laboratory, six mutations in body color have come to light. Three of these mutants, black, honey, and lemon, are fully fertile and can be kept as homozygous stocks with each col- or separate or in any desired combination. The remaining three mutants, cheese, silver, and sooty, have been discarded. Breeding tests have shown that the genes for the first three mutant body colors are linked and are, therefore, on the same chromosome. BLACK, bl (body). The recessive mutant black was found among descendants of X-rayed materi- al. Later a recurrence of the same gene ap- peared among the progeny of another X-rayed mated female (A. R. Whiting, 1939b). The fac- tor blackens the animal to an extreme degree, even at higher temperatures. The black pattern is similar to that of wild-type reared at 21- 22° C, but yellow areas are considerably light- er, almost cream in fact. The stemmaticum re- mains very black while the praescutum differs^ from that of wild-type at a lower temperature in the shadowy continuation of the median patch to the posterior edge. Dissection shows this pattern to be entirely within the cuticle. Legs are completely black and wings and anten- nae darkened. The whole animal presents a glistening jet-like appearance which becomes even more striking at 19° C. Dissection re- veals in the praescutum a ghost pattern corre- sponding to the light areas of higher tempera- tures, but no trace of the light spots charac- teristic of the mesoscutellum. Mandibular teeth are black. No difficulties are encountered in 34 GENE MUTATIONS separating black from wild-type whatever the conditions of rearing, for the final check is always extent and intensity of yellow regions on head and legs. They remain more extensive and a deeper yellow in wild-type. HONEY, ho (body). In 1932 a type female pro- duced eight daughters and six sons. One of these males lacked black pigment entirely. This trait was found to be hereditary and re- cessive, and it was called honey. Black pig- ment is everywhere absent, even in animals^ reared at low temperatures. A praescutal pat- tern similar to wild-type may be observed, but this is represented in darker yellow or red in- stead of black. This pattern is in the cuticle and not in the structures beneath it. At 30° C, honey has the same color as wild-type raised at a temperature high enough to prevent the formation of black in the body. This trait is associated with good viability, but females) are of reduced fertility. LEMON, le (body). In September, 1933, one male with lemon body color appeared in a fraternity of 124 males bred from a virgin female (P. W. Whitine, lQ34b). A wild-type female had been crossed with a gynoid (to be described later) male which had been X-rayed (dosage about 3500 R units). The trait is very striking, the ground color being pale lemon-yellow, rather than honey-yellow as in wild-type and honey. The black pigmentation of the feet, female gonapophyses, and wings appears perfectly nor- mal while the antennae are black except for the two basal segments which are lemon-yellow. 35 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD Lemon thus shows a striking contrast to honey, and is partially dominant to wild-type. Pale basal segments and characteristic lemon prae- scutal pattern are dominant whereas general body color of the heterozygote resembles wild-type. There are present on the praescutum two dis- tinct anterior bands sharply divided by a light line, and below these, irregular spots, often asymmetrical. Study of the cuticle of the lemon thorax by means of sections and removal from tissue underneath shows that the black pattern is in the cuticle itself. The spotted effect results from internal structures showing through the transparent portions which correspond to the yellow regions in the wild-type and other mutant forms. The two praescutal bands, char- acteristic of lemon, outline more accurately the muscle masses beneath than does the solid patch of non-lemon forms, for dissection re- veals a definite longitudinal division in the muscle masses of this region in all color types. This dividing line in the praescutal pattern is mentioned by Schlottke (1926) as appearing in some wild-type individuals at high temperature, and it sometimes shows in honey as a darker line in the center of the median praescutal patch. In lemon, however, it is consistently present and more extreme. Lower temperatures* change the black pigment of the lemon mutant less strikingly than that of wild^type or black. The black areas never become as intense or widespread, but in spite of this there is a striking lightening of yellow. The females of this mutant are fully fertile, and the males are of normal viability (A. R. Whiting, 1939b). 56 GENE MUTATIONS CHEESE, ch (body). This mutant appeared in the summer of 1934 in the offspring of an X-rayed female (dosage about 4500 R units) (P. W. Whit- ing, 1935b). Cheese is a pale, opaque, green- ish-yellow, and the color is especially notice- able in the head. It was discarded soon after its occurrence. SILVER, si (body). This mutant appeared in the summer of 1934 in the offspring of an X-rayed female (dosage about 4500 R units). In thisi mutant type pigmentation is apparently arrested since the body fails to darken. This mutant, has been discarded. Variation in general body pigmentation is) controlled largely by temperature, but influ- ence of hereditary factors is also obvious. V/hen reared at 30° C, the wasps show differ- ences in color of the mesosternum, SOOTY, s (mesosternum). A factor for sooty mesosternum was shown (P. W. Whiting, 1926a) toj be linked with defective (see wing mutations). This mutant has been discarded. A few mutations affecting body size and shape have occurred. MINIATURE, m (body). One mutation for bouy size occurred in March, 1928. Six sons from a female mated to an X-rayed male (dosage about 2950 R units) appeared with body size much re- duced. The primary wing is less rounded on the costal margin. The antennae are shortened, both by shortening of the segments and by re- duction of their number. There is also some irregularity in the way the segments are set 37 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD together. No marked disproportion appears in the reduction of the size of the legs. Minia- ture is a serailethal. Many die as pupae. Adult males are of good fertility, but adult females are almost sterile. (Discarded) BROAD, br (thorax). In August, 1930, a number of males were found with thorax abnormally broadened. There is some tendency toward re- duplication in the wings. The mutant type was first noticed in about 50 per cent of the sons of two different sisters. The character is somewhat irregular, and it cannot be stated with certainty that this represents the first appearance of the mutation (P. W. Whiting, 1932a). Broad may be called an irregular re- cessive, but about 50 per cent of the heterozy- gous females show the trait to a slight extent. Broad has good fertility and viability. (Dis- carded) SMALL-HEAD, sh (head). This appeared in the progeny of a mated female that had been X-rayed (P. W. Whiting, 1929b). Wasps exhibiting this^ mutation have very small heads. The mutant type is much more distinct in the homozygous^ females and shows considerable overlapping in the males. It is of good fertility and viabil- ity. (Discarded) EXTENDED-HEAD, Eh (prothorax). In July, 1931, an X-rayed mated wild-type female (dosage about 3840 R units) produced in her progeny one male with an extended head, which proved to be a dominant mutation (N. C. Bostian, 1931). In this mutant type, the membrane dorsal to the 3S GENE MUTATIONS presternum is much expanded so that the head is thrust forward. When dead and dried, the mu- tant type cannot be distinguished from normal. Heterozygous females have fertility somewhat reduced. Extended-head males have their via- bility reduced somewhat less than fifty per cent as compared with their v/ild-type sibs. (Discarded) By far the most numerous mutations in Habro- bracon, are those affecting the wings. Shape, size, position, and venation may be affected singly or in combination. The normal wild-type wing is fairly regular in outline with a few definite veins (Fig. 5). The v/ing mutations, like all others, have been named from the ef- fect most obvious to the discoverer. DEFECTIVE, d (veins). Defect most common of the wing varia nature. This mutation invol fourth branch of the radius pie genetic factors are invo ture influences the trait, th being correlated with higher Whiting, 1932a). A single ge in breeding experiments, poss wild-type stock (P. W. Whit isolated and found to give phenotypically defective unde tions, 30° C, Defective is o and fertility. (Discarded) ive is one of the tions occurring in ves a break in the vein (R4). Multi- Ived, and tempera- e greater defect temperature (P. W, ne occurring early ibly derived from ing, 1924b}, was about 95 per cent r standard condi- f normal viability WRINKLED, w (wings). Wrinkled occurred in a female, and is the result of a mutant factor causing difficulty in shedding pupal integument (P. W. Whiting, 1926b), As a consequence ir- 59 Pig. 5 • Right primary wing. C. Costal margin, 0. Outer margin, I. Inner margin. A. Apox. ^1> ^3* %• First, third and fourth branches of radius vein. m-cu. Medio-cubital cross-vein. 1, 4. First and fourth radial cells, p. W. "Ahlting, 1932c. X 47. 40 GENE MUTATIONS regular wrinkling of the wings results, and portions of the integument are found adhering to the antennae which show a tendency to frag- ment. Wrinkled is fertile in both sexes but is mechanically hampered in eclosion and in other activities. (Discarded). REDUCED, r (wing size). This mutation occurred in March, 1925 (P. W. Whiting, 1926b) in a sin- gle male which had small wings and irregular reduced venation (Fig. 7). The trait is varia- ble but always distinct from type. Reduced af- fects chiefly the middle area of the wings and usually causes disappearance of vein m-cu. It is of good viability and fertility in both sexes. SHORT, sh (wings). This mutation was first ob- served in cultures derived from some of the sibs of the miniature mutants (P. W. Whiting, 1932a). It is uncertain whether short occurred as a mutation at approximately the time of the mutation to miniature or whether it had been carried in the stock. Short is very suscepti- ble to temperature, overlapping with normal to a considerable extent. It is impossible to distinguish a culture of short from type when reared at room temperature. Under standard conditions, 30° C, wings of short average much smaller, but even in this case there is more or less overlapping. Females are fully fertile, and males are of normal viability. (Discarded) NARROW, n (wings). Narrow v/as first observed in April, 1930. Fourteen males appeared from a cross between a type female and a type male 41 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD that had been X-rayed (dosage about 2100 R units). Narrow cuts off irregular slices of both wings on costal and inner margins. It is somewhat variable but does not overlap with type. It is of good viability and fertility in the male, but females are apparently sterile. (Discarded) V/AVY, wa (wings). Wavy appeared in June, 1930, from a wild-type female that had been mated to an X-rayed wild-type male (dosage about 2740 R units). Wings of wavy are shortened, showing transverse waves which are especially notice- able near the distal part of the costal margin. Antennae are usually normal, but in many cases one or both become markedly depigmented dis- tally, the segments tending to drop off. Fer- tility and viability of males are good, but fe- males are somewhat weak. VESTIGIAL, v (wings). Vestigial is a wing mu- tation found in August, 1930. A virgin mother produced a number of vestigial winged males. Vestigial is semilethal, many wasps dying be- fore eclosion. Fertility in the males that eclose is good, and many of them live a normal length of time. (Discarded) SPREAD, sp (wings). This mutation appeared in May, 1930, from a male fraternity of orange stock. Eight males with wings spread out to the sides were observed. Further examination showed that these males had light areas on the raesopleura in the position of insertion of the directive wing muscles. The v;ings themselves are normal, and the defect is probably muscu- 41 GENE MUTATIONS lar. The trait grades into type. It was im- possible to obtain offspring from female spread. Males are of normal fertility. (Discarded) NOTCH, No (wings). Notch is a mutation affect- ing the outline of the wing. It appeared in April, 1930, in the progeny of a type female by a tapering male (see antennal mutations). Notch exerts its effect on the margin of either pri- mary or secondary wings; the notches may be terminal or lateral. The first branch of the radius vein is shortened, causing the usual rounded lobe of the wing to be notched; how- ever, there is much irregularity and asymmetry in the expression of this factor. Notch does not breed true, and it is very likely that there are more factors than one involved. In many cases the trait acts like a dominant lethal. (Discarded) SHOT-VEINS, sv (veins). Shot-veins is a semi- dominant factor that arose simultaneously in three different lines after extreme heat treat- ment of larvae. It causes veins of the wings) to be broken and distorted (Fig. 7). This mu- tant was found in August, 1930. Shot-vein stock has proved to be fully fertile and stable. In connection with studies of linkage made in 1931, shot-veins v/as crossed to white eyes, and the Fl females bred as virgins. The double re- cessive, shot-veins and v/hite eyes, showed a mottling of red spots in the posterior ventral region of the compound eye. It has since been observed from very large numbers that these spots are present in both eyes of all white- eyed individuals with shot-veins. Their dis- «y THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD tribution and intensity show some variation, but they can never be seen from the dorsal view. A homozygous, white shot-veins stock was de- rived and called variegated. This variegated stock has been found to breed true and to be of normal viability and fertility (A. R. Whiting, 1933f, 1934). BEMT-WINGS, bw (wings). In June, 1932, a vir- gin female produced a fraternity of eighteen males, five of which had bent-wings. In this^ mutation the wings are quite narrow and are bent anteriorly along the costal margin. The females are apparently unable to sting active caterpillars. However, when placed with para- lyzed caterpillars a few may sting, feed, and lay eggs, but the eggs fail to hatch. The males are of normal viability. (Discarded) GABLED, gb (wings). In March, 1932, an orange defective female (which had been mated with an X-rayed male) produced wild-type females. Sev- en of the azygous males obtained from them had wings which sloped when folded like gables of a roof. The mutant type is thus easily recog- nized in the vials. The wings have venation more reduced than in any other mutant type ex- cept veinless. The factor causes very poor vi- ability; gabled males are less than ten per cent as frequent as their normal wild-type sibs from heterozygous mothers. Gabled females have not been produced (P. W. Whiting, 1934b). (Discarded) FLARE, fl (wings). In February, 1932, a wild- type virgin female produced eleven males two of GENE MUTATIONS which had wings in which the costal margin flared forward distally. Flare overlaps with wild-type so that classification is uncertain. Flare females produce among their offspring some which are phenotypica.lly normal. Both sexes are fertile and of normal viability. (Discarded) INDENTED, in (wings). In December, 1931, an orange defective female. X-rayed (dosage about 1500 R units) as a five-day larva, was subse- quently mated to a wild-type male. An Fl fe- male produced eighteen males of which five had the primary wings somewhat indented toward the tip. Indented frequently shows irregular thin- ning of the radius veins beyond the stigma in primaries and irregular narrov/ing of the secon- daries. Viability is normal and females are fertile. (Discarded) CUT, ct (wings). In February, 1931, six azy- gous males with cut wings appeared in a frater- nity. The outer margin of the wing is greatly indented or straightened giving a cut appear- ance (Fig. 6). The trait is very variable but may be readily recognized. Viability and fer- tility are normal. STRAP, sr (wings). In January, 1932, three males with strap wings appeared in wild-type stock. The first and third radius veins are greatly shortened and the outer margin of the wing is, therefore, given a lobed appearance. The trait is very pronounced and of equal viability with wild-type. (Discarded) 45 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD CONFLUENT, cf (wings). This is a mutation af- fecting wing veins and antennal segments. It was found on August 2, 1930, in a male with shortened wings showing fusion between first and third branches of radius veins at their ex- tremities. There is more or less fusion and deficiency of segments distally in the anten- nae. Degree of abnormality in wings is corre- lated with that of antennae. At slightly in- creased temperature many confluents die in co- coons, and at lower temperature confluent over- laps with wild-type. Females are fully fertile and males are of normal viability. (Discarded) TRUNCATE, tr (wings). This mutation was found on August 7, 1Q30, in the offspring of an X- rayed female (dosage about 2600 R units). One of the male progeny had primary wings much shortened distally tending to cause coalescence of the first and third radius veins. Truncate is very irregular in appearance, apparently having a considerable overlap into type. Ter- minal antennal segments are very likely to be more or less fused, and v/ings are often but slightly affected. The variation suggests con- fluent in certain respects, but in combination with reduced it is more easily recognized. Both sexes are fertile with normal viability. (Discarded) UNEXPANDED, un (wings). This first appeared in the progeny of a type female by a wild-type male in August, 1930. Wasps with this mutant trait have primary and secondary wings unex- panded. This character was carried for several generations, but the wasps were highly invia- 46 GENE MUTATIONS ble, the majority dying in cocoons. Females were obtained, but were inviable and the stock was discarded. POINTED, p (wings). In July, 1931, N. C. Bostian (1931) X-rayed (dosage about 3200 .R units) a wild-type mated female. One of the daughters produced among her progeny twelve males with pointed wings. The tips of the wings of this, mutant type, both primaries and secondaries, are narrowed and wrinkled, the narrowing being especially, noticeable in the radial cell. Fe- males are sterile: they sting caterpillars, feed and lay eggs, living for several days, but the eggs fail to hatch. Pointed males appear in normal numbers from heterozygous mothers. (Discarded) TRUNCATED, td (wings). In December, 1931, an orange defective female, X-rayed (dosage about 2000 R units) as a five-day larva, was subse- quently mated to a wild-type male. An Fl fe- male produced among her progeny five males with truncated wings. The first radial cell in the primary wings of this mutant type is greatly reduced, the wing is narrowed slightly, and the outer margin assumes a squared appearance. The trait is variable but easily recognized and does not overlap with v/ild-type as does truncate, previously described (P. V/. Whiting, 1932a). Truncated females are weak, failing to sting caterpillars or to oviposit. Truncated males appear in normal numbers from heterozygous mothers. (Discarded) 47 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD SMALL-WINGS, sv7 (wings). In March, 1933, six males appeared showing a mutant trait, small- wings. These were among the progeny of an Fl female from a cross between a heterozygous orange female by a narrow winged male. In this mutant type venation appears perfectly normal but wing size is greatly reduced, and there is no overlapping with wild-type (Fig. 6). Small- wings is of good fertility and viability. CREPE-WINGS, cw (wings). In April, 1932, a heterozygous female produced a fraternity of seven males including two with wings irregular- ly wrinkled. The outer margin of the primary and secondary wings is very irregular, and the surface of the wings resembles crepe paper. The trait is variable but easily recognized. The mutant type is somewhat over fifty per cent viable as compared with wild-type sibs. Fe- males are of decreased fertility. (Discarded) DROOPY, dr (wings). In March, 1933, a mutant type with wings held out and sloping downward appeared. Droopy males occur in good numbers but are likely to die in cocoons. They are less viable when reared at 23° C. than at 30° C. Droopy females try to sting the caterpillars but have produced no offspring except in one case when droopy males only -appeared. (Discarded) EXTENDED-WINGS, ew (wings). In July, 1932, five males with extended wings were found. This mu- tant type is easily distinguished when the wasps are active, but if they are etherized there seems to be no observable difference from wild-type. They must, therefore, be separated GENE MUTATIONS in the active condition, which makes counting tedious. The secondaries are extended lateral- ly at an angle of 45 to 60 degrees from the midline, and they also droop somewhat. Both deviations from the normal are apparently the result of deformation at the axilla. The pri- maries may be held almost normally, or they may be extended laterally at an angle approaching or equal to that of the secondaries. The pri- maries are not held downward like the seconda- ries, but in the normal plane as though the ax- illa of the primary is normal, and their posi- tion is simply the result of a defect in the secondaries. The abdomen is slightly elevated as if for balance, especially in cases v/here both pairs of wings are widely spread. The fe- males are sterile but the males seem to be of normal viability. (Discarded) BROKEN, bk (wings). In 1934 a male with mutant wings appeared in the progeny of an X-rayed wild-type female (dosage about 4500 R units). In this mutation the outer margin of the pri- mary wings is broken and the v;ings are very fragile. The veins in the primary wings often resemble those of shot-veins. Broken and shot- veins have been tested together and found to segregate independently (Fig. 7). Both sexes are fertile and of normal viability. AEROPLANE, ae (wings and tarsi). This mutation appeared in the progeny of an X-rayed female (dosage about 3500 R units) in 1934. The wings are stiffly outstretched, and the tarsi are black and brittle. The males are unable to 49 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD mate, and the mutant was lost immediately after appearance. EXTENDED, ex (wings). In 1934 in the progeny of a wild-type female. X-rayed (dosage about 4000 R units), a male appeared with wings held back but failing to fold over the body. This mutant does not show when the wasps are bred at room temperature. This mutant was not tested with others and was discarded soon after its appearance. This mutation recurred in the sum- mer of 1945 in the progeny of an X-rayed wild- type female. Both sexes are fertile and of normal viability. PINCHED, pd (wings). In 1934 this mutant ap- peared in a single male among the offspring of an X-rayed wild-type female (dosage about 4000 R units). The fourth radius vein is very short so that the longitudinal veins are drawn to- gether giving the wing a pinched appearance. Both sexes are fully fertile and of normal vi- ability. (Discarded) REDUPLICATED, re (wings). In 1934 there ap- peared among the progeny of an X-rayed wild- type female (dosage about 4500 R units) a num- ber of males with the primary wings more or less doubled. The mutant is variable, some- times being indistinguishable from type. The males usually have drooping antennae, but the heterozygous females haVe normal antennae. This mutant seems to be semi-dominant since the het- erozygous females usually have wings redupli- cated. The females are fully fertile and the males are of normal viabi]itv, (Discarded) 50 GENE MUTATIONS ROUGH, ro (v/ings). This mutant appeared in the summer of 1934 in the progeny of a wild-type female that had been X-rayed (dosage about 3500 R units). In this mutant type the fourth radi- us vein is absent and the adjacent veins are roughened (Fig. 6). It is female fertile with males of normal viability. VEINLESS, vl (wings). The veins of the wing are partially or wholly missing in this mutant, so that the major portion of the wing appears as an unbroken translucent structure. There is some variation in this mutant, but it is always distinguishable from wild-type (Fig. 7). Both sexes are fertile and of normal viability. ELONGATE, eg (wings). In this mutant the first radial cell of the primary wings is elongated, and the outer margin of the wing is indented at the juncture of the third radius vein and the outer margin of the wing (Fig. 6). Both sexesi are fully fertile and viable. CRUMPLED, cp (wings). In- this mutation the in- ner margin of the primary v/ings is not rigid and the entire wing surface is folded in a wave-like pattern giving the appearance of be- ing crumpled (Fig. 6). The females are fully fertile, and the males are of normal viability. (Discarded) CLIPPED, cd (wings). Here the primary wings are mere stubs with venation completely broken up. There is no wing pattern or variation in the mutant as the wing is so reduced that it completely obscures any classification of the 51 WING TYPES ?lg» S . Right primary wings of wild-type and certain mutants. A. Wild-type. B. Criimpled. C. Small-wings. D. RouglTi. E. Cut. P. Elongate. X 22. 52 WING TYPES Pig. 7. Right primary wings of certain mutants. A, Shot-veins. B. Reduced. C. Clipped. D. Broken. E. Fused. P. Veinless. X 22. 55 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD wings. The wasps are easily distinguishable. The clipped females are sterile and the mutant must be introduced through the males which are of normal viability (Fig. 7). (Discarded) WST, wt (wings). This mutation occurred in the summer of 1945 in the progeny of an X-rayed female. In this mutation the raicrochaetae of the primary wings are irregular in length, giv- ing the wings a wet appearance. The females are fully fertile, and the males are of normal viability. Mutations for antennal length, structure, and carriage have occurred in Habrobracon. These factors may be found singly or in combi- nation. Many of the genes cited in the present work cause greater or less tendency toward an- tennal deficiency; hov/ever, only those that show marked deviations in antennal form are listed as antennal mutants. DEFICIENCY, de (antennae and posterior extrem- ity). In this mutant type the antennal seg- ments may be irregularly fused. Failure of normal development of structures at anterior or at posterior extremities sometimes occurs. Ex- ternal genitalia may be small or lacking, and the digestive tract may fail to open posterior- ly. No definite factors have been isolated for these deficiencies; however, they have been shown to be partially controlled by heredity and to be highly correlated with age of mothers (P. W. Whiting, 1926c). (Discarded) MINNESOTA YELLOW, My (base of antennae). In August, 1929, wild-type stock from Minneapolis,^ 54 GENE MUTATIONS Minnesota, included v/asps with three or four basal segments of antennae decidedly yellow when reared at 30° C. The difference evidently depends upon one gene v/ith various modifiers, determining the' extent of the trait v/hich may be increased by selection. Minnesota yellow is of normal viability and may be regarded as dom- inant since heterozygous females show the trait under standard conditions. (Discarded) YELLOW, Y (base of antennae). In February, 1929, two mutant males were found in the offspring of a female that had been treated with X-rays (dosage about 1460 R units) as a four day lar- va. The mutants had the three basal segments of the antennae strikingly yellow. The yellow color is more definitely confined to the three basal segments in this type than is the case in Minnesota yellov/, but lowering of temperature during development induces melanogenesis in both cases. Since heterozygous females show the trait, it may be called dominant. Yellow is fertile in both sexes but of lower viability than wild-type. Distal segments of antennae often appear pale and if temperature is but slightly increased over standard, yellow wasps become deformed and often die in cocoons. This is not the case with Minnesota yellow in which viability is normal (P. W. Whiting, 1932a). (Discarded) FUSED, fu (antennae and tarsi). A single male was found in February, 1929, with antennal seg- ments fused together and with tarsal segments lacking or fused together (Fig. 8). The wing of fused (Fig. 7) has a very characteristic and 55 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD constant indentation on the costal margin at the apex. The female cannot reproduce because of inability to feed from caterpillars. The fused males, however, are fertile to varying degrees, but they have some trouble in mating because of the defective tarsi (Fig. 9). Fused appears to be doubly effective in the duplex state since fused diploid males have antennae as short as those of the homozygous fused fe- males; while in the haploid males with fused antennae, the antennae are approximately the same length as the antennae of normal females. Two other occurrences of fused appeared in Feb- ruary, 1931. These two recurrences of fused in the same month are noteworthy as no fused was being bred at the time. The two recurrences' were moreover entirely independent (P. W. Whit- ing, 1932a). LONG, 1 (antennae and wings). In June, 1929, a number of males appeared with antennal segments) elongated and distal portion of wings shortened and curved ventrally (Fig. 8). Segments of the legs are somewhat longer and thinner than in wild-type. Males with the mutant long are of good viability and fertility but females are rather v/eak. SEMILONG, si (antennae and wings). A wild-type male was treated in June, 1930, with X-rays (dosage about 2280 R units) and then mated to three females. One of the daughters produced in her progeny six mutant males which have been called semilong because of their similarity to long. Semilong males are of good fertility and viability. Females are also viable and fertile 56 GENE MUTATIONS but rather weak. Antennal segments are length- ened but are not as attenuated as are those of long (Fig. 8). Leg segments are also somewhat lengthened but heavier than corresponding seg- ments of long. Distinction of semilong as of long from wild-type is clear in antennae and to a great extent in legs, but overlapping between long and semilong apparently takes place espe- cially in legs. The wing of semilong is quite flat. Distal shortening, however, takes place as is evidenced by the form of the outer margin and especially by the abbreviation of the first radial cell. A slight indentation is present at the apex of the wing, not however, as pro- nounced as in fused. It was first thought that, semilong might be allelic to long, but this) proves not to be the case. TAPERING, ta (antennae). In May, 1930, nine males with tapering antennae v/ere found in a wild-type culture. The antennae are very defi- cient with much fusion and irregularity of seg- ments distally (Fig. 8). Tapering does not overlap with type, but reverse mutations have occurred (P. W. Whiting, 1932a). Both sexes^ are fully fertile and of normal viability. ANTENNAPEDIA, ap (antennae). In the course of genetic work involving tapering, v/asps were ob- served with tarsal-like claws on the terminal segments of the antennae. A more or less well- formed foot may be present (P. W. Whiting, 1934b) . The distal segments may be modified to resemble a tarsus v/ith the terminal segment provided with claws, arolium, and calcanea as in the normal case. The foot may be present on 57 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD one or both antennae and is often imperfectly developed. The trait occurs in a large but variable proportion of the individuals of a se- lected strain. (Discarded) LEGLIKE, Ig (antennae). In April, 1933, males with leglike antennae were found in the progeny of a heterozygous female. Leglike is a much more extreme deviation than antennapedia. The entire antenna, except for the two basal seg- ments, has been changed so that the grooves for the sensillae are absent and yellow color simi- lar to that of the legs has replaced the black. Fusion of several segments distal to the two basal segments and swellings on the resulting mass suggest the malformed tibiae of certain types of inviable pupae (to be described later). The foot is provided with tarsal claws, aroli- um, and calcanea similar to a foot from the normal position. Leglike could not be repro- duced because the males would not mate, and no sisters were produced. (Discarded) ACIFORM, ac (antennae and female genitalia). In December, 1932, two sons of a wild-type fe- male were observed to possess antennae with segments of the terminal half much reduced in diameter (Fig. 8). The mutant type males are of normal viability. Gonapophyses of females are much shortened, and the sting is usually defective; so that the females are sterile, failing to sting and feed on active caterpil- lars. The antennal character is easily distin- guished, since the basal nine or ten segments are normal, but the terminal segments are much 58 GENE MUTATIONS narrower with more or less fusion and deletion in both sexes. DWINDLING, dw (antennae). In July, 1931, a mated wild-type female was X-rayed (dosage about 3200 R units). Among her offspring was one male with dwindling antennae. This mutant type shows much irregularity and fusion of antennal seg- ments in the male, but the proximal half of the antenna is not affected. Females of dwindling cannot be distinguished from wild-type pheno- typically. Dwindling males are numerically equal to somewhat over two-thirds of their nor- mal brothers. (Discarded) ATTENUATED, at (antennae and genitalia). In May, 1932, a fraternity was bred consisting of males, fifty per cent of which had antennae with much malformation and fusion terminally. Genitalia were also abnormal, and the type could, therefore, not be perpetuated. The mu- tation has been reported, and the mutant type figured (P. W. Whiting, and A. R. Whiting, 1934) . The antennae are similar'in appearance to those of dwindling. (Discarded) GYNOID, gy (antennae and abdominal sclerites). In April, 1932, among the offspring of a het- erozygous female, a male appeared having short antennae resembling those of a female. This mutant type, gynoid, the gene for which causes haploid males to be weakly intersexual was later described by P. W. Whiting, Greb, and Speicher (1934). Gynoid males are similar to normal males in internal structure and in ex- ternal genitalia. Their ocelli are large re- 59 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD sembling those of normal males. Sclerotization of the abdomen is progressively heavier anteri- orly, approximating the condition found in the female. Gynoid females are indistinguishable from wild-type. The trait acts as a recessive in heterozygous diploid males (P. W. V/hiting, 1943b). This mutant is highly fertile and vi- able in both sexes. SHORT, sh (antennae). In the summer of 1934, in the offspring of an X-rayed wild-type female (dosage about 4500 R units), a male was found with reduced antennal length. The mutant short resembles tapering, but the antennae are very much smaller. Both sexes are fertile and of normal viability. (Discarded) STUBBY, sb (antennae). Males with antennae seven to nine segments long v/ere observed in the offspring of an X-rayed wild-type female (dosage about 3500 R units). This mutant first appeared in the summer of 1934. The homozygous females for the mutant have antennae with five to seven segments. These few segments may be fused together giving the appearance of one or two large segments forming the antennae (Fig. 8). Both sexes are fertile with males of nor- mal viability. : STUBBY-ABNORMAL, sba (antennae). Males with ab- normally long stubby antennae were observed in a homozygous stubby stock. Breeding tests have shown that stubby and stubby-abnormal are allel- ic. Both males and females are fully fertile with normal viability. 60 ANTENNAL TYPES X 24. 61 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD COALESCENT, co (antennae). In a fraternity of v/ild-type stock several males were observed with antennae of approximately normal length but with the segments coalescent. The females are fully fertile with males of normal viability. A few mutations involving leg length and structure and foot structure have occurred in Habrobracon. These factors, like those for eye, v/ing, body, and antennal mutants, may be found singly or in combination. However, leg and foot mutants seem to cause reduced viability. This may be the result of a semilethal or their ina- bility to mate properly. BEADED, b (legs). In August, 1929, a fraterni- ty of males was observed containing a number of mutants with beaded legs. The proportions of the various parts of the wing of beaded are very aberrant, and wings often fail to expand. Antennae are very likely to show fusion of seg- ments and often more or less distal deficiency. Beaded is semi-lethal. The mutant types sur- vive much better at 23° C. than at 30° C. Even at higher temperature the majority may attain the pupal stage, but few emerge from the co- coons. Tibiae and femora are much shortened and swollen, the swelling of the tibiae being confined to the distal half (P. W. Whiting, 1934b). Males of beaded are fertile, but via- ble females have not been found (P. W. Whiting, 1932a). (Discarded) STUMPY, st (legs). In September, 1930, a sin- gle male v/ith stumpy legs was found among the offspring of an X-rayed female (dosage about 3250 R units). The most striking characteris- 62 GENE MUTATIONS tic of stumpy is the extreme reduction of tar- sal segments (Fig. 9). The appendages appear otherwise normal. Stumpy may be called a re- cessive, but a large proportion of heterozygous females bear a spur at the tip of their protho- racic metatarsi. More or less irregularity oc- curs in tarsal segments of all legs so that heterozygotes are not as sure footed as pure wild-type. No metatarsal spur has ever been found on middle or hind legs of heterozygoteS' nor has a prothoracic metatarsal spur ever been found in wild-type females. This spur closely resembles the spur normally occurring at the end of the prothoracic tibia. It may be pres- ent on either or both front legs, but many het- erozygotes fail to show it. Even in its ab- sence it is usually possible to identify het- erozveotes by tarsal irregularity. Stumpy is) of normal viability and fertility except that females have difficulty in feeding from cater- pillars and consequently may die without pro- ducing eggs. Apparently they cannot brace them- selves well enough to insert their stings for puncturing in order to feed. TWISTED, tw (legs). A male of wild-type stock was X-rayed in January, 1930 (dosage about 3560 R units), and then mated to four females. In the offspring of one of these females there ap- peared fourteen males v/ith twisted legs. Legs of twisted show much malformation and irregular bending. Many individuals fail to eclose, prob- ably on account of mechanical difficulties . Males are of normal fertility, but females are almost sterile (P. W. Whiting, 1934b). (Discarded) 63 LEG TYPES Pig. 9 • Right metathoracic legs of and certain mutants. A, Wild-type, C. Stumpy. D. Footless. wild- type B. Fused. X 26. GENE MUTATIONS CONSTRICTED, Cs (fe:Tiora). In August, r-)30, a single female was observed v/ith constricted femora. This mutant type is dominant and ap- parently lethal in the male. It is difficult to obtain offspring from constricted females, and fraternities are small. The difficulty is probably mechanical and concerned with feeding from the caterpillars. The trait appears in all three pairs of legs but is most obvious in the metathoracic pair (P. W. Whiting, 1932a). (Discarded) CLUB, cl (tarsi and wings). In July, 1931, a mated wild-type female X-rayed (dosage about 3270 R units) produced among her offspring eight males with club feet. The terminal seg- ments of the tarsi are much malformed, fused, and sv/ollen. The v;ings show more or less ab- normality with a tendency to form extra veins in the radial cell. Club males are about fifty per cent as frequent as their normal sibs. Fe- males fail to oviposit even v/hen caterpillars have been stung by another wasp. A recurrence of club appeared in the offspring of an X-rayed female (dosage about 3500 R units) in the sum- mer of 1934 (P. W. Whiting, 1935b). The hind feet of the club males are flattened and curved down. The antennae droop terminally with de- formation of three or four segments. There is a high percentage of pupal inviability, but the males proved fertile. (Discarded) FOOTLESS, fo (feet). In March, 1933, fifty-two males appeared in the offspring of a heterozy- gous virgin female which were footless. This mutant type is characterized through a defect 65 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD at the end of the fifth tarsal segment involv- ing lack of claws (Fig. 9). Wings of footless tend to be more or less wrinkled. Footless males appear in good numbers from heterozygous mothers. The females drink honey, but cannot ■feed on caterpillars or lay eggs. The males mate readily but experience much difficulty in walking and are unable to climb. A recurrence of this mutant is described by P. W, Whiting (1935b). This mutation again appeared in the summer of 1945 in the progeny of an X-rayed female, LUMPY, Ip (legs). In the summer of 1934 a sin- gle male, with legs resembling beaded, appeared in the offspring of a wild-type female that had been X-rayed (dosage about 4000 R units). The mutant was discarded soon after its appearance. WOOZY, wz (legs). A wild-type female X-rayed (dosage about 4500 R units) in the summer of 1934 produced in her offspring several males with leg segments abnormally dark. Some of the male offspring on the other hand were noticed to have leg segments practically without pig- ment. The wings in most cases were cupped over the body. This mutant type was found to be in- viable at 30° C. and to overlap with normal wild-type at room temperature. Both sexes were fertile and of normal viability. (Discarded) Many of the foregoing mutations, such as fused, are semi-lethal and would be lethal ex- cept for laboratory care in feeding and mating. However, a number of mutations have occurred which have their effect at a time or to such a degree that their possessor cannot eclose or 66 GENE MUTATIONS live as adults. The effects of such, factors are visible but completely lethal. LETHAL NAKED PUPAE, np (pupae). A single fe- male in a pure culture proved heterozygous for this factor. It caused metamorphosis of larvae into undersized naked pupae which attained ap- proximately normal coloration, but died before reaching maturity. This factor (P. W. Whiting, 1921f) proved to be linked with orange (P. W. Whiting, 1932a). INVIABLE PUPAE, ip (pupae). The following ex- amples will serve to illustrate some of the types of pupae differing in size and bodily proportions and dying on account of lethal or semi-lethal genetic factors (P. W. Whiting, 1934b). Records of mutant type males segregating from heterozygous mothers not infrequently show more or less numerical deficiency either of wild-type or of the mutant type. When this is statistically significant it is taken to indi- cate the presence of a linked lethal. Usually no record is made of inviable eggs or larvae, but in some cases dead pupae are recorded. The first case of linkage in Habrobracon was found (P. W. Whiting, 1921f) between orange eyes and a factor, lethal naked pupae, having a lethal effect during the pupal stage. Crossovers were 19.5 per cent and there were 142 pupae to 160 adults indicating that relatively few of the inviables died before pupation. No record was kept of eggs or larvae. The inviable pupae were of approximately normal proportions, per- haps slightly more slender, but they failed to 67 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD Spin cocoons and ranged in length up to 2.3 millimeters as compared with 2,9 millimeters for the normal wild-type wasp. A recurrence of this mutation occurred in the summer of 1934 (P. W. Whiting, 1935b) in the offspring of a mated wild-type X-rayed female (dosage about 4000 R units) . LETHAL, L (pupae). A locus of three allelic factors for lethal effects, wild-type, La, and Lb has been determined by Schaeffer (1945). The lethal effect is regarded as complementary and probably lies to the right of stubby. In September, 1943, P. W. Whiting (1943a) described a series of multiple alleles which seem to determine sex in Habrobracon. SEX GENE OR CHROMOSOME SEGMENT, x (sex). Nine factors are thus far known in the series, and they are designated by the symbols, xa, xb, xc, xd, xe, xf, xg, xh, xi. The tester stocks are designated as xa/xb, xc/xd, xe/xf, xg/xh, and xa/xi. Evidence of allelism of these sex fac- tors is furnished by the fact that they are all closely linked with the gene, fused. The mul- tiple alleles are regarded as differential chro- mosome segments which have been built up in the early evolution of the Hymenoptera. They may each consist of many genes, which determine the numerous sex differences, structural, function- al, and behaviorlstic , which characterize the Hymenoptera. These genes have the effect that all haploids or homozygous diploids are similar and male, and that heterozygous diploids or combinations of any two different alleles re- sult in females. 68 Chapter V RECORDING OF DATA In recording genetic data in Habrobracon juglandis, P. W. Whiting has simplified the con- ventional methods. Mutants are symbolized by the first letter or certain letters of the name given the mutant; for example, gl stands for glass, r for reduced, and gz for glaze. If the mutant is recessive to its normal wild-type al- lele, lower case letters are used; however, in a few cases, the mutant is dominant to its nor- mal allele, and it is then written with the first letter of the symbol capitalized; for ex- ample, Y stands for yellow, and Sk for speckled. Normal or the usual wild-type alleles are sym- bolized by the plus (+) sign. The plus sign is used at all times to indicate the dominant al- lele of any recessive character, but is omitted from the male and female formulas wherever possible. The following example will more clearly in- dicate the method of writing £?enetic formulas in Habrobracon studies. Let us, for example, cross a homozygous mottled female by a reduced male. The female would be indicated thus, +.mo/+.mo. She is type for reduced and there- fore the + sign need not be written in the for- mula. She is also homozygous for mottled so it would be permissible to merely write mo for her 69 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD genetic composition. The male would be indi- cated thus, r.+. He is type for mottled, there- fore, the + sign may be omitted from his for- mula leaving only the r to indicate that he is both phenotypically and genotypically a mutant with reduced wings. We may , therefore , indicate this cross by the formula mo X r. The Fl het- erozygous females produced from this mating v;ould be phenotypically type but carry in their genotype the recessive r and mo. Their formula would then be +.mo/r.+ which maybe written mo/r indicating that the mo gene was received from their mother and the r gene from their father. This again eliminates the + sign and simplifies the formula. The Fl males produced would, of course, all be mo. The Fl females, if set as virgins, will produce four classes of sons in gametic ratios, +, mo, r, mo.r. For clarity a period is used to separate the symbols of the double mutant. Since four classes of sons are produced by the Fl virgin females we may be certain that the mutants involved are not al- lelic. On the other hand if only two classes appeared as in some eye color crosses it would indicate that the mutants involved were allelic, each affecting the same character. In recording the F2 progeny t of the Fl fe- males, it is advisable to assign each female involved a number — 1, 2, 3, etc., and to letter each successive vial through which they are transferred — a, b, c, etc. Thus we have la, lb, Ic, etc., for the first Fl female of the origi- nal mo.X r cross. The progeny of each female and for each vial through which she passes may then be tabulated as in Fig. 10. The entire Drogeny from each female in the experiment is 70 EXFERIMEMT S646 mo X r Mo» 1 ^o/r P^ Virgin Set for P2 Sons Progeny Vial ^ mo T no«r Total «&©■ It) /if »!.rf U / ib 4 3 5 2 14 I0 5 8 6 ^™-«™«~— ^g-«-. Id 8 4 7 5 24 le 0 3 4 2 9 If 2 5 0 3 10 ig 5 6 S 1 15 Ih 3 4 2 G 15 .11 S 0 2 6 10 Total 33 55 31 23 127 Pig. la Indi¥idual F^^ f ©male ciiart showing F2 mala progeny pro'duced per vial and the total Pj, faraais' progan^fa 71 B^BEIMEHT 5546 % T@st for ULnkage of m.o and r BO X r F^ mo/r Virgins Set for Fg Sons Progeny Fraternities ^ mo r mo.r Total 3646.1 53 35 31 28 127 3646.2 39 26 32 28 ISS 3646.3 S9 26 20 52 107 3646,4 26 29 87 35 115 3646,5 35 36 37 57 155 3646.6 42 57 47 27 153 3646.7 29 23 35 86 lis 5646 o 8 ,54 24 41 31 150 ■.iimi— 267 231 270 242 1010 Conolusioa: mo and r segregate indepaadently. Fig. Hi MRst®r sho@t of F^ feaiales showing th© total progeny in th® ©sperimont and conclusions. 72 RECORDING OF DATA then tabulated on a master sheet (Fig. 11). Each female carries her ov;n number and to it is added the number of the experiment and the year in which it was performed. Thus in the number 3646.8 the 36 represents the experiment, the 46 the year in which the experiment was performed, and the .8 the eighth Fl female set in the experiment . This method of recording data is suggested by the writer as it offers the most convenient and accurate way of keeping track at all times of each Fl female and her progeny involved in any experiment. 73 Chapter VI SEX CONDITIONS Like other hymenopterans , Habrobracon juglan- dis is parthenogenetic in that males are pro- duced from unfertilized eggs, so that the males possess only a single set of chromosomes. Fe- males which normally develop only from ferti- lized eggs have a double set of chromosomes and are diploid. The haploid males are extremely useful in genetic studies, since their appear- ance indicates their exact genetic composition. The females, being diploid, may bear more fac- tors than they show, since the effects of cer- tain genes may mask those of others, which are recessive. In order to bring recessive alleles to light, females are set unmated on Ephestia caterpillars and allowed to lay eggs. The re- sulting sons exhibit the effects of all the genes carried by their mothers, since during egg maturation the paired chromosomes of the fe- males separate giving dominant genes to some eggs and their recessive alleles to others. For this reason, the wasp is an especially advanta- geous animal for genetic studies, since in one generation only, all of the genie characters possessed by the stock are brought to light. Occasionally females from unfertilized eggs (thelytoky) are found in fraternities which nor- mally produce males from unfertilized eggs 74 SEX CONDITIONS (arrhenotoky ) . These males and females from un- fertilized eggs have been termed "impaternate" by Ray Lankaster (1919) to differentiate them from the parthenogenetic virgin mother. Breed- ing tests show that impaternate females are nor- mal diploids. P. W. Whiting (1924a) has ex- plained their production from unfertilized eggs by the assumption that the second maturation division of the oocyte is suppressed. If this assumption is true and the impaternate females occur among the F2 from a cross involving mutant characters, their genotypes, as indicated by their appearance and that of their offspring, are of interest in ascertaining whether the first division of the oocyte from which they were formed was equational or reductional for the locus or loci involved (K. G. Speicher, 1934). If the Fl female producing an impater- nate daughter is heterozygous for a recessive factor and the first oocyte division is reduc- tional for the locus of that factor, the impa- ternate daughter will be homozygous for either the recessive or its type allele. If the first oocyte division is equational, the impaternate daughter will be phenotypically wild-type. Breeding tests are necessary to distinguish im- paternate females resulting from an equational division from those receiving the type allele from a reductional division. Diploid males of biparental inheritance reg- ularly occur in related stocks. These sterile (or near-sterile) males are diploid, developing like females from fertilized eggs. The haploii Tiales have been called uniparental and azygous in contrast to the biparental zygous males and females. The offspring of these diploid males 75 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD crossed with recessive females are triploid fe- males showing the dominant traits of their dip- loid fathers. There is no evidence of inter- sexuality in diploid males or in triploid fe- males unless the reduced size of ovaries and near sterility of the latter indicate this. Moreover their reproductive reactions are en- tirely normal, the diploid males mating readily and the triploid females stinging caterpillars and feeding and ovipositing upon them. Unmated females produce azygous sons only. Mated females produce azygous sons from unfer- tilized eggs but in smaller numbers than do un- mated females. They also produce zygous bipa- rental offspring from fertilized eggs. Whether the biparental offspring shall be daughters on- ly or both sons and daughters depends upon the relationship of the male and female used in the mating. If the male is from a stock unrelated to the female all the biparental offspring are daughters, but if the male comes from a related stock, biparental sons as well as daughters are produced. In general the azygous and biparental off- spring from a mated female occur in the same ratio from the different vials through which the female is transferred (a, b, c, d, etc,) until her supply of sperm is exhausted. In sub- sequent vials (e, f, g, etc.), azygous sons only appear. Biparental males cannot conveniently be sep- arated from their azygous brothers unless the mother has a recessive trait and the father has the allelic dominant. Orange-eyed females crossed with wild-type (black-eyed) males pro- duce black-eyed daughters and orange-eyed azy- 76 SEX CONDITIONS gous sons. If biparental sons are produced they maybe readily separated from their azygous brothers by their black eyes. Fertilized eggs may be "female-producing" or "male-producing," the latter occurring only if parents are related. "Male-producing" ferti- lized eggs are less likely to hatch than "fe- male-producing." Consequently there are more "bad eggs" and fewer biparental offspring if the mating has been v;ith a related male, since the percentage of eggs fertilized is the same wheth- er the male is related or unrelated. Biparen- tal male larvae are also less likely to mature than female larvae. This further reduces off- spring from related parents. Two types of male sterility may be distin- guished in Habrobracon. If the eggs are not fertilized, the mated female breeds like an un- mated female, producing a large number of azy- gous sons. This occurs in the case of matings>- with biparental males which produce diploid sperm rarely capable of fertilizing eggs. There* are also males with abortive sperm ducts or testes which may readily be mated but transmit no sperm. Recent evidence indicates that sperm may to some extent be inactivated by high dos- ages of X-rays, so that they are unable to pen- etrate the eggs. Cytological studies indicate that there are ten chromosomes in the normal haploid male (Fig. 12) and twenty in the biparental diploid male (Fig. 12). The normal female has twenty chro- mosomes (Fig. 12), while the daughters of normal mothers and biparental fathers have an as yet undetermined but considerably larger number, in all probability thirty. A spermatogonlal study 77 CHROMOSOIAES OF HABROBRACON 1 )> fjPtl 2 >> dati 4 €> (IUm 8 1935. A, 1-4. Spermatogonia! complexes of haploid males, B, 1-8. Second spermatocyte complexes of haploid males. C, 1-3. Spermatogonia! complexes of diploid males. D, 1-3. Second spermatocyte complexes of diploid males. E, 1-2. Odgonial com.plexes of diploid females, X 3,280 78 SEX CONDITIONS has been made in both the normal and biparental males. Following the spermatogonial divisions, both kinds of males show an abortive first mat- uration division--a small bit of the cytoplasm being pinched off at the narrow end of a pear- shaped cell. This division of the sperm cell in the male is comparable to the reduction or mei- otic division of the egg, at which time the paired chromosomes separate, forming abortive polar bodies and one haploid egg nucleus. Eggs if unfertilized by sperm will hatch into normal haploid males. If fertilized, they will pro- duce normal diploid females; and in abnormal cases, diploid biparental males. It has been shown (Speicher, 1935; Risman, 1941) that diploid males have much larger cells than diploid females, while haploid males have cells approaching the diploid female size. It seems possible that large cell size might be responsible for the high diploid male mortality and that a demonstrable difference in cell size might appear between diploid males differing in mortality. In developing Habrobracon it is observed that each bristle on a wing surface corresponds to and gives indication of the presence of a wing surface cell (Speicher, 1935). Since each cell of the single layer forming the upper surface of the wing bears a single bristle (microchaeta) , variations in cell size produce proportional variation in dispersal of bristles. Thus, if fewer michochaetae are noted per unit area larger cells are present in the wing surface. A tendency toward larger cell size in diploid male material with higher mortality is indi- cated in microchaetal evidence. The tendency is 19 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD not extreme in the adult ani'iials stuliei pre- sumably because extreme types did not survive to the adult stage. Evidence of selective ac- tion of mortality is shov/n by differences be- tween frequency distribution curves of diploid ■males and the curves of more viable types. In all material investigated (Grosch, 1945) diploid male cell size was significantly larger than female cell size. This is shown by the number of microchaetae per unit area of wing surface and by measurements of eye facets. To explain diploid male mortality it is suggested that doubled gene situations in the diploid male, homozygous for a sex allele, might func- tion poorly under the surface-volume relation- ship of the large diploid male cell. The large size of diploid male cells may itself be the result of gene duplication. The system which raises haploid male cells to a size comparable with diploid female cells may be increased in activity when its determiners are doubled in the homozygous sex allele condition of the diploid male. No statistically significant differences are shown in body length between diploid males and females, and observations indicate that diploid males do not exhibit gigantism. Haploid male mean body length is shown to be consistently less than that of diploid wasps, and this is taken as indication of the tendency toward dwarf- ism of the haploid male. Wing length corre- sponds to body length in all classes. Larger eyes of the male are a secondary sex character not affected by cell size differences. This sex character is not as clear cut as anten- nal length. Long antennae of the male are a SEX CONDITIONS secondary sex character. In no diploid males has there been a significant shortening of an- tennae which would be considered a tendency to- ward intersexuaLity . Such stability of second- ary sex characters is discussed on the basis of the sex allele being a differential chromosome segment. Female antennae are shorter chiefly because of fewer segments. Diploid males have a tendency toward fewer and loneer antennal seg- ments than haploid males. Fewer antennal seg- ments in the diploid male are not regarded as the result of intersexuality but as an indirect effect of chromosome number. Definite types of reactions characteristic of sex are found in Habrobracon. The females sting caterpillars on which they subsequently feed and lay their eggs. The males are entire- ly indifferent to caterpillars, but they show characteristic reactions towards females, such as flipping wings, mounting, and beating with wings and antennae in the process of mating. Gynanders (sex-mosaic individuals) occasion- ally appear in bisexual fraternities . Gynandro- morphic offspring from a .recessive female by a dominant male show the maternal trait in the male (haploid) parts, the paternal trait in the female (diploid) parts. This evidence is con- sistent with the theory of egg binuclearity . Fertilization of one nucleus results in the fe- male parts; development of the other without fertilization results in the male parts. Gynan- ders may have male heads, female abdomens, or the reverse; one side may be male, the other fe- nale; anterior left and posterior right may be of one sex, the remainder of the other sex; male islands may occur in female regions or female 81 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD islands in male regions. When genitalia are mixed in sax, there may be a full set of male structures and a half set of female structures. Two of these cases have been published (P. W. Whiting and A. R. Whiting, 1927). Gynanders in- dicate tlmt the various sexual reactions are determined by the head rather than by the repro- ductive organs of the animal. Those with male heads and female abdom^ens react towards females but are in lif f erent to caterpillars ; while those with female heads and male abdomens pay no at- tention to females but respond to caterpillars. Gynanders v;ith mixed heads act in general either like males or li^-^e females. Gynandromorphic be- havior is not all as clear-cut as the head and abdomen variations described above. Some show momentary sex-reversal for one or more brief periods of time, and therefore, behave sometimes like females and sometimes like males. There is also a bisexual type of individual which at- tempts to sting caterpillars as well as to mate with females; and there is also a type v/hich has been termed "wires crossed." These individuals attempt to mate with caterpillars and to sting females. All these various irregularities of behavior are thought to be caused by the mosaic character of the. sensory and nervous systems rather than to any hormonal action. Very little evidence of intersexuality has been found in Habrobracon so far. One mutant type, gynoid, has been found, the gene for which causes haploid males to be weakly intersexual. Gynoid females are indistinguishable from wild- type. The trait acts as a recessive in hetero- zygous diploid males. Gynoid males are similar to normal males in internal structure and in 82 SEX CONDITIONS external genitalia. Their ocelli are large re- sembling those of normal males. Their normal male instincts indicate that the brain is struc- turally as in .the male, since mating reactions in Habrobracon are determined by the brain. Sclerotization of the abdomen is progressively heavier anteriorly, approximating the condition found in the female. Antennae of normal males have about twenty-one segments in the f lagel lum, those of females usually not more than fourteen. In gynoid males the segments are reduced in num- ber to that of the female, although they are not quite as short and thick. Superficially a gyn- oid male suggests a sex-mosaic or gynander with female head and male abdomen. Certain struc- tures intergrade , the body is approximately sym- metrical with all parts presumably of the same genetic constitution, and the type is perpetu- ated as a pure-breeding form. Nine intersexual females have recently oc- curred among the offspring of a single female. Superficially these appear to be the reverse of the gynoid males, being more masculine anterior- ly and more feminine posteriorly. The heads are characteristically male having large ocelli and long antennae, the segments ranging from eight- een to twenty-one v/ith tv/enty as the mode. Tests made on five of the nine showed indiffer- ence to caterpillars and vigorous attempts to mate with females, indicating the brain to be structurally male. Abdominal sclerotization is male-like anteriorly. The first and second ter- gites are thin and the anterior sternal thick- enings small. Sclerotization is progressively heavier posteriorly and sternal thickenings be- come elongate, approximating the condition of 83 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD the female. Like gynoid, these intersexes dif- fer from sex mosaics in being approximately sym- metrical and similar to each other. They pos- sess intermediate sex characters and occur in a group in one fraternity as if caused by a sin- gle hereditary factor. The existing data must be regarded as inade- quate to prove whether these intersexes are the result of a modification of the normal sex-dif- ferentiating factor or whether, like gynoid, they are the result of an independent change. It is questionable whether the diverse effects of gynoid antennae and abdominal sclerotization should be regarded as multiple effects of a sin- gle gene. Gynoid may possibly be a transloca- tion from the differential segment determining sex. In a male with the sex allele in the nor- mal position this might give a complementary feminizing effect causing intersexuality . Mosaics also occur which apparently possess either one sex or the other; however, these too exhibit interesting peculiarities. Genetic ev- idence indicates that mosaics, v;ith but few ex- ceptions, arise from binucleate eggs. The two nuclei are assumed to be the products of the second oocyte division. In the case of gynan- ders one of the nuclei is fertilized and gives rise to the female parts while the unfertilized nucleus produces the male parts. Cell descend- ants of each of the two nuclei tend to be more or less segregated in different regions of the embryonic syncytium and consequently the genet- ically diverse regions may be roughly separated by a plane. This plane of division may appar- ently lie at any angle to the axis of the body and may separate proportions varying from ap- SEX CONDITIONS proximate equality to such decided inequality that tissues of either type may represent but a minute portion of the specimen. Furthermore, the region on either side of this plane may con- tain tissues of the opposite genetic constitu- tion indicating more or less wandering of nuclei in the embryonic syncytium. Occasionally lack of segregation may be so extreme that the re- sulting mosaic is an irregular patchwork. Read- justments during later embryonic development may, moreover, cause intermingling of adjacent tissues differing genetically. In certain normal wasps , there occur two non- allelic, recessive genes both of which cause the eyes to be whitened. For convenience, these genes are called white and ivory. One interest- ing combination of these colors occurs in the mosaic compound eye, one section of the eye be- ing genetically white, the other genetically ivory. Such an eye might be expected to look uniformly white; however, such is not the case. The white, non-ivory region remains white and isi sharply marked off from the ivory, non-white region by a black border which grades impercep- tibly into the ivory (Fig. 13) (A. R. Whiting, 1933b). The double dominant character is recon- stituted in the region where the two recessives are in contact even though there is no diploid tissue present there. This phenomenon is thought to be the result of the diffusion of some sub- stance from the white region into the ivory. Another instance of the effect of one gene upon another is exhibited in the progeny of a female heterozygous for ivory and cantaloup eye colors. Such a female set as a virgin may produce, in adiition to the four genotypes of sons regular- 85 Pig. U . Right compound ©yo of mal© mosaic for whit© and ivory. The double dominant cfcaract«^r is re cons ti tilted in the ragion where th8 two rec©saiv0S are in cojitact. X 208. 86 SEX CONDITIOiNS ly expected, a male with ivory, noii-catitaloup and non-ivory, cantaloup regions in the same eye. In this case the genetically ivory region bordering the cantaloup changes to wild-type black, a phenotypic complementary effect the result of soaking through into the ivory region of some product dependent upon the dominant al- lele to ivory in the cantaloup region. Similar- ly, feminization occurs in the genitalia of a high proportion of haploid mosaic males, the result of a complementary effect, an interaction between sex factors, differing in the two re- gions, X and Y. These "gynandroid" males (P. W. Whiting, Greb, and Speicher, 1934) develop from unfertilized binucleate eggs in which the sex alleles have been segregated at maturation into the different nuclei. They are , therefore , hap- loid throughout. They sugE^est gynanders (haplo- diploid male-female mosaics) which also arise from binucleate eggs, but in the formation of a gynander one of the nuclei must be fertilized to originate the female regions. Gynandroids are entirely male in appearance except for the relatively small feminized structures added to the male genitalia, usually on one side only (Fig. 14). The male genitalia may show redupli- cation of parts. Gynanders, on the other hand, have extensive female regions of the body, while the genitalia may be either male, female, or mixed. In the latter case there is usually a complete set of male parts but no reduplication. The female structures tend to be lateral on one side only and anterior to the male organs, v/ith appendages of full length. Interpretation of gynandroidism as a complementary effect led to the conclusion that females are heterozygous and 87 Fig, 14* Ventral view of the genitalia of a gynander, a male-female mosaic, A complete set of male genitalia is present. The left sensory gonapophysis of normal length, and two elements of the sting, are the female structures. X 120. P. W. Whiting, 1940a. Pig« 15, Dorsal view oi male, mosaic for long (antennae and ?/5„ngs)^ for narrow (wings), and for cantaloup (oyao). p. w. Whiting, 1954c. X 20. 89 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD that normal males are X and Y with equal fre- quency. The feminized structures just mentioned do not appear on all mosaics, for in many the line of mosaicism does not pass through the genital- ia. It has also been found, however, that these structures do not always occur in mosaics even when the line does bisect the body (Fig. 15). These facts have led to the supposition that in Habrobracon there are several kinds of males, genetically distinct for sex-determining fac- tors but phenotypically similar. When tissues differing in sex-determining factors adjoin in a haploid mosaic male, one influences the other, and traits characteristic of diploid tissue are the result. 90 CHAPTER VII SEX DETERMINATION Genetic research with the wasp Habrobracon juglandis was begun at the University of Penn- sylvania in 1916 by P. W. Whiting. Many phases of this work have been developed since that time but the central problem has always been that of sex determination. In 1845 Dzierzon put forth the theory that in the honey bee, drones (males) develop from unfertilized eggs while workers and queens (fe- males) come from fertilized eggs. This theory was based on the fact that unmated and old queens produce drone broods and that race-crossing re- sults in drones like the maternal race while the daughters are hybrid. Dzierzon (1854) stat- ed that the drones of the second generation from a cross resemble either the paternal or the maternal race and that thcce two types oc- cur in equal numbers. He, therefore, glimpsed the fundamental gametic ratio twelve years be- fore Mendel published his paper on peas. Dzier- zon's Law applies to other insects of the order Hymenoptera, including wasps, ants, ichneumon- flies, chalcis-f lies, and saw-flies, but many exceptions occur. P. W. Whiting (1918) working v/ith the ichneumonoid wasp, Habrobracon, found that in this form also haploid males are pro- 91 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD duced from unfertilized eggs, and females from fertilized eggs. Nachtsheim in 1913 had shown that somatic cells of the honey bee possess the haploid num- ber of chromosomes in the case of the male, and the diploid number in the case of the female. Sex determination in the Hymenoptera was then assumed to rest upon the possession of one sex chromosome in the haploid male or two in the diploid female. Bridges (1925), discussing sex determination in the bees and wasps stated that it was the outstanding unsolved puzzle, although before the genie balance theory it seemed one of the clearest and simplest of cases. The explanation of sex determination in Hab- robracon was further complicated when diploid males were found to occur occasionally. Tests made with the first mutant type, the recessive orange eye-color, demonstrated that three types of individuals, haploid males from unfertilized eggs and diploid males and females from fertil- ized eggs, occur regularly in the species when parents are related. When orange females were crossed with the wild-type black-eyed males, there appeared not only the expected black-eyed heterozye;ous females from fertilized eggs and orange haploid males from unfertilized eggs, but also a smaller group of "anomalous" sterile or almost sterile males which were black-eyed. Since males were then regarded as necessarily haploid, these "patroclinous" sons were thought to be either androgenetic or mosaic in origin, having arisen through failure of syngamy (P. W. Whiting, 1921a). This view persisted until 1925 when it was 92 SEX DETERMINATION shown (P. W. Whiting and A. R. Whiting, 1925; A. R. Whiting, 1926, 1927, 1928a) that "patroc- linous" males were really of biparental inheri- tance, receiving dominant traits of both par- ents. Genetic search was then made for a gene that they might possess in the simplex condi- tion, occupying an odd X chromosome or a dif- ferential X region in one of the pairs. No such gene was found. Later cytological work (Torvik- Greb, 1935) indicated no visible difference be- tween the ten pairs of chromosomes of these bi- parental males and the ten pairs characteristic of females. According to the principle of gen- ie balance, if the genetic complex of the fe- male were merely that of the male doubled, the ratio of male-producing to female-producing genes should remain the same. There should then be but one sex, the male, because any multiple of the haploid complex would make but a slight change in size or proportion of parts, not af- fecting such a radical transformation as is in- volved in sex difference. In 1933 (P. W. Whiting, 1933e,f) the hypoth- esis of complementary factors v;as suggested and shortly thereafter proved true by sex-linkage. According to this hypothesis the female is het- erozygous for a pair of sex factors, called X/Y at the time, and she produces two kinds of hap- loid males from unfertilized eggs, X and Y. Mating with a closely related X male gives dip- loid zygotes, male-producing X/X, female-produc- ing X/Y, and male-producing Y/Y. The male-pro- ducing homozygotes are much less viable than the female-producing heterozygotes , accounting for the low frequency of diploid males. A supplementary hypothesis of differential 93 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD maturation was proposed by P. W. Whiting (1933e) to explain the absence of diploid males in out- crosses. In these crosses, the egg, being in an indecisive stage of reduction division at the time of fertilization, V70uld eliminate into the polar body the same sex factor that was be- ing carried in by the sperm, and thus the fer- tilized egg would always be heterozygous for the sex factor, that is, female-producing. In close-crosses, however, some influence seemed to prevent this mechanism of differential matu- ration from affecting the disposal of the like sex factor; and so there were some diploids which, being homozygous for the sex factor, were males. It had been noted previously (A. R. Whiting, 1925) that females mated to related males were less fecund than those mated to unrelated males . This appeared to be the result of excess bad eggs in close-cross fraternities. On the basis of the theory of complementary factors, these eggs are considered to be male-producing zy- gotes, and increased fecundity in out-crosses the result of a replacement of these by female- producing zygotes. This hypothesis of complementary factors was suggested after study of complementary effects in eye-color and in genitalia of haploid mosaic males, which occur occasionally among the prog- eny of unmated females heterozygous for mutant traits (P. W. Whiting, Greb, and Speicher , 1934) . Interpretation of gynandroidism as a complemen- tary effect led to the conclusion that females are heterozygous and that normal males are X and Y with equal frequency. This hypothesis of complementary factors was proved true by sex- 94 SEX DETERMINATION linkage of the recessive gene j fu-sed (P. W. Whiting, 1935a, c) . Snell, in 1935, presented evidence based on Bostian's (1934) published data that would sup- port a theory of independently segregating mul- tiple sex factors. He suggested that heterozy- gosity of one or more of these factors results in a female, but homozygosity of all produces a diploid male (Snell, 1935). In 1939 P. V/. Whiting proposed a theory of sex determination in Habrobracon based on mul- tiple alleles (P. W. Whiting, 1939). In this theory, the explanation of sex inheritance in close-crosses is essentially the same as that proposed by P. W. Whiting in 1933. The females are heterozygous for the sex factor, the haploid males are of different types as regards the sex factor, and the diploid males are homozygous for the tv/o sex alleles involved. The impor- tance of the multiple allele theory is the fact that it gives an explanation of sex determina- tion in outcrosses, where no diploid males are produced. The upper case letters X and Y were then abandoned as sex symbols, and lower case x was taken to indicate the sex factor exclusive- ly. The X accompanied by certain letters of the alphabet now designates the various alleles in the series--xa, xb, xc, etc. According to the multiple allele theory any heterozygote for two members of the series, xa, xb, xc, etc., is female, any horaozygote or azygote (haploid) is male. Given n alleles in the series, there should be possible n different haploid males, n corresponding diploid males and ( n2-n) /2 females . With respect to the three classes of off- spring— females, diploid males, and haploid' 95 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD males, different ratios have been considered in the publications. At first, ratio of females among total progeny was used, but this was soon given up in favor of ratio of total diploids. Ratio of males among diploids was also calcu- lated. It was noted that these two ratios were negatively correlated and that outcrossed fe- males had higher fecundity than close-crossed. The latter fact, checked later by egg counts, showed that low viability of diploid males, to- gether with presence or absence of male-produc- ing zygotes rather than difference in ratios of eggs fertilized, lies at the basis of the nega- tive correlation. Since, according to the mul- tiple allele theory, male-producing zygotes are formed in close-crosses with frequency equal to female-producing, the ratio of diploid males to females may be used to express the relative viability of the diploid males. Bostian (1939) gave the first evidence sup- porting the multiple sex allele theory. He se- lected for independent segregation of fused and sex for twenty generations, while constantly inbreeding. He was unable to establish a line breeding true for independent segregation of sex and fused, although on the basis of Snell's hypothesis, the establishment of homozygous sex factors on the chromosome bearing fused would allow for such a condition. The results of this work indicate that there were triple sex alleles present. A review by P. W. Whiting (1940c) of the available data further supports and con- firms the multiple allele theory of sex deter- mination . The multiple allele theory of sex determina- tion in Habrobracon has been exhaustively tested 96 SEX DETERMINATION (P. W. Whiting, 1940c, 1943a). Laboratory stocks have been analyzed for sex factors. Technique consisted in selecting one stock, designating its sex alleles as xa/xb, and introducing into it a recessive gene. If all significantly large fraternities from crosses of recessive females of this stock to dominant males of an unknown stock contained dominant males (diploid), as well as the recessive males (haploid) and domi- nant females (diploid), the PI male stock was considered to have the same sex alleles, xa/xb. If no dominant males (diploid) appeared in any of the fraternities, the PI male stock was re- garded as having different alleles — for example, xc/xd. If only one-half the fraternities in- cluded dominant males, the PI male stock was considered to have one allele in common with xa/xb--for example, xa/xi. Recessive genes were introduced into each nev/ stock as it was ana- lyzed for sex factors, and "tester" stocks .were made up for future use. Nine factors are thus far known in the series (xa through xi), and the tester stocks are des- ignated xa/xb, xc/xd, xe/xf, xg/xh, and xa/xi. Evidence of allelism of these sex factors is furnished by the fact that they are all closely linked with the gene, fused. Lack of diploid sons and equality of fused and non-fused daugh- ters from crosses of stocks tentatively desig- nated as having different sex alleles in the x series proves the sex factors to be different, but their linkage with fused indicates their allelism. The multiple alleles are regarded as differ- ential chromosome segments which have been built up in the early evolution of the Lymenoptera. 91 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD They are necessarily associated with haploid parthenogenesis, and consist of many genes de- termining the numerous sex differences, struc- tural, functional, and behavioristic , charac- terizi'ng Hymenoptera. These genes have, in the aggregate, duplicate effects such that all hap- loids or homozygous diploids are similar and male, but combinations of any two different al- leles result in females (heterozygous domi- nants), likewise all similar. This principle of sex determination may be illustrated by the foilov/ing example. An xa/xb female homozygous for the autosomal recessive gene, veinless, but heterozygous for fused and having fused associated with xb may be crossed with a non-veinless xb fused male (Fig. 16A). The recessive veinless serves to mark the hap- loid sons which are fused and non-fused in equal numbers. The diploid offspring, whether males or females, will be non-veinless. Among the daughters, xa/xb, the non-crossovers will be wild-type, the crossovers fused, but among the diploid sons, xb/xb, the reverse condition ex- ists. Since crossing over between x and fused is about ten per cent, fused will show great deficiency among the daughters, great excess among the diploid sons. If both parents have fused associated with xa, the offspring occur in the same ratio (Fig. 16B) , but if fused is associated with xa in one parent and with xb in the other, the ratios of fused among the two types of diploid offspring are reversed (Fig. 16C and D) giving an excess of fused among the daughters, deficiency among the sons. Thus there are four different arrangements possible 98 GROSSES IffVOLVIKG SEX DETERMINATION MOTHER MFLOID FATHER SONS DAUGHTERS DIPLOID SONS MD EXCSS3 xa/xb OP BAD E0G3 Ko/xo and xb/xc- xa/3ta op xb/xb vi vlofu ^/tVL tvi/r\x */ta £\i/ru A vT xbfu xa'^ xbru B vl xafu xc ♦ xafu C vl vi' xa * xbfu xafu D vl xb + xalu xbfu vl xbfu xcfu K vl xa ■!• 9 9 1 1 10 1 1 9 9 10 Pig. 16, Ratios of offspring axpected according to varioufl arrangemants of sex alleles in crosses of vainless feisiales hQterosy(;;ous for fus«d by fuaod males, P. %. Whiting, 194Sftt 99 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD for X and fused but only two phenotypically separable ratios. Fraternities of the close-cross type may now be called two-allele fraternities, those of the outcross type three or four-allele fraternities. A third allele brought into the cross by a fused male, xc.fu for example, always masks sex-link- age, since all diploid offspring are hetero- zygous for X and hence female, fused and non- fused in approximately equal numbers (Fig. 16E) . Diploid males reappear with inbreeding, and their presence is always associated with obvious sex-linkage of fused as shown by Bostian ( 1939) . Sex determination, is in a sense, polygenic, but because of no crossing-over within the seg- ment, the various groups of genes act as a sin- gle series of allelic factors . Just as the many sex-producing genes distributed among the X-chromosome of Drosophila segregate as a unit from the Y without crossing-over, so the domi- nant female-producing genes and the recessive male-producing genes of the x factor in Habro- bracon segregate without crossing-over from their homologues. Males, as determined by the various alleles (whether azygous or homozygous) , or females as determined by any of the hetero- zygous combinations, are always phenotypically similar (P. W. Whiting, 1945b). Whether the sex alleles ever mutate has not been established with certainty. They appear to be very stable. Extensive breeding within a two-allele stock has resulted in two-allele fraternities almost exclusively, as evidenced by the presence of diploid males. With certain very rare exceptions, the fraternities lacking diploid males are small, so that the lack is 100 SEX DETERMINATION evidently the result of error in sampling. The rare exceptions, v/hich were not adequately tested, rr,ay have been the result of contamina- tion, though this is unlikely, or to some com- bination of factors reducing diploid male via- bility, or to mutation in a sex allele itself. The nature and evolution of the sex factor, X, the differential segment, may no\7 be con- sidered. Mutant genes are known to cross over both to the left and to the right of x, hence x is interstitial, but so far no crossing over within X has been noted. If, however, crossing over within x did occur, x would be inconstant, xa would not remain distinct from xb, or xb from xc, etc. The work of Horn ( 1943a, b) demon- strates that F2 males segregating from crosses of xa/xb with xe/xf stocks are still xa, xb, xe, or xf, that they all sire diploid sons, and that those that were xa or xb sire diploid sons by xa/xb females and not by xe/xf females and the reverse (as shown by linkage with fused). It may be concluded, therefore, that there is no crossing over within x; or perhaps the rare "mutations" of x, if such actually occur, may be crossovers. Whether or not Whiting's multiple allele theory of sex determination is applicable to the Hymenoptera in general is as yet a question. Were it not for parthenogenesis, this comple- mentary scheme might never have been proposed. Diploid males have been shown in two other spe- cies of Habrobracon (Speicher and Speicher, 1940; Inaba, 1939), but so far no one has ap- plied breeding and observational tests that 101 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD might reveal them elsewhere; and no evidence has yet appeared that prevents the extension of the multiple allele theory to the Hymenoptera in general. 102 Chapter VIII LINKAGE So far in Habrobracon juglandis there have occurred about one hundred known mutations each involving a different gene. Since the wasp has only ten pairs of chromosomes, it is apparent that each chromosome must bear a number of dif- ferent genes. The position of each gene and its allele or alleles is definite; that is, each pair of alleles always occupies the same locus on the same pair of chromosomes in dip- loid v/asps. Of course, in haploid males, only a single set of chromosomes is present. Crosses have been made involving these mutant types in various ways to test the principles of heredi- ty. Linkage tests are made by crossing various mutant types and counting the F2 haploid sons of the unmated Fl heterozygous females. The azygotic ratios thus obtained approximate gam- etic ratios except as they may be affected by differential viability of the various haploid genotypes. Linkage tests thus made show that maps are very long in crossover units. On this account, despite much work extending over sev- eral years, there is as yet no approximation of linkage groups to the number ten, corresponding to the haploid set of chromosomes. If certain gene mutations, such as speckled, reduced, and glass, occur more frequently in 103 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD combination than they do separately, the genes are said to be linked; and therefore, borne on the same chromosome. At the present time four linkage groups (Fig. 1^) are recognizable. These groups may occupy four different chromo- somes or they may be found to be linked and thus occupy fev/er than four chromosomes. In 1933 fifty genes v;ere known, and they comprised eight linkas^e groups. It vvas thought then that each group represented a different chromosome. However, since that time, nev; genes have come to light that have linked the second and fourth groups, others have linked the fourth and fifth, others have linked the fifth and eighth, and still others have linked the first and third. It is apparent, therefore, that while the num- ber of known genes has increased, the number of linkage groups' has decreased. There is one unusually long sex chromosome known to be at least five hundred units in length. Starting at the left end of this sex chromosome, designated linkage group one (I), the genes are arranged in the order: speckled, reduced, glass, etc. Helsel (1943, 1944) con- ducted experiments in order to check recombina- tion and interference in this region, and to compensate for any viability disturbance or po- sition effects introduced the genes in all pos- sible combinations. She reported 12.07 per cent crossing over betv/een speckled and re- duced, and 12.69 per cent crossing over between reduced and glass. Coincidence is 0.264, but varies markedly as the genes are introduced in different combinations. Linkage of these genes had been shown (P. W. V/hiting, and Benkert, 1934) with about 13 per cent crossing over be- 104 HA5H0BRAC0N LINKAGE GROUPS Group I Sk — 13 — r r — 15 — gl gl — 37 — . fu X — - 10 — fu fu — 24 — sb sb — 30 — bl bl — 14 — le 1© — 6 — c c — 15 — 1 1 — 3 — n n — 9 — ho lio — 8 — vl vl — 12 -« cw cw -- 5 -.« ro ro "- 12 — bu b\i' — > 37 -« dr dr — 17 ««, er cr -- 41 -«- si si — 0 — ' CO CO -- 33 — ct ct »- 22 — rd rd — 40 — gy gj -• - 4 — ac ao -« 4 ~- 8l el Group II k — 28 m — 8 o — 0 Group III bk — 25 St — 9 wh — 10 pi — ,3 at — • 22 — St — wh — at — wh — St Group IV sv — 12 — td td — 27 — ma sw — 24 — av ma Flg» !?• Linear arrangemQnt of genes with crossovar parc^ntagas. For additional data seo Table II. 105 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD tv/een speckled and reduced, and about 15 per cent crossing over betv/een reduced and glass. The sex gene lies to the right of glass and is followed by fused and stubby, which have been shown to be sex-linked. The strength of the sex linkage of fused has been shown to vary from 8,6 per cent to 17,6 per cent crossing over (P. W. Whiting, 1940e). Kager (1941) work- ing v/ith orange-eyed females heterozygous for stubby, showed crossing over to be 37.5 per cent between stubby and the sex gene, and 24.2 per cent betv/een stubby and fused. Linkage across the sex gene indicates that speckled and stubby appear to segregate independently, and no linkage between speckled and fused is noted. Stubby shows very slight, if any, link- age with reduced or with glass. A study by P. W. V/hiting and Benkert (1934) indicates link- age between reduced and fused with 45.5 per cent crossovers. Linkage between fused and glass, with 41.7 per cent recombinations, sug- gests that glass is nearer fused than is re- duced. Recent experiments bear out this con- clusion. No conclusive evidence is as yet available for the sex linkage of glass, which may be very loose or may be masked by a third allele and by viability differences. A linkage map of regions near the sex gene with approxi- mate intervals may be constructed as follows: Sk —13-- r ~-15'- gl x >-10«- fu —24— sb Anderson and P. W. Whiting (1939) reported a "cantaloup" group of 14 loci with a map dis- 106 LINKAGE tance of 187 units lying to the right of stubby. Klotz (1940) reported on linkage tests made with veinless, lying approximately in the cen- ter of this group. Veinless and honey are linked with a crossover value of approximately 7.5 per cent (Clark, 1942). Veinless and stub- by show no crossing over and are, therefore, thought to be not linked. Veinless and lemon show a linkage value of approximately 32,3 per cent. Veinless and black shov/ a linkage value of approximately 34.6 per cent. These data in- dicate that the factors , veinless , honey , lemon, and black, are located on the same chromosome. Crossover percentages are 14 between black and lemon, six between lemon and cantaloup, and 19 between cantaloup and honey (A. R. Whiting, 1939b) , Black is linked to stubby with a cross- over value of approximately 30 per cent. Vein- less and cantaloup are linked with approximate- ly 30,6 per cent crossing over. Long and vein- less show a linkage value of approximately 17,0 per cent. Long and cantaloup show a linkage value of approximately 14,5 per cent. Inter- ference is evident in the three point cross be- tween cantaloup, long, and veinless, with the ratio of coincidence being 0,537. Clark (1942) reported data from six loci scattered along about 70 units of the "cantaloup" group thought to be near the center of the sex chromosome. These data indicate that the black to lemon region does not interfere with the lemon to can- taloup region, but the cantaloup to honey re- gion does interfere with the lemon to cantaloup region. In testing the possibility that the length (23.0) of the black-lemon region might be responsible for the lack of interference, 107 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD the region was compared with a region of simi- lar length (22,3) to the right of lemon, namely, cantaloup to honey. Coincidence for black- lemon, and lemon-cantaloup, was ' found to be 1.07, while the lemon-cantaloup, and cantaloup- honey, was 0.28. Clark suggests that the reason for the lack of interference between the black- lemon, and the lemon-cantaloup, regions is' that the spindle fiber attachment center is located near lemon. If such is the case, then the re- gion of the sex gene is about 150 units away from the spindle fiber attachment region. A third group of genes designated as the "red" group (Anderson and P. W. Whiting, 1939) consists of five loci with a map distance of 74 units. This group forms the right end of the sex chromosome. Twelve other genes are also located on this first chromosome. For their position and crossover value see Fig. 17 or Table II. A second chromosome, or linkage group two (II) of three loci, 35 units in length, has been identified (P. W. Whiting and Benkert, 1934). This linkage group consists of three mutants, kidney, orange, and miniature. A three point experiment planned to determine the relative positions of kidney, orange, and miniature , gave 7.5 per cent crossing over between orange and miniature, and 27,5 per cent crossing over be- tween miniature and kidney, with 2.8 per cent double crossing over. From these data it may be concluded only that miniature lies about one-fourth the distance from orange to kidney (Fig. 17). The third chromosome, or linkage group three (III), of four loci is approximately 44 units 108 LINKAGE in length. Carson (1941) introduced three genes, broken, white, and stumpy, in the four possible combinations. Linkage between broken and stumpy gave a crossover value of approximately 25 per cent. While the linkage between stumpy and white gave a crossover value of approximately nine per cent. From these and other data Car- son concluded that broken, stumpy, and white, are arranged on the chromosome in that order. The gene, attenuated, since lost, showed link- age with white with 10 per cent crossing over, and with stumpy with 22 per cent crossing over. Its position, therefore, is to the right of white (Fig. 17). Recent work done by P. W. Whiting shows that v/hite and pellucid are very closely linked, only a few recombinations oc- curring out of several hundred wasps counted. There is also suggestive evidence for linkage between red and white with 41.5 per cent recom- bination (Helsel, 1942). Further breeding tests will be required to substantiate the evidence. If found valid, the known linkage groups will be reduced to three. The fourth chromosome, or linkage group four (IV), consists of four known loci and is ap- proximately 63 units in length (P. W. Whiting and Benkert, 1934). The four mutants in this linkage group are shot-veins, small-wings, trun- cated, and maroon, arranged in this order on the chromosome. Shot-veins and small-wings are linked with a crossover value of approximately 24 per cent. Matings between shot-veins and truncated show linkage with a crossover value of approximately 12 per cent. Maroon and trun- cated are linked with crossing over approxi- mately 27 per cent. 109 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD There are ten genes remaining to be located in the ten possible linkage groups, Table II. At present, breeding tests are being carried on in order to test the presence or absence of linkage among these unlocated genes. New genes are still coming to light. These may prove to be located on chromosomes as yet unmarked, or it may be that they will be found to be linked with known groups. The number of genes that may eventually show up through visible effects is incalculable. Table II shows which genes have been so far tested. In linkage tests, the x-factor acts as a single gene, occupying a point on the linkage map. The method of making a linkage test with ■the sex-factor may be illustrated by a single example (P. W. Whiting, 1945b). The mutant gene, fused, lies about ten units to the right of x. An orange-eyed female heterozygous for fused, o.xa.+/o.xb.fu, is crossed with a black-eyed fused male, +.xb.fu. The orange-eyed sons are haploid from unfertilized eggs and are fused and non-fused in equal numbers. Sex-linkage is not determinable among these because xa males and xb males are similar. The black-eyed zy- gous diploid or biparental offspring o/+, are either fused or non-fused, and either males or females. From the ratio of the different com- binations the percentage of crossovers may be determined. In the example given, the non- crossover offspring will be xa.+/xb.fu or non- fused females, and xb.fu/xb.fu or fused males; the crossovers, averaging in this case 10 per cent, will be xb.+/xb.fu or non-fused males, and xa.fu/ab.fu or fused females. This is called a tv;o-allele cross. 110 LINKAGE In crosses involving three sex-alleles, sex- linkage cannot be determined because all the zygous offspring are heterozygous for x and are, therefore, female; xa/xb X xc gives xa/xc, xb/xc. Fecundity is higher here, and the ratio of females to haploid males is doubled because the poorly viable diploid males are replaced by females. In this case, if the cross is made between a female heterozygous for fused and a fused male, fused and non-fused females are produced in equal numbers. Three kinds of fraternities from heterozy- gous females by fused males are therefore dis- tinguishable as follows: Diploid Haploid Females Males Males Non-fused Non-fused Non-fused fused fused fused (1) two-allele 9/1 1/9 1/1 (2) two-allele 1/9 9/1 1/1 (3) Three-allele 1/1 0 1/1 Linkage tests involving two or more mutant types fall into two general categories, (1} re- pulsion tests, (a X b), and (2) coupling tests, (a.b X +). The mutant factors may be intro- duced from different parents (repulsion tests) or the double miutant type may be crossed with wild-type (coupling tests). It is advisable to make these reverse crosses to check deviations that result from viability differences, but thus far most tests have been of the repulsion type as this is the more convenient way to test 111 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD new factors. No consistent differences have been found between F2 fraternities resulting from reciprocal crosses (a female X b male and b female X a male in the case of repulsion tests or + female X a.b male and a.b female X + male in the case of coupling tests). When two mu- tant types are female sterile, the female het- erozygous for one is crossed with the male of the other, in which case only those F2 frater- nities containing both mutants are of value. Unmated females resulting from crosses in- volving various mutant genes produce males par- thenogenetically . These segregate in gametic ratios. Thus a female, o/bl , from a cross of orange-eyed by black-bodied, shows independent segregation of these two differences. The four types of sons, wild-type, orange, black, and orange-black, occur in equal numbers. The same result is obtained from the reverse cross fe- male, +/o.bl, resulting from a cross of wild- type, by orange-black. Lemon body color and cantaloup eyes are linked with about ten per cent crossovers. Thus le/c females produce males, +, le, c, le.c, in 1:9:9:1 ratio while +/le.c females give the same types of male off- spring in reverse ratio, 9:1:1:9. A female may be heterozygous for several differences produc- ing more complex ratios. Factor interaction is shown, as in lemon, honey, and black, body col- or, giving characteristic and clearly distinct double mutant types, le.ho, le.bl, ho.bl, and the triple mutant, le. ho.bl. Masking effects are illustrated, as when white eyes prevent other eye color differences from showing. Via- bility differences appear when some mutant types are less frequent than wild-type. Dif- 112 LINKAGE ferent mutant combinations show different de- grees of reduced viability which may be deter- mined by relative numbers in counts of segre- gating fraternities. Linkage estimates are ordinarily made from double backcross data. Breeding out virgin Habrobracon Fl females gives ratios of the type obtained from. a double backcross. The formula for estimation of linkage is derived by using the method of Maximum Likelihood (Mather, 1938; Fisher, 1941). This method leads, in the theory of large samples, to an estimate having the smallest standard error which the data will allow. If P equals recombination fraction, r equals number of recombinations or crossovers, s equals number of straights or non-crossovers, then P = r/(r + s). It must be remembered that each double recombination represents two cases of a single recombination and must be added to the single recombinations in each region to ob- tain the total amount of recombinations in that region. The recombination fraction can never exceed 50 per cent since of the four available chromatids only two can produce detectable re- combinations. A higher recombination fraction indicates that the genes are relatively far apart; a low recombination fraction, that the genes are close. A variation of the formula, P = r/(r + s), was presented by P. W. Whiting and Benkert (1934). They let AB, aB, Ab, and ab represent the frequencies of the four phenotypes of off- spring expected from heterozygous females a.b/+ (coupling test) or a/b (repulsion test). It may be supposed that wild-type, AB, is the most vi- able, but somatic overlapping may increase the 113 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD relative number of AB and of aB phenotypes at the expense of Ab and ab respectively, or the reverse may occur. It may also happen that AB will be increased at the expense of aB, while AB is not increased at the expense of ab. This may be the result of the influence of factor B versus b or to a modifier of trait A versus a linked with factor B or b. A factor preventing overlapping acts as a "differentiator" (Bridges, 1919). AB may exceed aB as the result of differen- tial viability in which case we might expect a comparable excess of Ab over ab. However, the ratio ab/AB cannot be predicted from (aB/AB) X (Ab/AB) since in some cases the double mutant type fails to be reduced proportionally to the single while in others the double may be highly lethal although one or both of the singles may show viability equal to that of wild-type under the conditions of culturing. Linkage of one of the genes (A or a for ex- ample) with a lethal or semilethal may cause an excess or a deficiency of a mutant type of nor- mal viability. Thus aB may surpass AB or the reverse. In this case v;e may expect a corre- sponding shift between ab and Ab, but such may not be obvious because of viability differences or somatic overlaps between the various combi- nations of A and a with B and b, or because of linkage of a second lethal or semilethal with B or b. V/hile lethals are invisible in the mate- rial thus far presented on linkage, semilethals may be either visible or invisible. Complete lethals, dying as pupae, which may be identi- fied as to a second trait (eye color for exam- ple), have been shown in Habrobracon, Degree 114 LINKAGE of lethality of a semilethal ' factor differs widely under diverse environmental conditions and in different stocks. This has been shown in Habrobracon for a number of visible mutant types of low viability. The mutant genes are here serailethals which have a recognizable ef- fect on the surviving individuals. Factors or- dinarily designated as semilethals presumably differ from these only because of failure to find a conveniently visible trait difference (Schaeffer, 1945). In Habrobracon, with its haploid males, stocks very quickly become pure for modifying factors by lethal selection. It seems likely that a balance of factors should thus be readily attained tending toward greater viability. Relatively disharmonic combinations may be expected to enhance by their cumulative effect the influence of a semilethal among the progeny of hybrid females. A larger proportion of the La/Lb daughters than of the La/Lb sisters of the hybrid mother might then be inviable. In five tests involving reciprocal crosses of two orange-eyed stocks and in one test of one of these stocks having 'the loosely sex-linked gene stubby with a third stock, lethal effects v/ere present associated with the dominant allele to stubby. The lethal effect is regarded as complementary. It is probable that this lethal factor lies to the right of stubby (Schaeffer, 1945). Another lethal factor (Helsel, 1942) showing 25 per cent recombination with white and pellucid has been detected. In the case of linkage between a and b, AB/(AB + aB) might be expected to equal ab/(Ab + ab) were it not for one or more of the three disturbing factors--somatic overlapping, non- 115 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD proportionate dif f erential vlabil ity , and linked lethals or semilethals. It is obvious that v/hat has been said as to the relations of a and b may also apply to the relations of either a or b with a third factor c, or a fourth factor, d. It is sometimes nec- essary to make comparisons of pairs of frequen- cies as regards a and b, separately for groups CD, cD, or Cd since either c or d may affect phenotypic ratios of a or b. Table I has been prepared in order that the reader may readily estimate the statistical significance of various pairs of frequencies occurring in the summaries of linkage data in Habrobracon literature. By dividing the lower frequency by the sum (n) a percentage is ob- tained deviating more or less below 50 per cent. (P = .90) are those percentages at or below which 10 per cent of the samples of a given size (n) will be expected to fall , while (P = .99) are those percentages at or below which one per cent of the samples of corresponding size will fall. The reader may then be 90 per cent sure that there is a significant difference from equality between the two frequencies if the percentage is as low as or lower than (P = .90) and he may be 99 per cent sure if it is as low as or lower than (P = .99). 'Whether these sta- tistically significant deviations indicate so- matic overlapping, dif f erential viability , linked lethals, linkage of the two genes under consid- eration, or some combination of these condi- tions must be judged in the individual case from the nature and variability of the traits and comparison of the different percentages 116 LINKAGE with each other and with the relation of the factors in the Fl parent. Whether there is linkage and what may be the best estimate of the percentage of crossovers cannot necessarily be determined from the ratio (AB + ab)/(AB + aB + Ab + ab) . Muller (1916) has shown a convenient method, The Square Root of the Product Method, for determining gametic ratio, recombinations (r) to straights (s), which compensates for viability differences. If viability is affected proportionally by tv/o. pairs of alleles, regardless of their combina- tion, and if the numbers of the genotypes be. AB, aB, Ab, and ab respectively, the gametic ra- tio, r/s equals ( 1/aB X ab : 1/aB X Ab) for the repulsion test and ( VaB X Ab : 1/AB X ab) for the coupling test. If, however, the factors differ in their effects on viability according to their various combinations with each other and if genotypes from the repulsion cross be ABl, aBl, Abl, abl, and from the coupling cross AB2, aB2, Ab2, and ab2, then gametic ratio should be equal to any one of the four expressions: AB2 X Abl y ABg X aB^ y abg X Ab-j^ y ebg X aB^* 117 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD This second method, the method of reverse crosses, does not compensate for linked lethals , but comparison may be made of the six ratios obtainable (two by first method in case reverse crosses are made and four by second) and in case of differing results presence of linked lethals determined. Muller's method does not compensate for so- matic overlaps or for difficulty in determina- tion of types, in which case phenotypic ratio involves not only deficiency of certain types below gametic ratio but also excess of other types. If viability difference be absent be- tween A and a and if overlapping be from a to A in the same proportion whether B or b be pres- ent, then the phenotypic distribution AB, aB, Ab, ab, may be corrected as follows: Decrease AB by subtracting ( (AB + Ab - aB - ab)/2(AB + Ab + aB + ab)) X aB + ab and increase aB by the same amount. Also decrease Ab by subtracting ((AB + Ab - aB - ab)/2(AB + Ab + aB + ab) ) X ab + aB and increase ab by the same amount. In other words each of the two numerically defi- cient groups should be increased at the expense of its corresponding excess group by a propor- tion of itself equal to one-half the difference between the sum of the deficient groups and the sum of the excess groups to the total. The gametic ratio calculated after this cor- rection is made will in case of linkage always give fewer recombination types than the origi- nal data. If overlapping of one difference is always in the same direction and unaffected by the other difference or correlated differences, the gametic ratio obtained should be correct. Even if overlapping is in both directions (from 118 LINKAGE A to a as well as from a to A), recombinations after correction should be fewer than before. True crossover value will not be greater than that indicated by ratio thus obtained, but it may be considerably less. The value of the method lies in the fact that it gives a maximum which is lower than may be calculated from the frequencies directly. If the percentage of single recombinations is known for each of two regions, the probabil- ity of two recombinations occurring simultane- ously, one in each region, is the product of the percentage of singles in each region. The- oretically, the percentage of double recombina- tions observed should equal the product of the percentages of singles but actually, in most cases, the observed double percentage is lower than that expected because of interference. Stevens (1936) has derived the following formu- la for measuring interference in terms of coin- cidence using the method of Maximum Likelihood: c = wn/(w + x)(w + y) where c equals coincidence, w equals number of double recombinations, n equals total number in cross, x equals region (1) recombinations, and y equals region (2) recombinations. This for- mula makes possible the estimation of coinci- dence directly from the raw data. A coincidence value of one (1) indicates no interference; a value of zero (0) indicates absolute interfer- ence. There is no significant change in recombina- tion percentage because of aging of the female. vCoincidence values also show no significant 119 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD change because of aging of the female; however, all crosses indicate that the variation of the coincidence value from one (1) is less in the later vials, but not significantly so. 120 Chapter IX ENVIRONMENTAL EFFECTS It has been found that in Habrobracon ju- glandis,asin other experimental animals, cer- tain environmental factors have marked effects upon the appearance, fecundity, and behavior of the animal. The nature of mutations and the rate of their occurrence are also affected. In- vestigation of various factors and their effects is at present in progress. Temperature, humid- ity, and X-radiation, have been found to be im- portant in developmental studies. Ever since Habrobracon has been reared in the laboratory, the degree of temperature has been known to determine body color, to affect repro- duction, to affect the production of mosaics, and to cause mutations. Wild-type individuals vary from honey-yellow to almost black, higher temperatures producing more yellow pigment , low- er more black. Heredity plays some part, for races under constant temperature differ con- sistently in pigmentation. Schlottke (1926) from a carefully planned and controlled study of temperature and pigment in- terrelationships in wild-type Habrobracon draws the following conclusions: (1) deposition of pigment (black) decreases linearly with rising temperature; males are on the average, darker than females, (2) pigment is deposited especial- 121 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD ly at points of muscle attachment and the last parts to become light in higher temperatures are the regions where muscles attach vertical- ly, (3) animals bred at lov/er temperatures are larger and darker than those at higher, but at a given temperature the smaller animals are the darker, and (4) changes in temperature at any time between egg stage, four days before laying, and prepupal stage, affect adult coloration. In a later study (1934) he shows that increase or decrease of oxygen content of the atmosphere increases pigmentation; and he concludes that variation of oxygen concentration influences the oxidations concerned with pigment formation, indirectly as a non-specific stimulus on the organism as a whole. Kuhn (1927) has shown by selection and by crossing that heredity plays a considerable part in color determination. He argues for a hereditary cytoplasmic influence. P. W. Whiting (1926a) has demonstrated linkage of a gene affecting color of raesosternum with a gene determining defects in wing vein, R4. An extensive series of experiments indicates con- siderable genetic independence of various body regions as well as both linkage and physiologi- cal correlation with certain mutant genes. Despite the great range of variability in diverse regions of the body, bilateral symmetry is the rule with very rare exceptions. An oc- casional defective specimen with asymmetrical morphological malformation is likely to show correlated changes in pigmentation. There have likewise occurred a few specimens with an ir- regular mottling of color, but this appears to be pathological. Instances in which specimens of normal form and non-mosaic for mutant traits 122 ENVIRONMENTAL EFFECTS have shown pigmental asymmetry are exceedingly rare. Observation of specimens mosaic for var- ious mutant factors and for sex (gynanders), however, indicate greater or less asymmetry in body color. A certain quantity of black pigment in the given region is, of course, a prerequi- site for visible asymmetry. Total absence of color resulting from high temperature during development or to other causes obviously renders a genetic difference incapable of expressing itself. Likewise, if environmental conditions are such that the parts are very black, a rel- atively slight difference determined genetical- ly would not be apparent. Females are in gen- eral less deeply pigmented than males in any given strain, and consequently gynanders usual- ly show striking asymmetry in color correlated with asymmetry in sex. The pigments of the stemmaticum of Habrobracon are granular and present in hypodermal cells^ beneath clear cuticle. The other pigments , rang- ing from pale yellow through brown to black, are in the cuticle and appear to be non-granular. Both granular and diffuse pigments are similar- ly affected by temperature changes and undoubt- edly belong to the melanins. The melanins are formed from colorless substances or chromogens) with the aid of enzymes in the presence of oxy- gen. A. R. Whiting (1939b) points out that Wright's theory of pigment formation in mamma- lian hair is apparently applicable to the cu- ticular pigments of Habrobracon. According to Wright (1917) the colorless chromogen is formed in the cytoplasm, and two enzymes or their fore- runners are formed in the nucleus. The union of these substances in the cytoplasm in differ- 12J THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD ing amounts and under different conditions gives rise to different colors and intensities of color. Chromogen and enzyme I (and 02) are nec- essary for any color production ; acting together they produce yellow. Enzyme II has no effect alone, but in combination with I oxidizes chro- mogen to black and is effective below the thresh- old of I alone. In the absence of I no color can be produced, in the absence of II, no black. Figure 18, is an attempt to express in simple form the maximum potentiality of each genetic type in respect to each enzyme. Wild-type is used as a standard. Quantity of enzyme I is rep- resented on the left, of II on the right. Any environmental condition which lowers the ex- pression of II increases the expression of I. Lower temperatures change the black pigment of the lemon mutant less strikingly than that of wild-type or black. The black areas never be- come as intense or widespread, but in spite of this there is a striking lightening of yellow. This fact taken together with the lighter gen- eral color at higher temperatures and the ob- served transparency of thoracic cuticle at both temperatures suggests a deficiency of enzyme I. There is too little of I to combine with II to form as much black as in wild-type and likewise too little to produce the full amount of yellow even at high temperatures (A. R. Whiting, 1939b). The effect of temperature on the eye colors, carrot, maroon, and the combination, carrot-ma- roon, has been tested (David, 1938). Limited experiments have also been made with the eye color, cantaloup, and with the body color, lem- on. The temperature ranged from 37° C. to 15° C. Except for cantaloup, which showed little_ 124 lemon black honey-black honey ENZYME I ENZYME II Fig. 18o Relative potencies of enr^yines I and II in each genetic type. A, R, Siting, 1939b. 125 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD if any change, the intensity of the eye colors varies inversely with the temperature, in con- trast to that of body color (type and lemon) which varies directly with the temperature. Carrot showed wide variation from white at low temperature to a deep reddish carrot at high temperature, maroon showed less variation, from a bright red to a dark red or black, and the com- bination, carrot-maroon, showed still less vari- ation from white to pale yellow. White-eyed carrot wasps were phenotypically like geneti- cally white-eyed. The bright red of the maroon was similar to the deep reddish orange of the carrot. Body color is determined earlier in de- velopment than is eye color. Temperature treatment affects fecundity al- though short time exposure to cold has only a slight effect. Constant high temperature de- creases fecundity by a little less than a third and constant low temperature lowers it to less than half that of control wasps. Constant cold and extreme heat both have very significant ef- fects on fecundity though they apparently act in different v/ays. Cold acts by slowing down all the activities of the adult wasps thereby decreasing the rate of egg production. Cold al- so keeps many of the eggs from hatching and kills many of the young larval wasps. Heat treatment apparently has little or no effect on rate of egg production. It affects the young, the consequences being especially noted in the pupal stage. There are many naked pupae and many dead in the cocoons. Maercks (1933) investigated the influence of temperature and relative humidity on the eggs of Habrobracon. He found the optimal tempera- 126 ENVIRONMENTAL EFFECTS ture for hatching to be 29° C, the optimal rel- ative humidity, 80 per cent. The time required for hatching is increased by lower temperatures but is not noticeably influenced by relative humidity except at 19° C. and below, when lower relative humidity slows up development. At op- timal temperature egg mortality is not signifi- cantly influenced by relative humidity; at low or high temperature it is increased by low rel- ative humidity; at optimal relative humidity it is low through a wide range of temperatures (16- 35° C.) but rises quickly to 100 per cent near the lower and upper limits (12° C. and 38° C). Temperature treatment of mated females slight- ly affects the sex ratio of offspring, the most significant decrease in female ratio being ob- served at constant low or high temperatures. Short time exposure of heterozygous females to extreme cold does not significantly affect the rate of production of male mosaics and gynan- ders. However, when such females are placed at a constant low temperature, no mosaic offspring appear. On the other hand if females are kept at 35° C. to 37° C. a significantly greater num- ber of mosaics appear (Greb, 1933c). Experiments with crosses of closely related stocks show that the percentage of diploid males decreases as culture temperatures are increased (P. W. Whiting and Anderson, 1932). Females mated with related males produce fewer biparen- tal offspring if kept at 30° C. than if kept at 20° C. However, at the higher temperature, the percentage of diploid males is greater. Trans- fer of young to other temperatures appears to -lave no effect on ratios except that the ratio qtf biparentals is increased if transfers are 127 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD made from 20° C. to 30° C. or 35° C. This is probably to be explained by a differential le- thal effect on males in general. At all temper- atures percentages of males among biparentals change significantly v/ith increasing age of mothers. An experiment in which eggs were count- ed showed that at the higher temperature the percentage of eggs producing impaternate males was decreased. Among biparental offspring males were increased and females correspondingly de- creased.. The conclusion is drawn that increased temperature increases mortality of males and favors homeosyngamy (male-producing combina- tions) at the expense of heterosyngamy (female- producing) (Anderson, 1935, 1936). On the whole, however, sex ratio is less dependent upon tem- perature than is fecundity. High temperature may cause or, at least, bring to light, various mutations. The mutant, shot-veins, a semidominant factor, appeared ac- cidentally. It arose simultaneously in three different lines after extreme heat treatment of larvae. It causes veins of the wings to be bro- ken up and distorted. The trait proved to be hereditary, and shot-veins is fully fertile and stable . A number of mutations have occurred following X-radiation in Habrobracon. Treatment is given at various stages, to eggs in the mother before they are laid, to eggs after laying, to larvae, to pupae, and to sperms in the adult male before mating. The offspring of the treated wasps are examined, and the un.isual individuals are al- lov/ed to produce progeny to show v/hether or not. the questionable traits are hereditary. Unlaid eggs of Habrobracon are most sensi- 128 ENVIRONMENTAL EFFECTS tive to irradiation in late metaphase I, almost equally so in latest prophase I ( diakinesis ) and least so in prophase stages before diakinesis. Adult (haploid male) survival from unfertilized eggs laid by treated females is used as the criterion (A. R. Whiting, 1939a, 1940a, c). Hatchability percentages of unfertilized Habrobracon eggs X-rayed in late prophase I with doses up to 400 R units are significantly high- er than those of controls, and the lethal dose is about 35,000 R units (A. R. Whiting, 1941). Eggs treated in late metaphase I are sensitive to 50 R units, and the lethal dose is abo^it 1,250 R units. There is no evidence of recovery •in this stage. The dose-hatchability curves of the two stages differ, that of metaphase I show- ing a linear relationship to dose. In 1942 (A. R. Whiting, 1942a) evidence against "physiolog- ical effect" as an important factor for meta- phase I sensitivity was presented as well as preliminary cytological observations. Two criteria for judging injury are used (A. R. Whiting, 1945a). One of these, hatchability , gives degree of lethal effect, both dominant and recessive, and is a very accurate measurement in Habrobracon where unfertilized haploid eggs are capable of development, where every egg can be accounted for, where the ideal environmental conditions are known and easily controlled, and where there is available a stable wild-type stock consistently giving 96 per cent hatchabi 1- ity or higher. The second criterion, chromosome changes in successive stages of the treated oo- cytes, is less satisfactory because Habrobracon 3:hromosomes are too small (1 u in diameter) for very careful analysis. Giant salivary chromo- 129 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD somes are lacking. Enough has been learned from cytological observation, however, to make this method of some value in connection with hatcha- bility data. The chromosome aberrations ob- served are necessarily gross ones and undoubt- edly represent lethal effects in most cases. Unmated mature Habrobracon females, when well-fed on host caterpillars and restrained from ovipositing for several hours by removal from host, store in each of their four egg sacs from two to five fully mature eggs which may be retained for at least thirty-six hours without injury. After a much longer period of storage they undergo resorption. They are all in late irietaphase I with spindle attachment regions sep- arated and about half way to the poles. The ends of the dyads are still in contact so that the chromosomes appear to be under tension. Oc- casionally, an egg in late diakinesis or in ear- ly metaphase I can be seen entering an egg sac. Successively younger prophase oocytes occupy the ovarioles anteriorly. Synapsis occurs as soon as the youngest oocytes can be distinguished from oogonia. An ovariole is represented sem- idiagrammatically in Fig. 19. Dominant lethal effects are induced in all eggs treated in metaphase I at about 1,000 R units, in spermatozoa at 10,000 R units, and in eggs treated in prophase I at 45,000 R units. Spermatozoa are not actually inactivated by doses much larger than 10,000 R units. Eggs are also not inactivated by doses much larger than lethal since they continue meiotic phenomena under such conditions. These facts strongly support the theory that the sex of the cell has nothing to do with its response to X-rays but 130 2. 0, b. 1* c* b. lo a. Pig. 19. A Single ovariole (semidiagrainmatic) showing regions used In classification of treated eggsj la, b and c Include eggs In metaphase I stored in egg sac; 2a, b and c include eggs in metaphase I, late 'and mid prophase I; 3 includes eggs in early proohase I. All are post-synaptic. A. R. Whiting, 1945b. X 62 131 TfTE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD rathsr that the condition of the chromatin de- termines cell sensitivity. The extreme and unexpected sensitivity of metaphase I in Habrobracon oocytes has caused some investigators to suggest that the effect is "physiological" and not the result of direct chromosome change. It has been found that hap- loid (male) larvae are more sensitive to irra- diation than diploid (female) larvae of Habro- bracon, as expected if chromosome change is re- sponsible. Eggs treated in metaphase I and in prophase I all have chromosomes and fragments , when pres- ent, distinct in outline in stages follov/ing oviposition and no fusion bridges have been seen. Absence of a low dose threshold for in- jury and absence of a high one for clumping and retardation of meiosis and early cleavage fur- nish additional evidence against any serious "physiological" effect in this stage. Since both metaphase I and prophase I eggs, after their re- spective lethal doses, present the same pattern in regard to degree of development before death, causes of death would appear to be of the same nature for each. The linear proportionality between dose and hatchability as well as the other facts about metaphase I oocytes outlined abo/e can be ex^ plained by the folio v; in g hypothesis (A. R. Whiting, lQ45a). At the time of treatment, dy- ads are unier tension since spindle fiber re- gions have started toward the poles while ends are still held in contact by chiasmata. When a hit breaks a pair of chromatids (a dyad) proxi- mal to a chiasma the parts will separate, too far to rejoin but not far enough for the fracture to 152 ENVIRONMENTAL EFFECTS be seen. Subsequent complete terminalization and separation of dyads in anaphase I leaves fragments and there are no bridges. A broken dyad produces a bridge in division II in either spindle with equal frequency. If a single chro- matid is broken the fragment remains attached to its uninjured partner and appears in division II. An egg with a single deletion will fail to hatch if unfertilized, and, even if fertilized, when the deletion is large.* If two chromatids in a "lug" (distal to chiasma) were broken and the chromatids fused laterally, an occasional bridge in division I might be expected. Tension is much less in this region so that restitution usually occurs when a hit causes a break in this short length of chromosome. As explained above, an egg with one double fragment has one chance in two of hatching, an egg with two such fragments, one chance in four (if the possibil- ity of two breaks in one dyad be left out of consideration) . In Habrobracon, bridges in division II fol- lowing treatment in late- metaphase I cannot be explained by chromosome splitting following breakage in view of the structure of the meta- phase tetrad. It therefore seems probable that a single hit has broken two chromatids in late metaphase I. It is known that many deficiencies are viable in the heterozygous condition, le- thality being dependent on gene content of the segment lost. Since diploid larvae are most re- sistant to irradiation than haploid, recessive deficiencies must be induced in somatic cells. It is surprising, in view of these facts, that all the lethal effects of irradiation appear to be dominant. If breaks proximal to the chias- 135 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD mata are the ones which tend to be permanent, these would necessarily result in large (and therefore dominant) terminal deletions. Dele- tions small enough to act as recessives might not have a lethal effect on the haploid embryo until after hatching and therefore would not be detected with methods used in present work. In the limited tests made with fertilized eggs it is also possible that a few deficiency hetero- zygotes surviving would not noticeably affect hatchability ratios. Genetic literature includes much discussion of lethal factors. In Habrobracon the occur- rence of dominant lethals in the sperm can be readily distinguished from direct killing of the male gametes. The most striking result of X-ra- diation of males is, however, the production of sterility (or partial sterility) of a second type. In this case the sperm are not inactivat- ed but are fully capable of fertilizing the eggs. Such fertilized eggs, however, do not hatch be- cause the sperm have a dominant lethal effect. Females mated to males with this type of steril- ity produce azygous sons only and in numbers equal to those produced by females mated to nor- mal males; in other words, although they produce no females, as the result of dominant lethal ef- fects of the sperm which have entered the eggs, they behave like mated rather than unmated fe- males in respect to number of azygous sons. Habrobracon, then, is especially well suited for separating dominant lethal male sterility from sterility resulting from inactivated sperm on the basis simply of numbers of azygous sons pro- duced by females mated to the males to be tested (P. W. Whiting, 1938a, b). 154 ENVIRONMENTAL EFFECTS As to the nature of dominant lethals it is probable that they are the result of extensive chromosomal alterations rather than to changes in restricted regions or in single genes. Ale- thai effect from a single gamete may be called dominant in contrast to a condition v/hich must be present in both gametes and hence recessive. Since in Habrobracon an unfertilized egg pro- vided with a single set of genes develops nor- mally into a male and since the addition of a second complete set by fertilization likewise results in normal development into a female, it might be thought that the addition of a defi- .cient set should not be lethal to the resulting zygote. In other words, if both In and 2n may develop normally, why should not In + (n-x) also develop normally? The explanation is doubtless to be found in genie balance. If n-x is too small to act as a recessive, containing rela- tively extensive deletions for example, the bal- ance should be so disturbed that development would be prevented. Theoretically the genie set in a sperm might become so extremely deleted by X-radiation that the fertilized egg would devel- op as an unfertilized egg into a male. No sex intergrades have ever been found in the treated material that might be interpreted as hyperploid males or hypoploid females. The sex types sur- viving after X-radiation have been fully as nor- mal as in untreated stock. Recessive lethal (or semilethal) factors may be a cause of "bad eggs" (P. W. Whiting, 1929a; Maxwell, 1935). Half of the unfertilized eggs of a female heterozygous for a recessive lethal do not hatch. Eggs may likewise be defective in their gross morphological or non-nuclear as- 135 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD pects because of unfavorable cultural conditions (very low humidity) or inadequate nutriment of females (feeding v/ith honey instead of caterpil- lar juice) v/hile females of certain genetic types lay v/ithered eggs of irregular form. Non- hatchability may then be the result of non-nu- clear causes, recessive lethals, dominant le- thals, or male-producing fertilization (P. W. Whiting, 1938a, b) . Dominant lethals may be induced in the sperm of Habrobracon by X-radiation of the males. At 10,000 to 20,000 R units all sperm have at least one lethal. With very high dosages, 41,000 to 142,000 R units, some sperm are directly inacti- vated while many still remain active and able to carry dominant lethals into the eggs (P. W. Whiting, 1937a, b). Neutrons have also been shown to produce dominant lethals (P. W. Whiting, 1936), being actually much more effective than X-rays. Ultra-short (1 meter) radio waves ap- pear to be ineffective (P. W. Whiting, i937a,b). Biparental males have been obtained (Stancati, 1932; Bishop, 1937) after X-radiation of sperm from related males, but the data are not suffi- cient to indicate their decrease. X-radiation of sperm from unrelated males has never resulted in biparental males. Experiments from crosses with closely related males (treated and untreat- ed) indicate that while the number of haploid males produced per day is not changed by X-radi- ation of sperm, both kinds of biparental off- spring (males and females) are decreased with increasing dosages and that they are decreased at approximately the same rate. This indicates that X-radiation of sperm fails to modify the type of fertilization and that the changes in- 136 ENVIRONMENTAL EFFECTS duced in the sperm are just as lethal to the biparental males as to the females (P. W. Whiting, 1933a, b). If females are treated and subsequently mated, there is no appreciable reduction in male ratio, indicating that few, if any recessive lethals are induced in the egg. While treatment of mated females causes a radical lowering of fe- male ratio indicating that more dominant lethals* are induced in the sperm than recessive lethals) in the egg. It has also been noted (P.W. Whit- ing, 1929a; N. C. Bostian, 1931) that the per- centage of female offspring from X-radiated .mothers, mated previous to treatment, is marked- ly decreased (Greb, 1933). P. W. Whiting ( 1937a) demonstrated that treat- ment of sperm with X-ray dosages of 20,000, 40,000, and 75,000 R units produces at least one dominant lethal in every sperm cell. No daugh- ters occur in the progeny and the average number of males produced per day does not equal that to be expected from virgin females, indicating that many eggs are fertilized and die. Sperm treated with 75,000 R units fertilized almost as many eggs as untreated sperm; therefore, the treatment apparently did not cause inactivation. However, a slight increase in the average males per day from mates of males treated with 75,000 R units as compared with those from mates of untreated controls suggested the possibility that spermatogenesis might to some extent be stopped and sperm supply decreased. Thus partial male sterility results from dom- inant lethals at relatively v/eak dosages, com- plete male sterility results from dominant le- 137 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD thals at stronger dosages, and partial sperii inactivation at very high dosages. Treatment of Habrobracon larvae with X-rays was begun in January 1929. Progeny from crosses of different stocks were used with pure stocks sometimes treated as checks. Treatments ranging from 730 to 4378 R units were given. No treat- ment had complete lethal or sterilizing effect, although the highest dosage approached this. Younger larvae were found to be more susceptible to X-radiation than older, and male larvae more susceptible than female larvae. Irradiation tended to kill the younger larvae immediately while the older ones continued development and often died after metamorphosis and before ec- losion (A. R. Whiting and Bostian, 1931). There have been some fifty mutant types de- rived from X-ray experiments on Habrobracon, Dosages of 2,000 to 5,000 R units have produced the majority of these mutations. After 10,000 R units there is a dominant lethal change of some sort produced in practically every sperm cell. Dosage for egg cells must be much higher for the chromosomes are not so closely packed together and apparently the closer the packing the better chance for irregular recombinations. Table III has been prepared to summarize the effect of •various dosages of X-rays on eggs, sperm, and larvae. Considerable experimentation is still being carried on along this line, and various dosages of X-rays are now being combined with treatment at various degrees of temperature. 138 Chapter X CONCLUSION The body of information assembled thus far establishes Habrobracon juglandis as a genetic animal of unusual merit. In addition to its possession of all the prerequisites for a ge- netic form it has the advantages offered by par- thenogenesis. Since the normal males are hap- loid, their genotypes are phenotypically ex- pressed; thus the need for the traditional back- cross is eliminated. While it has been shown that a considerable amount of information has been accumulated along various lines, in no case is the knowledge complete, and numerous avenues^ of research are open for investigation. Habrobracon has ten. pairs of chromosomes. Only four and perhaps fewer than four are as yet known; so that at least six await exploration and mapping. Many mutations have come to light, but unpredictable numbers await the chance to^ sho¥ their effects in haploid males. Numerous' mutations have occurred whose effects are le- thal to their owners; these when linked with non-lethals may disturb expected ratios in such a manner as to betray their presence. These may be located on given chromosomes. Regarding the development of the mutants, very little is known. They are known in their final adult form, but exactly when and where 139 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD deviation from normal development takes place is yet largely a mystery. In some mutants, there is a suggestion of accompanying differ- ences in behavior. These remain to be studied. Sex determination in Habrobracon has been shown to be complementary, but further experi- ment is necessary before this hypothesis can be shown to hold for the Hymenoptera in general. The effects of environmental changes, X-radia- tion, and neutron treatment are being tested, and these are bringing up new questions. All these problems await investigation, and their solution holds promise of interesting and sig- nificant contributions to the field of genetics. 140 APPENDIX TABJuB I 'Percentages deviating below 50 per cent with probability ©quai to c90 or to .99 that trus value is no lower in population from which sampls of a given size, n, is taken, (Whiting and Benkort, 1934) n Per cent Par cent (P. 90) (P. 99) 27 35 27 32 37 29 37 37 30 42 58 31 47 39 32 55 40 34 65 40 35 n Per cent Per cent (P. 90) (P. 99) 75 41 36 85 41 36 95 42 37 110 42 38 150 44 40 170 44 40 190 44 41 n Per cent Per cent (P. 90) (P. 99) 250 45 42 350 46 43 450 46 44 550 47 45 800 47 45 1500 48 47 2500 48 47 143 TABLS II 144 Dosage TABLE III X-RAY RESPONSES OF HABROSRACOH Sperm Metaphaae Prophase I^arvae I Egg I Egg Adult 50 R Approz, Viable 15% killed Viable Yoiin£ killed Viable mutations Approx. Decline 1,400 a Viable lethal in doae hatcii4ibillty Old active Viable mutations 2,500 R Meiosia Dcminaiit and ^^% lethala cleavage hatch Old pupate Viable mutations 4,060 E Dominant , 78^ lethala Meioala ^^^^ch Afiprox, LB thai 4056 4,378 Viable mutaCions 10,000 R Dominant lethala 60^ hatch 20,000 R 100^ Dominant lethala 20% hatch 25,000 R lOOjg Dominant lethala 12^ hatch 55,000 R 100^ Dofliinant lethala Lethal dose 44,600 R 200,000 R Still active All killed 250,000 R Still alive inactive 300,000 R All killed 145 BIBLIOGRAPHY BIBLIOGRAPHY Anderson, R. L., 1935. Offspring obtained from males reared at different temperatures in Habrobracon. THE AMERICAN NATURAL- IST, vol. 69, pp. 183-187. , 1936. Effects of temperature on fer- tilization in Habrobracon. GENETICS, vol. 21, pp. 467-472. , 1941. Non-autonomous development of transplanted eyes in Habrobracon. PRO- CEEDINGS OF THE SEVENTH INTERNATIONAL CONGRESS OF GENETICS, 1941, p. 47. Anderson, R. L.,and P. W. Whiting, 1939. 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Linkage test of elon- gate, a wing factor in Habrobracon. University of Pittsburgh Library. M.S. Thesis. Dunning, W. F., 1931. A study of the effect of X-ray radiation on occurrence of abnor- mal individuals, mutation rate, viabil- ity, and fertility of the parasitic wasp, Habrobracon juglandis (Ashmead). GENETICS, vol. 16, pp. 505-531. Dzierzon, J., 1845. EICHSTADTE BIENENZEITUNG, pp. 1-113. , 1854. BIENENFREUND AUS SCHLESIEN, p. 54. Fahringer, J., 1928. Opscula braconologica . Palaearktische region, vol. 1, pp. 61-- 606. Aethiopische region, vol. 2, pp. 1-224. Fritz Wagner, Wiener. Finkenbrink, Walter, 1933. Experimentel le Un- tersuchungen zur Dewitzschen Hypothese des Apterismus bei Insekten. ZETT- SCHRIFT FUR WISSENSCKAFTLICHE BIOLOGIE ■ ABT. A, ZEITSCHRIFT MORPHOLOGIE UND OK- . OLOGIE DER TIERE, vol. 26, pp. 385-426. Fisher, R. A., 1941. STATISTICAL METHODS FOR RESEARCH WORKERS. Edinburgh and London, England. 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PROCEEDINGS OF THE PENN- SYLVANIA ACADEMY OF SCIENCE, vol. 13, pp. 118-119. Greb, R. J., and M. T. Greb, 1938. Further stud- ies of the unstable gene of Habrobracon. PROCEEDINGS OF THE PENNSYLVANIA ACADEMY OF SCIENCE, vol. 12, pp. 56-59. Greb, R. J., and M. T. Greb, 1940. The linkage relations of sex factors to two adjacent mutant factors in Habrobracon. PROCEED- INGS OF THE SOUTH DAKOTA ACADEMY OF SCIENCE, vol. 20, pp. 79-83. Greb. R. J., and P. W. Whiting, 1933. Stumpy mosaics of Habrobracon and their bear- ing on interaction and reduction. THE AMERICAN NATURALIST, vol. 67, p. 66. Greenshields, F., 1939. Whiting's hypothesis* and Pteromalus: A critique of Dozor- ceva's (1936) study. THE AMERICAN NAT- URALIST, vol. 73, pp. 89-91. ■ 15S THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD Grosch, Daniel S., 1945. The relation of cell size and organ size to mortality in Habrobracon. GROWTH, vol. 9, pp. 1-7. Hager, Russell P., 1941. Sex-linkage of stubby (sb) in Habrobracon. 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Production of dominant lethal genetic effects by X-radiation of sperm in Habrobracon. SCIENCE, vol, 76, pp. 197-198. , 1933. X-radiation and sex ratio in Habrobracon. PROCEEDINGS OF THE PENN- SYLVANIA ACADEMY OF SCIENCE, vol. 7, p. 121. 163 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD Stevens, W. L., 1936. An analysis of interfer- ence. THE JOURNAL OF GENETICS, vol. 32, pp. 51-64. Torvik, Magnhild M., 1929a. Are Habrobracon nalas diploid for the X-ray mutation "short"? PROCEEDINGS OF THE PENNSYLVA- NIA ACADEMY OF SCIENCE, vol. 3, pp. 31- 32. „_,, 1929b. Are biparental males of Habro- bracon diploid for all chromosomes? ANATOMICAL RECORD, vol. 44, p. 280. ^_, 1930, The genetic composition of bi- parental males. PROCEEDINGS OF THE PENNSYLVANIA ACADEMY OF SCIENCE, vol. 4, pp. 87-83. , 1931. Genetic evidences for diploidism of biparental males in Habrobracon. THE BIOLOGICAL BULLETIN, vol. 61, pp. 139-146. Torvik, Magnhild M. , 1933. The Chromosomes of Habrobracon. PROCEEDINGS OF THE PENN- SYLVANIA ACADEMY OF SCIENCE, vol. 7, pp. 119-120. Torvik-Greb, Magnhild, 1934. Chromosome numbers') in Habrobracon. THE AMERICAN NATURAL- IST, vol. 68, p. 68. „„_> 1935. The chromosomes of Habrobracon. THE BIOLOGICAL BULLETIN, vol. 68, pp. 25-34. Viereck, H. L., 1911. Descriptions of six new genera and thirty-one new species of ichneumon flies. PROCEEDINGS OF THE UNITED STATES NATIONAL MUSEUM, vol. 40, p. 182. 164 BIBLIOGRAPHY Washburn, 1904. The Mediterranean flour moth. SPECIAL REPORT OF THE STATE ENTOMOLO- GISTS OF MINNESOTA, 1904. Watanabe, Chihsa, 1935. On some species of Bra- conidae from North China and Korea. INSECTA MATSUMURANA, vol. 10, pp. 43-51. Wenstrup, Edward Joseph, 1931. Male biparental- ism and reciprocal crosses in Habrobra- con. UNIVERSITY OF PITTSBURGH BULLETIN, vol. 28, pp. 230-237. Whiting, Anna R., 1925. The inheritance of sterility and of other defects induced by abnormal fertilization in the para- sitic wasp Habrobracon juglandis (Ash- mead). GENETICS, vol. 10, pp. 33-58. » 1926. Further data on diploid males of Habrobracon. ANATOMICAL RECORD, vol. 34, p. 210. > 1927. Genetic evidence for diploid males in Habrobracon. THE BIOLOGICAL BULLETIN, vol. 53, pp. 438-449. » 1928a. Genetic evidence for diploid males in Habrobracon. THE AMERICAN NATURALIST, vol. 62, pp. 55-58. > 1928b. Genetic evidence for diploid males in Habrobracon. ZEITSCHRIFT FUR INDUKTIVE ABSTAMMUNGSUND VERERBUNGSLEHRE SUPPLEMENTB, vol. 2, pp. 1587-1590. » 1929a. X-rays and heredity of wasps at the University of Pittsburgh. PROCEED- INGS OF THE PENNSYLVANIA ACADEMY OF SCIENCE, vol. 3, pp. 27-36. -»» 1929b. Diploid males in Habrobracon. PROCEEDINGS OF THE PENNSYLVANIA ACADEMY OF SCIENCE, vol. 3, pp. 28-29. 165 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD Whiting, Anna R., 1Q30. Disorganization of wing veins caused by X-raying larvae of Hab- robracon. ANATOMICAL RECORD, vol. 47, p. 384. _, 1933a. A simple device for the study of small opaque objects. THE COLLECTING NET (Woods Hole), vol. 8, p. 11. , 1933b. Eye colors in the parasitic wasp Habrobracon and their behavior in mosa- ics and in multiple recessives. THE COLLECTING NET (Woods Hole), vol. 8, p. 339. , 1933c. Variegated eye color in the par- asitic wasp Habrobracon. THE COLLECT- ING NET (Woods Hole), vol. 8, p. 398. , 1933d. Variegated eyes in Habrobracon. PROCEEDINGS OF THE PENNSYLVANIA ACADEMY OF SCIENCE, vol. 7, p. 118. , 1933e. How we draw Habrobracon. PRO- CEEDINGS OF THE PENNSYLVANIA ACADEMY OF SCIENCE, vol. 7, p. 119. , 1933f. Eye colors in the parasitic wasp Habrobracon and their behavior in mosa- ics and in multiple recessives. THE BIOLOGICAL BULLETIN, vol. 65, p. 362. , 1933g. Variegated eye color in Habro- bracon. THE BIOLOGICAL BULLETIN, vol. 65, p. 369. , 1934. Eye colors in the parasitic wasp Habrobracon and their behavior in mul- tiple recessives and in mosaics. JOUR- NAL OF GENETICS, vol. 29, pp. 99-107. , 1938. Mutant body colors in Habrobracon and their mosaics. GENETICS, vol. 23, p. 175. 166 BIBLIOGRAPHY Whiting, Anna R., 1939a. Sensitivity to X-rays of stages in oogenesis of Habrobracon. GENETICS, vol. 24, pp. 89-90. > 1939b. Mutant body colors in the para- sitic wasp Habrobracon juglandis (Ash- mead) and their behavior in multiple recessives and in mosaics. PROCEEDINGS OF THE AMERICAN PHILOSOPHICAL SOCIETY, vol. 80, pp. 65-85. > 1940a. Do Habrobracon females sting their eggs? THE AMERICAN NATURALIST, vol. 74, pp. 468-471. > 1940b. Further data on sensitivity to- X-rays of metaphase I eggs in Habrobra- con. THE COLLECTING NET (Woods Hole), vol. 15, p. 30. » 1940c. Temperature effects on sensi- tivity to X-rays of different meiotic stages in Habrobracon eggs. THE COL- LECTING NET (Woods Hole), vol. 15, p. 9. » 1940d. Sensitivity to X-rays of differ- ent meiotic stages in unlaid eggs of Habrobracon. JOURNAL OF EXPERIMENTAL ZOOLOGY, vol. 83,' pp. 249-269. » 1941. Susceptibility to X-rays of mei- otic stages in eggs of Habrobracon. PROCEEDINGS OF THE SEVENTH INTERNATION- AL CONGRESS OF GENETICS, 1941, p. 314. » 1942. X-ray sensitivity of first mei- otic prophase and metaphase in Habro- bracon eggs. GENETICS, vol. 27, pp. 174-175. » 1943. Hatchability and chromosome changes of Habrobracon eggs X-rayed in prophase I and metaphase 1. GENETICS, vol. 28, pp. 94-95. 167 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD Whiting, Anna R. , 1945a. Effects of X-rays on hatchability and on chromosomes of Hab- robracon eggs treated in first meiotic prophase and metaphase. THE AMERICAN NATURALIST, vol. 79, pp. 193-227. , 1945b. Dominant lethality and corre- lated chromosome effects in Habrobracon eggs X-rayed in diplotene and in late metaphase I. THE BIOLOGICAL BULLETIN, vol. 89, pp. 61-71. , 1945c. Differences in sensitivity, hatchability curves, and cytological effects, between Habrobracon eggs X- rayed in first meiotic prophase and metaphase. THE BIOLOGICAL BULLETIN, vol. 89, p. 191. , 1946a. Immaternate males and high vis- ible mutation rate from eggs irradiated in prophase I. GENETICS, vol. 31, pp. 235-236. _, 1946b. Motherless males from irradiated eggs. SCIENCE, vol. 103, pp. 219-220. Whiting, A. R., and C. H. Bostian, 1930. A study of mutations, mosaics, and non- inherited abnormalities, among progeny of individuals X-rayed as larvae in Habrobracon. ANATOMICAL RECORD, vol. 47, p. 384. Whiting, A. R., and C. H. Bostian, 1931. The effects of X-radiation of larvae in Habrobracon. GENETICS, vol. 16, pp. 659-680. Whiting, A. R., and R. H. Burton, 1926. Quad- ruple allelomorphs affecting eye color in Habrobracon. THE AMERICAN NATURAL- IST, vol, 60, pp. 285-290. 168 BIBLIOGRAPHY Whiting, A. R., and M. M. Torvik, 1932. Male biparentalism in Habrobracon. PROCEED- INGS OF THE SIXTH INTERNATIONAL CONGRESS OF GENETICS, vol. 2, pp. 209-210. Whiting, A. R., and P. W. Whiting, 1923. Facts indicating abnormal fertilization in Habrobracon. ANATOMICAL RECORD, vol. 24, pp. 411-412. Whiting, P. W., 1918. Sex determination and bi- ology of a parasitic wasp, Hairobracon brevicornis (Wesmael). THE BIOLOGICAL BULLETIN, vol. 34, pp. 250-256. » 1921a. The production of mosaic males from fertilized eggs in Hymenoptera. ANATOMICAL RECORD, vol. 20, p. 2. » 1921b. Parasitic v/asps as material for genetic research. SAINT STEPHENS COL- LEGE BULLETIN, May, 1921, pp. 21-24. > 1921c. Studies on the parasitic wasp, Hadrobracon brevicornis (Wesmael). I. Genetics of an orange-eyed mutation and the production of mosaic males from fertilized eggs. THE BIOLOGICAL BULLE- TIN, vol. 41, pp. 42-54. > 1921d. Rearing meal moths and parasitic wasps for experimental purposes. THE JOURNAL OF HEREDITY, vol. 12, pp. 255- 261. » 1921e. Heredity in wasps, a study of heredity in a parthenogenetic insect, Hadrobracon. THE JOURNAL OF HEREDITY, vol. 12, pp. 262-266. 169 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD Whiting, P. W., 1921f. Studies on the parasitic wasp Hadrobracon brevicornis (Wesmael). II. A lethal factor linked with orange. THE BIOLOGICAL BULLETIN, vol. 41, pp. 143-155. , 1922. Genetic mosaics and ontogenetic abnormalities in the parasitic wasp, Hadrobracon. ANATOMICAL RECORD, vol. 23, p. 94. , 1923a. Analysis of contamination in Habrobracon. ANATOMICAL RECORD, vol. 24, p. 411. , 1923b. The analysis of genetic differ- ences through haploid parthenogenesis. EUGENICS, GENETICS, AND THE FAMILY, vol. 1, pp. 102-105. , 1923c. Conflict of instincts in gynan- dromorphs of Habrobracon. ANATOMICAL RECORD, vol. 26, p. 395. , 1924a. Three different types of genetic sterility in the parasitic wasp, Habro- bracon. lOlVA ACADEMY OF SCIENCE, vol. 31, 1924. , 1924b. A study of heredity and environ- mental factors determining a variable character. Defective and freak vena- tion in the parasitic wasp Habrobracon juglandis Ashmead. UNIVERSITY OF IOWA STUDIES IN CHILD WELFARE, vol. 3, pp. 11-58. , 1924c. Some anomalies in Habrobracon and their bearing on maturation, fer- tilization, and cleavage. ANATOMICAL RECORD, vol. 29, p. 146. 170 BIBLIOGRAPHY Whiting, P. W., 1926a. Heredity of two variable characters in Habrobracon. GENETICS vol. 11, pp. 305-316. » 1926b.- Two wing mutations in Habrobra- con and their method of inheritance. THE AMERICAN NATURALIST, vol. 60, dd 443-434. » 1926c. Influence of age of mother on appearance of an hereditary variation in Habrobracon. THE BIOLOGICAL BULLE- TIN, vol. 51, pp. 371-385. » 1927. The relation between gynandro- morphism and mutation in Habrobracon. ZEITSCHRIFT FUR INDUKTIVE ABSTAMMUNGS- UND VERERBUNGSLEHRE SUPPLEMENTS, vol 2 pp. 1591-1593. ' ' » 1928a. The relation between gynandro- morphism and mutation in Habrobracon. THE AMERICAN NATURALIST, vol. 62. dd 59-62. ' 1928b. Mosaicism and mutation in Habro- bracon. THE BIOLOGICAL BULLETIN, vol 54, pp. 289-307.. ' 1928c. Production of mutations by X- rays in Habrobracon. THE COLLECTING NET (Woods Hole), vol. 3, pp. 5-6. ' 1928d. The production of mutations by X-rays in Habrobracon. SCIENCE, vol. 68, pp. 58-59. » 1929a. X-rays and parasitic wasps. THE JOURNAL OF HEREDITY, vol. 20, pp. 268-276. » 1929b. X-ray mutations in Habrobracon. PROCEEDINGS OF THE PENNSYLVANIA ACADEMY OF SCIENCE, vol. 3, p. 30. 171 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD Whiting, P. W., 1929c. Linkage of the semi- lethal X-ray mutation "miniature." PRO- CEEDINGS OF THE PENNSYLVANIA ACADEMY OF SCIENCE, vol. 3, pp. 30-31. , 1929d. The mutant types, "X-ray" and "Natural" of Habrobraoon. ANATOMICAL RECORD, vol. 44, p. 135. , 1930. Local and correlative gene ef- fects in mosaics of Habrobracon. THE COLLECTING NET (Woods Hole), vol. 6, p. 268. , 1931a. New Mutants in Habrobracon. ANATOMICAL RECORD, vol. 51, p. 120. , 1931b. Diploid male parts in gynandro- morphs of Habrobracon. THE BIOLOGICAL BULLETIN, vol. 61, pp. 478-480. , 1932a. Mutants in Habrobracon. GENET- ICS, vol. 17, pp. 1-30. , 1932b. Diploid mosaics in Habrobracon. THE AMERICAN NATURALIST, vol. 66, pp. 75-81. , 1932c. Asymmetry of v/ild-type traitsi and of secondary sexual characters in mosaics of Habrobracon. JOURNAL OF EX- PERIMENTAL ZOOLOGY, vol. 62, pp. 259-267. , 1932d. Exhibits for the Sixth Interna- tional Congress of Genetics. PROCEED- INGS OF THE SIXTH INTERNATIONAL CONGRESS OF GENETICS, vol. 2, pp. 232-233. , 1932e. Modification of traits in mosa- ics from binucleate eggs of Habrobracon. THE BIOLOGICAL BULLETIN, vol. 63, pp. 296-309. 172 BIBLIOGRAPHY Whiting, P. W., 1932f. Reproductive reactions of sex mosaics of a parasitic wasp, Hab- robracon juglandis. JOURNAL OF COMPAR- ATIVE PSYCHOLOGY, vol. 14, pp. 345-363. , 1933a. Sex-determination in Hymenop- tera. THE COLLECTING NET (Woods Hole), vol. 8, pp. 113, 121-122. , 1933b. Egg-trinuclearity in Habrobra- con. THE COLLECTING NET (Woods Hole), vol. 8, p. 399. , 1933c. The parasitic v/asp, Habrobracon juglandis (Ashmead),as laboratory class material. PROCEEDINGS OF THE PENNSYL- ■ VANIA ACADEMY OF SCIENCE, vol. 7, pp. 116-121. , 1933d. Reproductive reactions of gynan- dromorphs. PROCEEDINGS OF THE PENNSYL- VANIA ACADEMY OF SCIENCE, vol. 7, p. 117. , 1933e. Sex-determination in Hymenop- tera. THE BIOLOGICAL BULLETIN, vol. 65, p. 357. , 1933f. Selective fertilization and sex- determination in Hymenoptera. SCIENCE, vol. 78, pp. 537-538. , 1934a, Selective fertilization and sex- determination in Hymenoptera. THE AMER- ICAN NATURALIST, vol. 68, p. 68. , 1934b. Mutants in Habrobracon II. GE- NETICS, vol. 19, pp. 268-291. , 1934c. Egg-trinuclearity in Habrobra- con. THE BIOLOGICAL BULLETIN, vol. 66, pp. 145-151. , 1934d. A method of preparing small in- sects for photographing. THE COLLECTING NET (Woods Hole), vol. 9, p. 30. 173 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD Whiting, P. W., 1934e. Sex-determination in bees and wasps. PROCEEDINGS OF THE PENNSYL- VANIA ACADEMY OF SCIENCE, vol. 8, pp. 103-106. , 1935a. Sex-determination in bees and wasps. THE JOURNAL OF HEREDITY, vol. 26, pp. 263-278. , 1935b. Recent X-ray mutations in Habro- bracon. PROCEEDINGS OF THE PENNSYLVANIA ACADEMY OF SCIENCE, vol. 9, pp. 60-63. , 1935c. Genie balance, sex-determina- tion, and selective fertilization in Hymenoptera. PROCEEDINGS OF THE AMERI- CAN PHILOSOPHICAL SOCIETY, vol. 75, pp. 517-520. , 1936. Dominant lethal genetic effects caused by neutrons. SCIENCE, vol. 84, p. 68. , 1937a. A convenient test of physical agents as producers of dominant lethals. THE COLLECTING NET (Woods Hole), vol. 12, pp. 129-130. , 1937b. Habrobracon as a means for test- ing the effectiveness of physical agents in causing mutations. PROCEEDINGS OF THE PENNSYLVANIA ACADEMY OF SCIENCE, vol. 11, pp. 50-52. , 1938a. Decrease in biparental males by X-raying sperm in Habrobracon. PROCEED- INGS OF THE PENNSYLVANIA ACADEMY OF SCIENCE, vol. 12, pp. 74-76. , 1938b. The induction of dominant and recessive lethals by radiation in Habro- bracon. GENETICS, vol. 23, pp. 562-572. 174 BIBLIOGRAPHY Whiting, P. W., 1938c. The development of Hab- robracon eggs in different aqueous me- dia. ANATOMICAL RECORD, vol. 72, p. 94. > 1939. Sex-determination and reproduc- tive economy in Habrobracon. GENETICS, vol. 24, pp. IIO-IU. > 1940a. Radiation, mutation, and the gene. THE AMERICAN JOURNAL OF ROENTGE- NOLOGY AND RADIUM THERAPY, vol. 63, pp. 271-274. , 1940b. Multiple alleles in sex-deter- mination of Habrobracon. JOURNAL OF MORPHOLOGY, vol. 66, pp. 323-355. , 1940c. Investigations on genetics and sex-determination in the parasitic wasp, Habrobracon. YEAR BOOK OF THE AMERICAN PHILOSOPHICAL SOCIETY, 1939, pp. 306-307. , 1940d. Sex-linkage in Pteromalus. THE AMERICAN NATURALIST, vol. 74, pp. 377- 379. , 1940e. Proof of quadruple alleles in sex-differentiation of Habrobracon. THE COLLECTING NET (Woods Hole), vol. 15, p. 206. , 1940f. A single series vs. many pairs of sex factors in the wasp, Habrobracon. SCIENCE, vol. 92, p. 418. , 1941a. The cytogenetics of sex-deter- mination. PROCEEDINGS OF THE SEVENTH INTERNATIONAL CONGRESS OF GENETICS, 1941, pp. 314-315. , 1941b. Sex-determination in Habrobra- con. PROCEEDINGS OF THE SEVENTH INTER- NATIONAL CONGRESS OF GENETICS, 1941, pp. 315-316. 17S THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD ' Whiting, P. W., 1941c. Investigations on genet- ics and sex-determination in the para- sitic wasp, Habrobracon. YEAR BOOK OF THE AMERICAN PHILOSOPHICAL SOCIETY, 1940, pp. 274-276. , 1943a. Multiple alleles in complemen- tary sex-determination of Habrobracon. GENETICS, vol. 28, pp. 365-382. , 1943b. Intersexual females and inter- sexuality in Habrobracon. THE BIOLOGI- CAL BULLETIN, vol. 85, pp. 238-243. , 1943c. Androgenesis in the parasitic wasp, Habrobracon. THE JOURNAL OF HE- REDITY, vol. 34, pp. 355-366. , 1945a. The problem of reversal of male haploidy by selection. THE BIOLOGICAL BULLETIN, vol. 89, p. 191. __„» 1945b. The evolution of male haploidy. THE QUARTERLY REVIEW OF BIOLOGY, vol. 20, pp. 231-257. Whiting, P. W., and R. L. Anderson, 1932. Tem- perature and other factors concerned in male biparentalism in Habrobracon. THE AMERICAN NATURALIST, vol. 66, pp. 420- 432. Whiting, P. W., and R. L. Anderson, 1939. Red, rd (eyes), a second non-autonomus locus in Habrobracon. GENETICS, vol. 24, p. 90. Whiting, P. W., and L. H.'Benkert, 1931. Link- age groups in Habrobracon. ANATOMICAL RECORD, vol. 51, p. 113. Whiting, P. W., and L. H. Benkert, 1934. Asy- gotic ratios in Habrobracon. GENETICS, vol. 19, pp. 237-267. 176 BIBLIOGRAPHY Whiting, P. W.,and K. A. Gilmore , 1932. Genetic analysis of synapsis and maturation in eggs of Habrobracon. PROCEEDINGS OF THE SIXTH INTERNATIONAL CONGRESS OF GENETICS, vol. 2, pp. 210-211. Whiting, P. W., R. J. Greb, and B. R. Speicher, 1934. A nev/ type of sex-intergrade . THE BIOLOGICAL BULLETIN, vol. -66, pp. 152-165. Whiting, P. W., and Mary B. Lefevre, 1946. A possible difference determined by sex alleles in haploid males of Habrobracon. ANATOMICAL RECORD, vol. 94, p. 71. Whiting, P. W., and B. R. Speicher, 1933. Sex- linkage and selective fertilization in Habrobracon. Mimeographed by P. W. Whit- ing, University of Pennsylvania. Whiting, P. W., and B. R. Speicher, 1935. Im- paternate daughters of females heterozy- gous for a sex-linked gene in Habrobra- con. THE AMERICAN NATURALIST, vol. 69, pp. 82-83. Whiting, P. W., and M. F. Stancati, 1931. A gynandromorph of Habrobracon from a post-reduced binucleate egg, THE BIO- LOGICAL BULLETIN, vol. 61, pp. 481-484. Whiting, P. W., and E. J. Wenstrup, 1932. Fer- tile gynandromorphs in Habrobracon. THE JOURNAL OF HEREDITY, vol. 23, pp. 31-38. Whiting, P. W., and A. R. Whiting," 1925. Dip- loid males from fertilized eggs in Hy- menoptera. SCIENCE, vol. 63, pp. 437- 438. 177 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD Whiting, P. W., and A. R. Whiting, 1927. Gy- nandromorphs and other irre2;ular tyoes in Habrobracon. THE BIOLOGICAL BULLE- TIN, vol. 52, pp. 8Q-I21. Whiting, P. W., and A. R. Whiting, IQ28. Link- age of ivory eye color and minor fac- tors for defective venation in Habrobra- con. THE AMERICAN NATURALIST, vol. 62, pp. 479-480. Whiting, P. W., and A. R. Whiting, 1930. Genet- ic studies on the parasitic wasp, Habro- bracon juglandis (Ashmead). PROCEED- INGS OF THE PENNSYLVANIA ACADEMY OF SCIENCE, vol. 4, pp. 79-80. Whiting, P. W., and A. R. Whiting, 1934. A unique fraternity in Habrobracon. JOUR- NAL OF GENETICS, vol. 29, pp. 311-316. Wooldridge, Margaret A., 1933. Effects of tem- perature on eye size in Habrobracon. UNIVERSITY OF PITTSBURGH BULLETIN, vol. 30, p. 614. Wright, Sewall, 1917. Color inheritance in Mam- mals. THE JOURNAL OF HEREDITY, vol. 8, pp. 224-235. 178 GLOSSARY GLOSSARY OF TERMS MOST COMMONLY USED IN HABROBRACONOLOGY ALLELE, one of two or more alternative heredi- tary units or genes, or of the characters associated with the genes; for example, the gene responsible for orange eyes in Habrobracon is an allele of the alterna- tive normal gene for black eyes (Synony- mous with ALLELOMORPH). ANDROGENESIS, development from a sperm. AROLIUM, an appendage between the claws of insects . ARRHENOTOKY, production of impaternate males. AXILLA (AXILLARIES) , the articular scleriteS' of insect wings (excepting the tegula, which is at the front edge of the wing- joint) . AZYGOUS MALE, male produced by haploid arrhe- notoky in the regularly established par- thenogenesis of Dzierzon's Law. B BACK-CROSS, the mating of a hybrid to one of the parental varieties or species which produced the hybrid. 181 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD CALCANEA, largest segment of the insect foot, frequently designated the metatarsus. CENTROMERE, a definite region of a chromosome which takes the lead in activity during mitosis and meiosis. It is the point of spindle-fiber attachment. CHARACTER, a term used to designate any struc- ture, function, or trait of an organism. A Mendelian character represents the end product of development, in which a partic- ular gene has a specific effect. CHIASMA, cross-like figure formed by chromatids in meiosis. CHORION, tough shell or covering of the insect egg. CHROMATID, either one of the two identical strands into which a chromosome splits in anticipation of cell division. CHROMOGEN, a substance that under proper condi- tions may give rise to color. COINCIDENCE, the ratio of the actual number of double breaks to the expected number of double breaks which should occur if there were no interference. COSTA, the first principal insect wing vein. It constitutes the anterior edge of the wing. CROSSING-OVER, the interchange of blocks or chains of genes between two homologous chromatids; also applied to characters which show recombination as a result of such exchange. 182 GLOSSARY CUTICLE (CUTICULA), firm layer outside the hy- poderrais which protects the insect body and serves as a support for the internal organs. Formed from chitin. DELETION, the loss of a chromosomal segment. DIAKINESIS, the final stages of meiotic pro- phase in whi-ch the homologous chromosomes are in intimate association. DIFFERENTIATOR, a factor that prevents somatic overlapping of traits. DIPLOID, referring to the double set of chromo- somes, as found in the body cells of ani- mals and the sporophyte generation of plants, as distinguished from the single (haploid) set, found in the mature repro- ductive cells. DISPERMIC, an egg fertilized by two sperms. DOMINANT, a character which appears as the re- sult of the presence of either a single or a double dose of a particular gene, as contrasted with the recessive, which de- velops only when both members of a pair of genes are alike. Applied also to the genes. DOUBLES, double breaks in chromatids that occur during diakinesis, and the resulting geno- types, and the individuals possessing them. DYAD, each of the two chromatids formed from a single chromosome during meiosis. E ECLOSION, emergence of adult insect from the pupal case. 183 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD ECTOPARASITE, a parasitic animal infesting the outside of its host as opposed to endopar- asite, which lives inside the host. FACET, external face of an insect ommatidium. FEMUR, the third part of the insect leg, fol- lowing the coxa and trochanter, usually the largest part. FIRST RADIAL CELL, region of the insect wing enclosed by radius veins one and three. FLAGELLUM, the terminal part of an insect an- tenna. FOLLICLE CELLS, cells surrounding the develop- ing insect egg which are responsible for the formation of the chorion. G GAMETIC RATIO, the ratio of all types of eggs produced by a heterozygous female, ex- pressed phenotypically in the male off- spring of Habrobracon. GONAPOPHYSES, the elements that constitute the insect ovipositor. GYNANDER, an individual, mosaic for sex, GYNANDROID, an entirely haploid individual which, however, exhibits characteristics of both sexes. GYNOGENESIS, development from an unfertilized egg. 184 GLOSSARY H HAPLOID, referring to the reduced or single number of chromosomes, and to the struc- tures and individuals that bear that num- ber, such as eggs, sperms, and, in Habro- bracon, azygous males. HETEROSYNGAMY, union of dissimilar gametes, or gametes with unlike genotypes. HOMEOSYNGAMY, union of similar gametes, or gam- etes with like genotypes. HOST CATERPILLAR, in Habrobraconology , the larva of Ephestia kuehniella. HYPERPLOID MALES, males bearing more than the normal haploid set of chromosomes. HYPODERMAL CELLS, cells of the living layer of the insect body wall. HYPOPLOID FEMALES, females bearing fewer than the normal diploid set of chromosomes. IMMATERNATE, originating from a sperm and an egg whose nucleus has been destroyed. IMPATERNATE, originating without involvement of male parent or sperm, as the normal males of Habrobracon. INTERFERENCE, the inhibitory effect of one crossover upon the possibility of another. INTERSEXUALITY, a condition in which individu- als show characters intermediate between those of two sexes. 185 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD LINKAGE, the tendency of certain characters to remain together in heredity, since the genes responsible for their expression are located on the same chromosome. LOCUS, the position of a given gene or any one of its alleles upon a chromosome. LUG, either end of a chromosome. M MELANINS, black pigments. MESOPHRAGMA, the transverse partition between the prothorax and the mesothorax in insects. MESOPLEURON, the mid-region of an insect pleuron (see PLEURA) . MESOSCUTELLUM, the mid-region of an insect scu- tellum (see SCUTELLUM) . MESOSTERNUM, the mid-region of an insect sternum (see STERNUM) . METAMORPHOSIS, the series of marked changes through which an individual passes from the egg stage to the adult stage. In in- sects, complete metamorphosis includes egg, larva, pupa, and adult stages. METATARSUS, the first segment of the insect foot. MICROCHAETAE, minute bristles on an insect body . Those on the wings are of particular use in Habrobraconology . MIMICS, individuals assuming the appearance of individuals v/ith dissimilar genotypes , such as ivory and white eyes in Habrobracon. 186 GLOSSARY MOSAIC, an individual whose body indicates the possession of more than one genotype by the appearance of varying phenotypes in different regions; for example, in Habro- bracon, individuals with one white and one black eye occur. MUTANT, an individual possessing or exhibiting the effect of a changed gene. MUTATION, a sudden change in a gene resulting in a new hereditary variation. MULTIPLE RECESSIVE, the genotype or phenotype of an individual possessing the recessive alleles of two or more different pairs of genes; or the individual itself. N NURSE CELLS, cells that alternate in masses with the eggs in the ovarioles of Habro- bracon and other insects. OCELLUS, a single eye of an insect borne sepa- rately from others. OMMATIDIUM, a single eye of the compound eye of an insect. OOGONIUM, a descendant of a primordial repro- ductive cell which will develop into an oocyte. OVARIOLE, an egg tube in an insect. PARTHENOGENESIS, the development of an Individ ual from an unfertilized egg. 187 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD PATROCLINOUS, having certain characteristics derived from the father, as diploid males in Habrobracon. PEDICEL, the second segment of an insect antenna. PLEURA, the lateral divisions of an insect segment. PRAESGUTUM, the anterior chitinized line of an insect notum, or prothoracic sclerite (see SCLERITE) . PRIMARY WINGS, the anterior wings of an insect. R RADIUS VEIN, the vein that forms the distal costal margin in Habrobracon primary wings. RADIUS VEINS, Rl, R3, and R4, are branches of the radius vein. RECOMBINATION, a new combination of linked char- acters in an individual resulting from crossing-over in one or both of the par- ents; also the individual possessing the new combination. SCAPE, the first segment of an insect antenna. SCLERITE, any definite area of chitinization in the cuticle of an insect. SCUTELLUM, the posterior chitinized line of an insect notum or prothoracic sclerite (see SCLERITE) . SCUTUM, the central chitinized line of an in- sect notum or prothoracic sclerite (see SCLERITE). SECONDARY WINGS, the posterior wings of an insect. 188 GLOSSARY SEN3ILLAE, the sense organs of touch in insects, particularly hairs and cones. SEX-MOSAIC, an individual that is part one sex and part . the other. Tn Habrobracon, sex mosaics are part haploid and part diploid. SIBS, related individuals , particularly brothers and sisters. SINGLES, a term applied to genotypes resulting from one crossover, and to the individuals possessing them. SOMATIC OVERLAPPING, the intergrading of one phenotype into another so that neither are clearly distinguishable. STEMMATICUM, the triangular patch between the . simple eyes or ocelli of an insect. STERNITE, an area of chitinization in an insect sternum. STERNUM, the ventral division of an insect segment. STIGMA, a thickened, opaque spot on the anteri- or margin of the primary v/ing of an insect. STING, a pointed, hollow, needle-like organ at the posterior end of the abdomen in cer- tain female insects; for example, bees and wasps . STOCK, organisms reared and cared for for ex- perimental purposes. STRAIGHTS, gametes containing genes in the same combinations that were present in the par- ents; the opposite of crossovers. SYNAPSIS, the conjugation of pairs of homologous chromosomes in meiosis. SYNCYTIUM, a mass of protoplasm in which the nuclei are free and not separated from each other by cell membranes or walls, SYNCAMY, the union of gametes o 189 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD TARSUS, the foot of an insect. TERGITE, an area of chitinization in a tergum (see TERGUM) . TERGUM, the dorsal division of an insect segment. THELYTOKY, production of impaternate females. TIBIA, the fourth part of' an insect leg, fol- lowing the femur. TYPE, the kind of a species most commonly found in nature in a given locality; also ap- plied to the genes whose effects are ex- hibited by such an individual. TYPE SPECIMEN, a specimen considered to possess the characters of a group. In order to be classified as belonging to that group, all other specimens must exhibit the same characters as the type specimen. U UTERUS, the lower expanded end of an insect egg-tube. W V/ILD-TYPE, the kind of species most commonly found in nature in a given locality. 190 INDEX INDEX Abdomen, 9 Aciform, ac, 58, (105) , (144) Adults, (7), (8) and X-radiation, 136, 137 Aeroplane, ae, 49 Alleles, multiple, 23, 68 Anaesthesia, 19 Anaphase I, 133 ANDERSON, 106, 108, 128 Androgenetic, 92 Antennae, 2, 6, (61) length of, 80, 81 Antennal mutations , 54- 62, (61) Antennapedia, ap, 57 Ants, 91 Apis, 1 Arolium, 58 Arrhenotoky, 75 ASHMEAD, 1, 2, 3 Attenuated, at, 59, (105), 109 Axilla, 49 Azygotic ratios, 103 Azygous males, 76 B Bad eggs, 135 Bar-eyes, be, 33 Beaded, b, 62 Bee, Honey, 1, 91 sex -de termination, 91, 92 Behavior, and muta- tion, 140 BENKERT, 104, 106, 108, 109, 113, (143) Bent-wings, bv/, 44 Bilateral symmetry, 6 Biparental offspring, 76 BISHOP, 136 Black, bl, 34, (105), 107, 108, (125), (144) 193 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD Body, color, 6 color mutations, 33- 37 length, 80 shape, 37 size, 37 BOSTIAN, 38, 47, 96, 100, 137, 138 Bracon brevicornis, 1, 3 Bracon dorsator, 1, 2 Bracon hebetor, 1, 2 Bracon honestor, 1 Bracon juglandis, 1, 2 Braconidae, 1 BRIDGES, 92, 114 Broad, br, 38 Broken, bk, 49, (53), (105), 109, (144) Bulge, bu, 33 BURTON, 23 Calcanea, 58 Cantaloup, c, 25, (105), 106, 107, 108, 124, (144) Cantaloup-ivory combi- nation, 85, (86), 87 Carrot, wh^, 26, 124, 126 mimicry of, 29 CARSON, 109 Cell size in diploid males, 79, 80, 81 Cellophane disc, 18 Chalcis-f lies, 91 Cheese, ch, 37 Chiasmata, 132, 133 Chorion, 10 Chromosome, changes, 129 number, 77, (78) , 139 sex, 104, (105), 107 Chromosomes, (78), 103 CLARK, 107, 108 Clipped, cd, 51, (53), 54 Close-crossing, 19, 94, 100 Club, cl, 65 Coalescent, co, 62, (105), (144) Coincidence, 108, 119, 120 Color pattern, 6 Complementary factor hypothesis, 93 Compound eye, 6, 9 male mosaic, (86) Confluent, cf, 46 Constricted, cs, 65 Corn meal, 20 Costa, 9 Coupling tests. 111, 113, 117 Crepe wings, cw, 48 Crescent, cr, 33 194 INDEX Crossing-over, 104, (105), (144) Crossover percentage, (105), 109, 110, (144) formulas, 113, 117, 118 Crumpled, cp, 51, (52) Culture, 12-21 CUSHMAN, 1, 2, 3 Cut, ct, 45, (52), (105), (144) Cuticle, 9 Cytological studies, 77 Dahlia, o^, 24 mimicry of, 29, 30 Data, experimental, 69- 73 linkage, (72) master sheet, (71) , (72), 73 recording of, 69-73 Defective, d, 39 Deficiency, de, 54 Deletion, 133, 134 Determination of sex, 91-102 Development of wasp, 14-15 Diakinesis and X-radi^ ation, 130 Differential, matura- tion, 94 viability, 114, 116, 117 Diploid males, 4, 9, 75, 77, 79 cell size, 79, 80 in orange series, 23 microchaetae, 79 sex linkage. 111 sex determination, 92, 93 temperature, 128 Dominant lethal ef- fects, 130, 134 Dominant lethals and X-radiation, (145) DOTEN, 2 Double recessives, 30 Droopy, dr, 48, (105) Drosophila, 1, 19, 100 DUNNING, 30, 31 Dwindling, dw, 59 DZIERZON, 91 E Eclosion, 17, 21 Eggs, bad, 135 Elongate, eg, 51, 52, (105), (144) Environmental effects, 121-138, 140 Enzymes, 123, 124, (125) 19S THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD Ephestia kuehniella, 2, 3, 4, 12, 74 culture of, 20, 21 Ephestia cautella, 4 Extended, ex, 50, (144) Extended-head, eh, 38 Extended-wings, ew, 48 Extreme small, k"" , 30, (32) Eye, color, 23-30 mosaic, 85, (86) mutations, 22-33 shape, 22, 30-33 size, 30-33, (32) size in sexes, 80 Eyeless, el, 31, (32)i, (105), (144) Facet, 9 FAHRINGER, 4 Fecundity, 126 Female, 6, (7), 9-11, 17, 18 aging of, 119 Fl from mimics, 29 Fl formulas, 69, 70 data sheets, (71), (72) hypoploid, 135 Femora, 62 FISHER, 113 Flagellum, 6 Flare, fl, 44, 45 Follicle cells, 10 Foot mutations, 62-66, (64) Footless, fo, (64) , 65, (105), (144) Formulas, genetic, 69, 70 Fused, fu, 55, (61), (64), (105), (144) and sex, 96, 98, '.(99), 101 Gabled, gb, 44 Galleria mellonella, 4 Gametic ratio, 91, 117-118 symbols for, 70 Gene mutations, 22-68 in sex, 68 Genie balance, 92 and lethality, 135 Genitalia, 90 female, 9 male, 10 male-female m.osaic, 87, (88) Glass, gl, 28, 103, (105), (144) Glaze, gz, 28, (144) Gonads, 9 Gonapophyses, 9 GOIDANICH, 4 GRANDI, 4 196 INDEX G.ISB, 59, 87, 94, 127, 137 GROSCH, 9, 80 Gynander, 5, 81, 82, 127 appearance, 87, (89) origin of, 84 Gynandroid, 87 Gynandroidism, 87 Gynoid, gy, 59, (105), (144) H Habrobracon benefici- entior, 1, 2 Habrobracon brevicor- nis, 1, 2, 3 Habrobracon, female, (7) male, (8) Hadrobracon, 2, 3 Hadrobracon brevicor- nis , 2 HAGER, 106 Haploid males, 4, 5, 9, 10, 74, 75 phenotypes of, 22 sex -determination of, 92 Hatchability, 129 and X-radiation, 129, 132, (145) HELSEL, 104, 109, 115 Honey, ho, 35, (105), 107, (125), (144) HORN, 101 HOWARD, 1 Humidity effect, 127 Hymenoptera, 68, 140 sex-determination of, 92, 101 Hymenopteran, 1 Ichneumon flies, 91 Ichneumonoidea, 1 Impaternate females, 5, 75 v/asps, 75 INABA, 3, 101 Incubation, 12 Indented, in, 45 Independent assort- ment, 112 Interaction of fac- tors, 112 Interference, 104, 119, 120 Intersexuality , 76, 82-84 Inviable pupae, ip, 67 Ivory, oi, 23, 24 mimicry, 29 Ivory-white combina- tion, 85, (86) 87 JOHNSON, 1 197 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD K Kidney, k, 30, (32), (105), 108, (144) KIRBY, 1 KLOTZ, 107 KUHN, 122 LANKESTER, 75 Larvae, wasp, 15, 16 and X-radiation, 138, (145) Leg mutations, 62-66, (64) Leglike, Ig, 58 Lemon, le, 35, 36, (105), 107, 124, (125, (144) Length, body, 80 wing, 80 Lethal, L, 68 Lethal naked pupae, np, 67 Lethality, 115 Lethals, 66, 115 dominant, 130, 134, 135 effect of, 19 recessive, 135 Light ocelli, c'-^, 24 lo, 29 Linkage, 103-120 absence of, 110 data, (71), 72) estimation of, 113 groups, 104-110, (105), (144) Linkage map, ( 105) , (144)' sex region of map, 106 tests, 103 Locus, orange series, 24 Long, 1, 56, (61), (105), 107, (144) Lumpy, Ip, 66 MAERCKS, 14, 126 Male, (8), 13, 17, 18 azygous, 44, 75 hyperploid, 135 Maroon, ma, 25, ( 105) , 109, 124, 126, (144) mimicry of, 29 MARSHALL, 1 Masking effects, 112 Master sheet for data, (72), 73 MATHER, 113 Maturation, 10, 11 division, 79 effect on sex, 75 Maximum Likelihood, 113, 119 MAXWELL, 135 198 INDEX Meiosis, 10 MENDEL, 91 Mesophragma, 6 Mesopleura, 42 Mesoscutellum, 6 Mesosternura, 37 Metamorphosis, 4 Metaphase I, 129-133 and X-radiation, (145) Metatarsus, 63 Microbracon brevicor- nis, 2, 3 Microbracon hebetor, 1, 2, 3 Microbracon pectino- phorae, 3 Microchaetae, 9, 79, 80 Microscopic examina- tion of preserved wasps, 20 of progeny, 19 for sex, 18 Mimics, 29, 30 Miniature, m, 37, 103 Minnesota yellow, Ely, 54-55 Mosaic, 5, (39) eye, 85, (36) sex, 81-84, (88) and temperature, 127 Moth, bee, 4 fig, 4 flour, 4 meal, 4 Mediterranean flour, 2, 3, 4 Mottled, mo, 27 MUESEBECK, 1, 2, 4 MULLER, 117,- US Multiple alleles, 96, 97 orange series, 23, 24 sex series, 63, 95- 102 Multiple, recessives, 29 sex factors, 95 Mutants, development of, 139-140 Mutations, antennal, 22, 54-62, (61) appearance in males, 22 and behavior, 139 body, 22, 34-39 body color, 34-37 eyes, 22-34, (32) feet, 22, 62-66, (64) gene, 22-68 legs, 22, 63-66, (64) 199 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD Mutations, lethality, 22, 67-68 and linkage, 103-120, (105), (144) and sex, 22, 68, 104, (105), 106-108, (144) and temperature, 128 v/ings, 22, 39-54, (52), (53) N Orange, o, 4, 23, (105), 108, (144) locus, 25, 108 mimicry of, 29 series, 23-25 sex-determination, 92 Outcrossing, 19, 96 Ovariole, 9, 10, (131 Oviposition, 10, 13 optimum conditions for, 21 NACHTSHEIM, 92 Naked pupae, 67 Narrow, n, 41, 42 NEAVE, 3 Neutron effects, 136 Non-hatchability , 136 Notch, no, 43 Nujol, 14 Nurse cells, 9, 10 0 Ocellus, 6, 24 Ommatidia, 9 Oogonia, 10 Optimum, relative humidity, 14, 15 temperature, 14, 15 Parasitism, 4 Parthenogenesis, 4, 5, 74, 139 Pebbled, pb, 33 Pedicel, 6 Pellucid, pi, 27, (105), 109, 115, (144) Pettijohn's Breakfast Food, 20 Physiological effect of X-radiation, 129, 132 Pigmentation, 6, 37, 122-126, (125) development of, 16 and temperature, 121, 124, (125) Pinched, pd , 50 Fink, pk, 27 Plodia interp'.inctella, 200 INDEX Pointed, p, 47 Port, pt, 29 Praescutum, 6 Preserving fluid, 20 Primary wing, (40) Probability table, (143) Prophase I, 129, 130, (131), 132 and X-radiation, (145) Pupa, 16-18 Pupae, inviable, ip, 67 lethal naked, np, 67 wasp, 16-18 Pyrausta nubilalis, 2 Reduced, r, 41, 103, 104, 106, (105), (144) Reduplicated, Re, 50 REED, 4 Related stocks, 19 Relative humidity, 14, 15, 126, 127 Repulsion tests. 111, 112, 117 Reverse crosses, 118 RILEY, 1 RISMAN, 79 Rough, ro, 51, (52), (105), (144) R Radial cell, (40) Radio waves, 136 Radius veins, 9, (40) Ratio, azygotic, 103 gametic, 103, 118 sex, 18, 96 Reactions, sex, 11, 81 Recess ives, double, 29 multiple, 29 Reciprocal crosses, 115, 117 Recombination, 104, 106, 113, 117-119 Red, rd, 27, (105), 108-109, (144) SAY, 1, 2, 3 Saw-flies, 91 Scape, 6 SCHAEFFER, 68, 115 SCHLOTTKE, 36, 121 Sclerite, 9 Sclerotization, 16, 60, 83 Scutum, 6 Secondary v/ing, 46 Semllethals, 114, 115, 116 effect of, 19 Serailong, si, 56, 57, (61), (105), (144) 201 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD Sex, 6, (7), (8) . alleles, 95-102, (99) antennal length, 6, 80, 81 characters, 6-11, 79, 81 chromosome, 104, (105), 106-107, (144) chromosome segment, 68 conditions, 74-90 determination, 91- 102, (99), 140 distinction, 17 factor, X, 101, (105), 106, (144) factors, 93-102 and fused, 96 gene, 68, 106 linkage, (105), 106, (144) microscopic examina- tion for, 17-18 mosaic, 81, 87, (88) multiple alleles, 63 mutation, 101 ratio, 18, 97, 127, 128 reactions, 11, 81, 82, 84 temperature, 127, 128 X-radiation, 137 Shells, 14 Shiny, sy, 28 Shot-veins, sv, 43, 44, (105), 109, 128, (144) with v/hite, 26 Silver, si, 34, 37 SIMMONS, 4 Small, sm, 31 Small-eyes, k^ , 30, 31, (32) Small-head, sh, 38 Small-wings, sw, 48, (52), (105), (144) SNELL, 95 Somatic overlapping, 114, 116, 118 Sooty, s, 37 Speckled, Sk, 26, 103 linkage of, 104, (105), 106, (144) with white, 26 SPEICHER, 3, 9, 10, 31, 59, 79, 87, 94, 101 Sperm and X-radiation, 136, (145) Spermatogonia, 11 Snread, sp, 42, 43 Square Root of Product Method, 117-119 STANCATI, 136 Statistical signifi- cance of linkage, 116 Stemmaticum, 34 Sterile ova, 14 Short, 41 202 INDEX Sterility, male, 77 production of, 134 and X-radiation, 138, (145). Sternite, 9 STEVENS, 119 Stigma, (40), 45 Sting, 9, 17 Stinging, of caterpil lars, 12 of eggs, 14 Strap, sr, 45 Stubby, sb, 60, (61), (105), 106, 115, (144) Testes, 10 Thelytoky, 74 Tibia, 63 Tinted, tn, 27, (144) T0RVIK-GRE3, 93 Transparent, tp, 29, (144) Triploid females, 76 Truncate, tr, 46 Truncated, td, 47, 1C9 Tv/isted, tv/, 63 Type specimen, 2 Type, symbol for, 69 U Stubby-abnormal, sb^-, 60, (61) Stumpy, st, 62, 109 Symbols, 69 Syngamy, 92 Unexpanded, un, 46 UoS. National Museum, 2 Uterus, 10 V Tapering, ta, 57, (61) , (144) Temperature, 4, 6, 14, 15, 23 effects of, 121, 122, 126 optimum, 15 and mutation, 128 and pigmentation, 129 Tergite, 9 Tester stocks for sex, 97 Vagina, 9 Variegated, 26,(26>,.44 Veinless, vl, 51, (52), (105), 107 (144) and sex. 98, (99) Veins, radius, 9, (40), 43 shot, 26, 43, (105), 109, (144) Vestigial, v, 42 Viability, 111 differences, 112 VIERICK, 1, 2 Vipiinae, 1 203 THE GENETICS OF HABROBRACON JUGLANDIS ASHMEAD Virgin females, 17, 18 offspring of, 22 W WALKER, 4 WASHBURN, 3 Wasps, 91 WATAMA35, 3, 4 Wavy, wa, 42 WESMAEL, 2, 3 Wheat, rolled, 20 White, wh, 25, 26, (105), 109, 126, (144) ivory-white combina- tion, 85, (86) mimicry of, 29 WHITING, A. R., 14, 20, 23, 30, 34, 36, 44, 59, 82, 85, 93, 107, 123, 124, 129, (131), 132, 138 WHITING, P. W., 1, 3, 4, 24, 30, 35, 37-39, 44, 47, 54-57, 59, 60, 62, 63, 65-b9, 75, 82, 91-97, (99), 100, 104, 106, 108- 110, 113, 122, 127, 134-137, (143) Wild-type, stock num- ber one, 3, 4 stock number eleven, 3 stock number twenty- five, 3 stock num/oer thirty- two , 3 stock number thirty- three, 3 symbol for, 69 wing, (40) Wing, mutations, 39-54, (52), (53) primary, (40) , 45, 48 secondary, 45, 48 wild-type, (40) Wings, 79 length of, 80 Woozy, wz. , 66 Wrinkled, w, 39, 41 WRIGHT, 123 X-radiation, 23 X-radiation, and adults, (145) 204 INDEX -radiation, and ana- Y phase I, 133 chiasmata, 132, 133 Yellc/, Y, 55 chromosomes,, 130 Yellow, Minnesota, My, chromatids, 133 54-55 division II, 133 effects, 133, 134 Z eggs, 130, 132, (145) hatchability, 129, Zellor, 2, 3 132, (145) larvae, 138, (145) lethality, 130, (145) male sterility, 137 metaphase I, 130- 134, (145) mutation, 138, (145) prophase I, 132, (145) responses, (145) sex ratio, 137 spermatozoa, 130, (145) fsterility, 137 summary, (145) synapsis, 130 205