836 X\NNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA [Vol. 60, No. 4

tory, natural enemies and the poisoned bait spray as a of the Diptera of America North of Mexico. USDA method of control of the imported onion (Phorbia Agr. Handbook 276. 1696 p. scpctorum Meade) with notes on other onion pests. Vos de Wilde, B. 1935. Contribution a l'etude des lar- J. Econ. Entomol. 8: 342-50. ves de Dipteres Cyclorraphes, plus specialament des Steyskal, G. C. 1947. The genus Diacrita Gerstaecker larvae d'Anthomyides. (Reference in Hennig 1939, (Diptera, Otitidae). Bull. Brooklyn Entomol. Soc. p. 11.) 41: 149-54. Wahlberg, P. F. 1839. Bidrag till Svenska dipternas 1951. The dipterous fauna of tree trunks. Papers Kannedom. Kungl. Vetensk.-Akad. Handl. 1838: Mich. Acad. Sci., Arts, Letters (1949). 35: 121-34. 1-23. 1961. The genera of Platystomatidae and Otitidae Weiss, A. 1912. Sur un diptere du genre Chrysoviyaa known to occur in America north of Mexico (Diptera, nuisable a l'etat de larve a la cultur du dattier dans Acalyptratae). Ann. Entomol. Soc. Amer. 54: 401-10. l'Afrique de Nord. Bull. Soc. Hist. Nat. Afrique 1962. The American species of the genera Melieria Nord. 4: 68-69. and Pseudotcphritis (Diptera: Otitidae). Papers Weiss, H. B., and B. West. 1920. Fungus and Mich. Acad. Sci., Arts, Letters (1961) 47: 247-62. their hosts. Proc. Biol. Soc. Wash. 33: 1-19. 1963. The genus Notogramma Loew (Diptera Acalyp- Wolcott, G. N. 1921. The minor sugar-cane insects of tratae, Otitidae). Proc. Entomol. Soc. Wash. 65: Porto Rico. J. Dep. Agr. Porto Rico. 5: 46 p. 195-200. Zetterstedt, J. W. 1847. Diptera Scandinaviae. 6: 2163 1965. Otitidae p. 642-54. In A. Stone et al. A Catalog 2580.

Some Irradiation Studies and Related Biological Data for variipennis (Diptera: )1

ROBERT HENRY JONES2 Entomology Research Division, Agricultural Research Service, USDA, Kerrville, Texas ABSTRACT Colony specimens of the biting midge Culicoides vari- creased, but even 30,000 R failed to sterilize completely. ipennis (Coquillett) were treated with gamma radiation Treated females did not recover and rarely laid more than as larvae, pupae, and newly emerged adults at 5000, 10,000, 1 egg batch, though those batches that were laid often had and 15,000 R, and as pupae at 20,000 and 30,000 R. The rather low percentages of sterility. With treated females, criterion for the sterility of males was the hatch of eggs the number of eggs per egg batch decreased as the radi- deposited by untreated females with which they mated. ation dose increased, until at 30,000 R no eggs were laid. Males treated at doses above 5000 R were, with few ex- Miscellaneous biological information for colony C. rari- ceptions, 95%—100% sterile in first matings, but males ipennis, principally relating to reproductive performance, treated at all levels tended to recover fertility in later was obtained in testing the treated specimens and the matings. Recovery decreased as the radiation dose in- accompanying controls.

The biting midge Culicoides variipennis (Coquil- worm fly, Cochliomyia hominivorax (Coquerel), lett) is a common and sometimes serious pest of from both the Southeastern and Southwestern United livestock. In addition, Foster et al. (1963) indicated States, and is being used with several other that the species is a vector of bluetongue disease of species (Smith 1963). in the United States. Nevertheless, C. vari- In the winter of 1961-62, I had the opportunity to ipennis, like most Culicoides, has not been very thor- study the effects of gamma radiation, the sterilizing oughly studied. Our knowledge of its biology is agent used for the screw-worm fly, on C. variipennis. fragmentary, and few practical methods have been At that time, a colony (Jones 1966) was being main- proposed for large-scale control if it should become tained at Kerrville, Texas, at the same time a cobalt80 necessary. The larval habitats of the species are source was available there (Jefferson 1960). It was diverse (Jones 1961), and although the large breed- thus possible to investigate the general patterns of ing sites that sometimes occur in very favorable areas radiosensitivity for the species, and at the same time can be located and treated with insecticides, typical to gather some miscellaneous biological information. sites are small and occur in such obscure places that it would be virtually impossible to find and treat MATERIALS AND METHODS them all. Under these circumstances, 1 method of control to consider would be the sterile-male tech- In an experimental program designed to test effects nique, in which artificially sterilized individuals are on reproduction, certain biological characteristics of released throughout the mating season in numbers C. variipennis tend to make handling procedures large enough to overwhelm the native population, rather involved. The life cycle is short—at the time thereby greatly reducing the number of offspring of this work, with colony specimens (C\ v. sonorcn- available for each succeeding generation. This tech- sis Wirth and Jones, isolated December 1957) the nique has been successful in eradicating the screw- egg developed in about 2 clays, the larva in 16 days, and the pupa in 2^2 days. Because of the short pupal stage, test pupae had to be separated on the 1 Accepted for publication October 3, 1966. 3 Present address: Livestock Insects Investigations; Entomol- basis of quite small differences in age so that there ogy Research Division, ARS, USDA; Building 45, Denver Federal Center; Denver, Colo. 80225. would be enough groups for comparison. Sexual July 1967] JONES : IRRADIATION OF Culicoides variipennis 837 development in the species occurs very early, and during a single period, the were separated at 4- mating is commonplace in cages of colony adults 12- hr intervals as usual, but were later combined with 24 hr old. (Before setting up this study it was deter- flies that had emerged slightly earlier or later). For mined that adult males as young as 8 hr old can suc- adults, age at treatment was designated by the num- cessfully inseminate females, and that females as ber of hours after emergence; for immature forms, young as 4 hr old can be inseminated.) In addition, by the number of hours between irradiation and emer- both sexes can mate repeatedly and remate immedi- gence—thus, pupae in a 12- to 8-hr emergence group ately. However, even though mating frequently oc- were more nearly mature at irradiation than those in curs in cages that contain numerous adults, it is not a 28- to 24-hr group. certain, or even likely, that a standard percentage of The day after emergence, groups of females were females in such cages will be inseminated, and to given their first blood meal (on the belly of a rabbit) guarantee mating it is necessary to isolate a pair and and those that engorged fully were then used for induce copulation. For these reasons, the sexes had mating. A female and a male were drawn into a IX to be separated within about 4 hr after emergence to 3-in. aspirator vial and gently tapped together to in- prevent undesired matings, the adults had to be duce copulation. The time in copula was recorded mated a pair at a time, and each adult had to be for each mating, and the pair was separated immedi- uniquely designated and handled as an individual. In ately afterward to prevent remating. After mating, ('. t'uriipcmiis the female is nonautogenous, and ap- each individual was caged separately in a standard parently without exception requires a blood meal for holding cage made of a J^-pt ice cream carton with a each batch of eggs deposited. Although she can use cloth lid. A homeopathic vial was inserted through a sperm from more than 1 mating, she can also store hole in the side to provide liquid through a cotton enough sperm from a single mating to fertilize sev- wick. Cages for males had plain cardboard bottoms; eral egg batches. Thus, to determine the permanence cages for females had holes in the bottom fitted with of any sterility induced, test females had to be given small plastic dishes containing moist cotton covered a series of blood meals to permit recovery of a series with circles of paper toweling for oviposition (these of egg batches and test males had to be mated over a same cages served as emergence cages when pupae period of time with a series of untreated females. were placed on moist cotton in the plastic dishes). The first tests were conducted at 5000 and 10,000 All flies received an alternating diet of 5% honey solu- R. When these doses did not completely sterilize, tion for 1 day and distilled water for 2 days, but the further tests were conducted at 15,000, 20,000, and females began their first day with water and the 30,000 R. Just before irradiation, each test group to males with honey. For later blood feedings, a female be used was divided in half, and 1 of the halves was was transferred to a feeding cage and then returned kept untreated to serve as normal controls; these to her holding cage. In feeding procedures, the fe- normal specimens were handled throughout the test male was always observed for engorgement, since in exactly the same way as the treated ones. Only mere presence in a feeding cage did not automatically flies from the same test and age group were mated mean that she would take a blood meal. with one another, and each mating series began with Females almost invariably laid their eggs during all 4 of the possible combinations between the treated the night, attaching them to the circles of paper (T) flies and their corresponding normal (N) con- toweling. (Egg dishes were always kept in the cages trols: T$ X T9, T<5 X N9, N$ X T9, and N4 to keep the females from laying eggs on the cotton X N 9. These series were replicated as many times wick on the water vial.) The paper circles with the as the number of flies or the available facilities per- eggs were collected the next morning, kept moist at mitted. However, variations in emergence times, the room temperature, and examined daily under the disinclination of the females to blood feed or the microscope for hatching, which usually took place males to mate on certain days, or the accumulation of between 2 and 5 days after oviposition and was al- small events such as the escape or death of individual ways completed by 7 days. Eggs that had hatched flies caused many test groups to end up incomplete or were counted and discarded. During the first experi- without replication. ments, eggs that looked as if they might hatch, judged Adults were irradiated in air in standard holding by the eyespots of the developed larvae inside, were cages (2 in. high, cardboard with cloth lids) ; larvae kept for several weeks, in case hatching might be and pupae were irradiated while barely covered with merely delayed, but none hatched. Thereafter, all water in open plexiglass dishes. Immediately after eggs remaining on a paper circle at 7 or 8 days after treatment, pupae were placed in holding cages for oviposition were discarded as sterile. Discarded eggs emergence. Test larvae were selected on the basis of were classified as very light (teneral), moderate, or uniform size; after treatment they were put back in dark brown, and with or without visible eyespots. larval medium, and all pupae that developed were Teneral or varicolored eggs were always sterile. collected every 48 hr and placed for emergence. Generally, moderately light brown eggs also were Throughout the study each emergence cage was sterile, but occasionally even a whole batch of them checked at about 4-hr intervals. If flies had emerged would hatch. Although some sterile eggs were split during the preceding 4 hr they were separated by sex down the side, eggs that had hatched were clearly into cages labeled with their test group and time of recognizable by the Y-shaped slit forming a cap at emergence (sometimes if only a few flies emerged the anterior end. Because of this characteristic pat- 838 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA [Vol.60, No. 4 Table 1.—Comparison of number of eggs laid by nor- batches. Normal females usually laid eggs 3-6 days mal females when mated with normal vs. treated males. after a blood meal. The interval between blood feed- ing and oviposition was never less than 3 days, but Ni X N 9 matings J- 0 /\ N 9 matings was occasionally quite a bit longer—1 lasted 16 days. Total Number Total Number Sometimes after a blood meal a female did not lay egg of eggs egg of eggs eggs at all, but would oviposit when fed a second Egg batches • batches time—evidently the original blood meal did not batches counted Avg Range counted Avg Range "take." This may also explain why some normal fe- 1st 105 123 11-243 188 121 1-239 males that had engorged only once failed to oviposit. 2nd 52 129 30-210 98 124 5-232 (No record was kept of possible "aborted" blood 3rd 29 105 13-165 52 107 2-201 meals, but blood spots were occasionally noted on the 4th 15 72 49-127 25 99 10-174 egging dish within a day or two after a blood meal. 5th 6 60 15-90 15 85 57-137 6th 1 46 10 63 13-110 These spots may have been related to a failure to 7th - - 4 47 38-62 oviposit or may merely have been residues of unused blood.) In general, the number of eggs laid by all females tern, no particular effort was made to confirm hatch- from N<5 X N9 matings decreased with successive ing by finding the larva. egg batches. This pattern of gradual reduction oc- Females sometimes laid eggs from the same blood curred also with females from T$ X N2 matings, meal a day or two apart, and when this occurred the which presumably received largely sterile sperm, and groups of eggs were handled and recorded separately it did not change even in the few instances in which but the figures were later totaled as belonging to 1 these females were remated with normal males and egg batch. One day (or rarely, 2 or 3 days) after a subsequently laid egg batches containing larger per- female had finished laying an egg batch she was centages of viable eggs. In fact, in number of eggs given another blood meal. Sometimes a female that per egg batch, T$ X N2 matings seemed in all re- had laid several egg batches with high percentages spects very similar to N# X N2 matings. Compari- of sterility, or that would not lay eggs after 1 or more sons of all females in these 2 categories are given in blood meals, was remated with a normal male to see Table 1. whether the pattern would change. All males used in such rematings were 1 day old and previously un- Table 2.—Results of a separate experiment with a sin- mated. Many test males, particularly those that were gle, untreated colony male, mated on 3 successive days treated, after their first mating with test females were with a series of colony females. periodically remated with normal, 1-day-old, newly engorged virgin females. Rematings were usually Time Time (sec) in ------Eggs laid made on a daily or alternate-day schedule and con- copula Be- For tinued until the male's death. The females used in Female (min: tween $ to Percent these additional matings were then handled exactly no. sec) mate" No. sterile as the females from first matings, including being given additional blood meals to obtain additional egg First day batches. 1 2:05 _ 20 179 2 2 2:55 50 15 0 (D-3)c 3 6:05 60 10 91 1 RESULTS 4 8:20 65 20 94 7 These rather complex methods of handling the 5 21:05 165 85 0 (D-17) flies provided several different categories of data. 6 130:50 45 15 0 (D-18) Potentially, for a test group of 4 mating pairs, both Second day the numbers of eggs and the percentage of sterility 7 2:45 _ 325 158 0 8 5:00 100 50 37 0 could be obtained for 3 categories: all egg batches 9 9:10 105 35 70 10 produced by the original test females, treated or nor- 10 9:40 125 50 0 (D-16) mal; all egg batches produced by additional normal 11 16:10 160 70 150 99 females with which the test males were mated; and 12 18:05 95 55 147 100 13 99:05 85 25 0 (D-4) occasionally, egg batches produced by additional or 14 1:15 65 35 88 100 test females after they had been remated with addi- 15 3:20 210 20 0 (D-6) tional normal males. Because nearly half of these 16 95:40 165 25 121 100 data came from matings of normal flies (not only the 17 3:10 120 5 0 (D-6) test matings in which both male and female were nor- 18 132:45 190 25 0 (D-6) mal, but all additional matings of normal males)-, a Third day fairly good picture of some aspects of the behavior of 19 3:40 - 145 0 (D-6) 20 8:10 215 100 0 (D-12) normal flies emerged. 21 40:45 610 240 102 100 In this study, normal females lived up to 44 days. 22 164:00 95 20 109 99 The maximum number of eggs deposited in 1 egg a Time taken to remove 1 ? and introduce the next. batch was 243, and the maximum number from 1 2 b Time between introduction of female and beginning of mating. c D = died without ovipositing; numbers indicate age (days) at over her lifetime was 1143, a total from 7 egg death. July 1%7] JONES : IRRADIATION OF Culicoides variipennis 839 The sterility of eggs in successive egg hatches best be explained by assuming that the matings were from normal females mated with normal mates was for some reason partially or completely ineffective, rather variable, but generally tended to increase, espe- and not that the flies were inherently sterile. The cially about the fourth egg batch. For example, with possibility of ineffective matings must, of course, be the 15 9 from Ni X N$ matings that laid 4 or kept in mind in considering the data from the irradi- more egg batches, the average percentage of sterile ation tests. In view of it, one cannot consider a eggs in the first egg batch was 2.5%; in the second, single, unreplicated, sterile egg batch as positive 3.4% ; in the third, 7.37<>: and in the fourth, 30.8% proof of sterility. (corresponding average numbers of eggs were 118, The results of the irradiation tests are summarized 124, 106, and 72). It is not surprising that a female in Tables 3-6. Not all the possible categories of data that had been mated only once should produce in- have been included because of limitations of space creasingly sterile egg batches over a lifetime as the and because some of them, for the purpose of judging available sperm supply was depleted. A similar situ- radiation effects, were repetitious or not very indica- ation seems to have occurred with most females from tive. For one thing, since the number of eggs laid by Tit X N9 matings: most of them produced most of normal females mated with treated males apparently their viable eggs in the first few egg batches. (How- did not differ from that laid by normal females mated ever, when a female produced only a small percentage with normal males, the percentage of sterile eggs, of viable eggs, it was not uncommon for a few to rather than the number of eggs, from a T $ X N 9 occur in each egg batch.) mating was the only criterion for radiation effects on A much more obvious instance of loss of reproduc- the males. Furthermore, the sterility of additional tive capacity occurred in a related experiment in egg batches produced by the normal female without which a single untreated colony male, beginning at remating served only to corroborate the sterility of 1 day of age, was successively mated on 3 consecutive her first egg batch. For these reasons, neither the days with a series of 1-day-old, newly engorged vir- number nor the percentage of sterility of eggs in gin females. The results are shown in Table 2. Al- additional egg batches from normal females has been though nearly half the females died without laying included, but only the percentage of sterile eggs in eggs, enough egg batches were produced to show a first egg batches from successive rematings of males. clear pattern. It appears that this male's sperm sup- On the other hand, the number of eggs laid by treated ply was exhausted by the repeated matings, although females was often considerably reduced—in some some recovery took place overnight. Rather inter- cases so much so that a percentage of sterility was estingly, the apparent decrease in sperm supply was meaningless. The number of eggs in additional egg accompanied by an increasing length of time in batches from these females should logically have been copula. the indicator of continuing radiation effects, but re- In the main series of tests, in which males were re- markably few additional egg batches were produced. mated no more often than once a day and were there- For the 3 earliest groups tested, pupae and adults at fore not "stressed" as was the male just cited, the 5000 R and pupae at 10,000 R, no additional blood pattern of progressively longer mating times was meals were given, and so no additional egg batches only slightly apparent at best. The times in copula were obtained from any of the females, treated or generally ranged from 1 min 35 sec to 4 min (a very normal. However, with later groups a great effort few, all for later matings of males, were between 4 was often made to induce the treated females to lay and 5 min), and were of essentially the same order, eggs: They were given additional blood meals and whether the male was treated (Tables 3 and 4) or sometimes remated, 1 or several times; but from all normal (Tables 5 and 6). In addition, the normal later groups only 6 treated $ laid more than 1 egg males apparently did not produce fewer viable sperm batch, and the largest of these had only 17 eggs. at later matings than at the first. The data from first Most of them laid no eggs. Thus, radiation effects egg batches for as many as 5 matings of a single nor- on a treated female could be measured not only by mal male (Tables 5 and 6) seem to show the same her laying a very small number of eggs, but by a pattern in all of them: percentages of 0%-4% sterile respectable life-span in which no eggs were produced. eggs were the rule, but some egg batches had slightly For these reasons, Table 3-6 omit the additional egg to considerably higher percentages. Of the 34 first batches of treated females, except as notes, but in- egg batches from Nc? X N? first matings, about clude a notation for the life-span of the many females 76% were 0%-4% sterile, 9% were 100% sterile, and that lived more than 5 days without ovipositing. 15% fell between the 2 extremes. The 100% sterility figures are the most surprising; out of 104 matings DISCUSSION in which both male and female were normal there The data in Tables 3-6 show a few rather clear were 5 such figures, each from a different mating trends. Treated females laid reduced numbers of pair. One of the 5 9 was remated with another nor- eggs, or none. Often, treated males were nearly or mal male and then produced an egg batch that was completely sterile in first matings but recovered a only 4% sterile; likewise, 1 $ was remated and the good degree of fertility in later matings. Both sexes, resulting egg batch was 3% sterile. If these results even after treatment with quite high doses of radi- are typical, the 100% figures and other uncharacter- ation, were able to produce a few offspring. How- istically high percentages of sterility can probably ever, although the sterilizing effects naturally in- 840 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA [Vol. 60, No. 4

Table 3.—T $ X T 9 matings : Eggs in first batches when each treated male (T $ ) was mated first with a treated female (T$ ) and subsequently with normal, untreated females (N 2 ).

Sterile eggs (%) in first batches'" No. first- First Subsequent (T $ X N 9 ) Age (hr) batch eggs (T$ X matings at irra- from first T9) diation0 mating mating 2nd 3rd 4th 5th Comments

Larvae irradiated at 5000 R 261-245 146 Pupae irradiated at 5000 R 30-24 103 28 34 24 27-24 53 72 D-22 33 0 L-13 24-22 100 79 22-18 0 t 34 123 90 56 D-22 23 6th mating, D-17; 7th, 5% ; 8th, D-19 8-0 Adults irradiated at 5000 R l-2/2 77 94 t 72 t 47 6th mating, 11% 87 78 Pupae irradiated at 10,000 R 56-48 0 D-8 50-46 19 95' 95 100 71 99 24-16 45 100 Adults irradiated at 10,000 R 24 100* •Batches 2-3: 28, 3 eggs ; both 1007<> sterile 20 100 6 100 97C •Batches 1-3: 95, 105, 84 eggs; 98%, 97%;, 96% sterile. Female remated; batch 4: 76 eggs, 10% sterile D-12 Pupae irradiated at 15,000 R 53-50 24 100 50-46 0 D-29d 46-42 16 100 95 94 82* •Batches 1-7: 56, 91, 74, 63, 57, 39, 43 eggs; 82%, 65%, 72%, 82%, 95%, 1007c 95%, sterile 42-37 26 100 33-30 0 D-41* 100 •Female refused additional blood meals 30-26 60 100 100 100 100* •Batches 1-3: 73, 47, 121 eggs; all 100% sterile. Female remated; batches 4-7: 74, 70, 68, 48 eggs; 93%, 30%, 66%, 797* sterile 26-22 100 100 98* 100 61 6th mating, 16%. •Batches 2-3: 1007o sterile. Female remated; batch 4: 100% sterile 36 100 22-17 7 100* 91 •Batch 2: 15 eggs, 1007o sterile 2 100d 17-11 35 100d 97* 96 L-8 •Batches 1-3: 90, 126, 112 eggs; 977o, 957*, 100% sterile. Female remated; batch 4: 92 eggs, 5% sterile Adults irradiated at 15,000 R / X Vi- 2/ 2 0 t 100 1 100 0 D-17 98 Pupae irradiated at 20,000 R 44-40 0 D-18 t 100 98* 100 *6 additional egg batches, all 1007> sterile 40-34 0 D-26d 100 99* •1 additional egg batch, 100% sterile 27 100* 100 100 98 100 •Batch 2: 4 eggs, 1007

Sterile eggs (%) in first batches'* No. first- First Subsequent (T«J X N9) Age (hr) hatch eggs (T$ X matings at irra- from first T9) diation" mating mating 2nd 3rd 4th 5th Comments

Pupae irradiated at 30,000 R 23-18 D-7 D-18 100 6th mating, 100% ; 7th, 100% D-22 ia-13 D-13 t D-18 100 D-34'1 6th mating: batch 1 lost; batch 2, 100% sterile 13-9 D-2S 89* *Batch contained 9 eggs D-27 100 100 100 J. 6th mating, 100%; 7th, D-ll; 8th, 100%; 9th, D-30; 10th, 100% D-16 100 100 100 100 6th mating, 100% 9-4 D-21 4-1 D-36d t 100 D-21 100 100 100 100 6th mating, 100%; 7th, 100%; 8th, t; 9th, 100%; 10th, 100%; 11th, 100%; 12th, 100%

" (liven as hours between irradiation and emergence; thus, higher numbers indicate younger larvae and pupae, but older adults. b D == female died, L = female lost (including escape, accidental death, or discard), without ovipositing; age (days) at time of death or loss shown by associated numbers, t indicates that the female died or was lost when less than 6 days old and thus could not have ovi- posited. * refers to comments. c Italic figures indicate that the next 1 or more egg batches from the same female were corroborative; i.e., sterility was within a few percentage points of the first batch from the indicated mating; or, if the first batch was 100% sterile, so were all subsequent batches. d This female was given additional blood meals and/or matings, but no eggs (or no additional eggs) were laid. creased as the radiation dose increased, no clear tend- may have been a sterile female—she laid 5 sterile ency was evident for 1 stage or age group to be more eggs but died the next day. sensitive to treatment than another. Particularly At 15,000 R, groups of older larvae were included among pupae and y2- to 3-hr-old adults, which made for treatment, and of the treated specimens the only up the majority of the groups treated, data for all ones that lived to produce offspring, and that con- emergence groups were similar at any given dose, sequently appear in Tables 3-6, were far along in showing a more or less comparable reduction in the development at irradiation—nearly pupae. All the number of eggs laid by treated females and a com- younger larvae treated were clearly damaged somati- parable degree of both initial sterility and later re- cally. Those in 5 younger groups (treated at 92-71 covery by treated males. hr before emergence) emerged as weak adults; the Male recovery was one of the most striking re- females would not feed and the males would not mate. sults of the tests. Although 10,000 R were sufficient The youngest, those treated at more than 92 hr be- to produce 95%-1007o sterility for the first 2 matings fore emergence, did not reach the adult stage. Tn of males treated as pupae or young adults, even 20,000 another treatment series at 15,000 R the records are R failed to guarantee sterility over a male's lifetime, incomplete, but do show that males, except for the and viable sperm were produced by 2 $ treated at oldest group treated, would also not mate. In the 30,000 R. In the female, 30,000 R did seem to guar- younger emergence groups of this second series, flies antee sterility, since no eggs were laid, though at of both sexes were often found dead a few steps from lower doses those egg batches that were laid some- their puparia. [It was also in one of these younger times had rather low percentages of sterility. Further- groups that a gynandromorph was found, trans- more, unlike the males, treated females did not re- versely divided into male and female. Only 1 other cover. The rare occurrence of more than 1 egg batch such gynandromorph has been reported for C. vari- from any treated female, and the small size of the ipennis (Curtis 1962).] few that were laid, indicate that the irradiation did In contrast, with pupae and adults the only indica- not affect merely the first egg batches per se, but tions of possible somatic damage were with younger interfered with the reproductive system as a whole. pupae treated at 30,000 R. Those treated specimens These general patterns of sensitivity were evident that emerged as adults appeared vigorous, and their mainly in pupae and young adults; the small groups longevity was comparable to (or actually, better of larvae tested were more difficult to judge. In gen- than) that of the normal controls for the group; but eral, doses of 5000 and 10,000 R seemed to have little there was no emergence later than 33 hr after treat- sterilizing effect, though 1 thoroughly sterile male ment, as compared with 52-56 hr for pupae treated did emerge from the 7 larvae treated at 5000 R, and at other doses. 2 more from the 4 larvae treated at 10,000 R (Tables As has been pointed out, even after treatment with 3 and 4). In the 10,000-R group there was also what 30,000 R, 2 $ produced a few viable sperm. At 842 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA [Vol. 60, No. 4

Table 4.—TcJ X N9 matings: Eggs in first egg batches when each treated male (T$) was mated with normal, untreated females (N2 ).

Sterile eggs (%) in first batches'* No. first- First Subsequent (T $ X N 9 ) Age(hr) batch eggs (T $ X matings at irra- from first N 9 ) diation" mating mating 2nd 3rd 4th 5th Comments

Larvae irradiated to 5000 R 261-245 137 2 145-130 74 100e 100 100 130-119 66 2 Pupae irradiated at 5000 R 56-48 0 t 8 7 27 100 6th mating, D-8 48-40 158 94 40-32 0 D-25 30-24 87 34 123 27-24 60 149 41 109 L-17 51 29 22-18 50 L-31 100 0 L-9 150 16-8 49 194 93 32 100 0 L-23 4-0 59 78 Adults irradiated at 5000 R 129 77 D-8 L-9 t D-7 6th mating, 32% 125 83 77 t t 80 Larvae irradiated at 10,000 R 146-131 95 23 131-120 145 100 L-ll 100 0 D-23 100 Pupae irradiated at 10,000 R 56-48 69 97 100 50-46 21 100 87 82 89 100 40-32 128 100 26-22 86 99 99 89* *Batch 2: 157 eggs, 98% sterile 24-16 0 L-26 t 75 8-0 0 t t t 90 11 Adults irradiated at 10,000 R /2-2 87 97 97 98 97 100 142 98 99 100* *Batch 2: 100 eggs, 99% sterile Larvae irradiated at 15,000 R 71-67 0 D-9 67-63 43 100 56* 175 80 *Estimated figure 173 28 t 100 Pupae irradiated at 15,000 R 53-50 0 D-29d 50-46 134 96 46-42 56 96 88 42-37 92 100* *Batch 2: 73 eggs, 99% sterile 33-30 203 100 100* *Batches 1-3: 194, 129, 122 eggs; all 100% sterile. Female remated; batch 4: 62 eggs, 100% sterile 30-26 231 95 94 93 72 49 26-22 167 100 •143 99* •Batches 2-4: 160, 136, 124 eggs; 99%, 100%, 100% sterile. Female remated; batch 5: 91 eggs, 1% sterile 22-17 157 99 17-11 0 D-33d Adults irradiated at 15,000 R 134 100 100 100 100 100 6th mating, 100%; 7th, 100% 91 100 July 1%7 JONES: IRRADIATION OF Cidicoides variipennis 843

Table 4.—T<$ X N9 matings: Eggs in first egg batches when each treated male (T $ ) was mated with normal, untreated females (N9). (Continued.)

Sterile eggs (%) in first batches'* Subsequent (T $ X N $ ) \gc (hr) batch eggs (T«J X matings at irra- from first N9) — ._ diation* mating mating 2nd 3rd 4th 5th Comments

Pupae irradiated at 20,000 R 52-48 195 83 Si 77 44-40 161 100 40-34 177 98 161 100 16-11 110 98 t t 95 11-7 104 100 100 100 100 t 6th mating, 100% 33 100 98 7-3 149 100* •Batches 2-4: 148, 113, 93 eggs; all 100% sterile. Female remated; batch 5: 76 eggs, 12% sterile 109 100 Pupae irradiated at 30,000 R 33-28 194 100 100 100 100 100 134 100 J. 100 23-18 94 100 t 100 131 100 13-9 95 100 100 100 •Batch 1 lost; batch 2: 128 eggs, 99% ster- ile ; batches 3-5 : 100% sterile 59 100 100 9-4 0 L-6 4-1 109 100 t 100 112 100 1 100 6th mating, 100% ; 7th, 100%

11 (liven as hours between irradiation and emergence; thus, higher numbers indicate younger larvae and pupae, but older adults. h 1) ~ female died, L :=: female lost (including escape, accidental death, or discard), without ovipositing; age (days) at time of death or loss shown by associated numbers, t indicates that the female died or was lost when less than 6 days old and thus could not have ovi- posited. * refers to comments. •' Italic figures indicate that the next 1 or more egg batches from the same female were corroborative; i.e., sterility was within a few percentage points of the first batch from the indicated mating; or, if the first batch was 100% sterile, so were all subsequent batches. 11 This female was given additional blood meals and/or matings, but no eggs (or no additional eggs) were laid.

20,000 R, 9 of the 17 $ treated produced viable very large decreases in sterility—their first egg sperm, 3 of them in first matings. However, at this batches had all been 95%-100% sterile, but after re- dosage the percentage of viability was small, amount- mating the percentage dropped to 1%-12%, figures ing, with 1 exception, to l%-6% of the egg batch; within the normal range for first N $ X N 9 matings. furthermore, recovery of fertility was slight. At The fact that practically complete sterility was re- 15,000 and 10,000 R, the initial percentage of viabil- placed by normal fertility, and not by an intermediate ity was similarly small, but by the time of the fifth or percentage, suggests that the sperm of the treated sixth mating considerable recovery had often taken males were either absent or in one way or another place. If an average of 95% sterility were adequate not competitive with those of the normal males. In a for males released in a control program, any of these control program, this possibility of the female's mak- doses might be suitable for producing males that were ing full use of a normal sperm supply could mean sterile for the first 1 or 2 matings. However, the that any number of matings with sterile males could colony male in Table 2 shows that males are inher- be undone by 1 mating with a normal male; and the ently capable of numerous matings; and in the crowded more times she remated, the greater would be her conditions of colony cages they often do mate repeat- chances of finding a normal male. edly. Whether field conditions produce situations Until these and similar questions are answered, the similarly favorable for such repeated mating is un- feasibility of the sterile-male technique for C. vari- known. However, if they do, and if 5 or 6 matings ipennis cannot really be evaluated. It is true that an are common, the recovery observed at 10,000 and initial sterility of 95%—100%, without apparent so- 15,000 R suggests that these doses would not be matic damage, can be induced by gamma irradiation, sufficient. If 100% sterility were required, of course, although the doses required are unusually high for even 20,000 R would be too small. Diptera (LaChance et al. 1967). Other methods-of At least 1 other question to consider in a control sterilization might give even better, or more perma- program would be the number of times a female could nent, results. Still, some of the basic characteristics be expected to mate. In these tests, 4 of the 7 normal of the species suggest that application of any kind of 9 that were originally mated with treated males and biological control in the field is not likely to be a were later remated with normal males then showed simple matter. 844 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA LVol. 60. No. 4

Table 5.—N <$ X T2 matings: Eggs in first batches when each normal, untreated male (Nc5) was mated first with a treated female (T 2 ) and subsequently with untreated females (N $ ).

Sterile eggs (%) in first batches'1 No. first- First Subsequent (N $ X N 2 ) Age (hr) batch eggs •(N$ X matings at irra- from first T2) diation" mating mating 2nd 3rd 4th 5th Comments Larvae irradiated at 5000 R 311-293 91 1 130-119 72 3 2 7 5C 49 22 Pupae irradiated at 5000 R 56-48 73 44 30-24 104 1 27-24 116 1 10 2 t D-9 0 L-6 22-18 125 35 4-0 35 3 Adults irradiated at 5000 R l-2# 130 12 130 60 154 7 0 L-12 Larvae irradiated at 10,000 R 131-120 5 100 Pupae irradiated at 10,000 R 56-48 0 L-21 50-46 0 f 100 14 21 48-40 32 62 40-32 34 44 24-16 0 L-22 8-0 0 D-16 t 8 2 Adults irradiated at 10,000 R Yz-2 0 ! D-8 0 7 100* *Female refused additional blood meals 44 30* •Batch 2: 17 eggs, 82% sterile 41 98* •Batch 2: 4 eggs, 100% sterile Larvae irradiated at 15,000 R 67-63 0 D-10 Pupae irradiated at 15,000 R 53-50 0 D-12 1 1 50-46 14 100 46-42 0 D-12 1 0 D-34d 42-37 0 D-29* *Female refused additional blood meals 33-30 30 100 100 3 30-26 40 65d 26-22 17 88 0 * *Female refed and remated; batch 1: 30 eg 83% sterile. Female remated ; batches 2- 14, 2 eggs; 64%, 0% sterile 22-17 23 87 1 11 73d 17-11 0 D-19* *Female refused additional blood meals 7-2 0 L-14 Adults irradiated at 15,000 R Y2-2Y2 0 D-32d 1 3-6 103 89d 52 100d Pupae irradiated at 20,000 R 44-40 8 50d 2 40-34 25 92d L-6 7 0* *Batches 1-4: 79, 118, 71, 52 eggs; 0%, 2 18%, 90% sterile 0 D-24d 20-16 0 D-23 8 2 59 0 16-11 0 D-17 10 3 0 2 11-7 0 D-17 2 7-3 0 L-10 fulv 1%7 JONES: IRRADIATION OF Culicoidcs variipoinis 845 Table 5.—X

Sterile eggs (%) in first batches" Xo. first- First Subsequent (X $ X N 9 ) Age (hr) batch eggs (N<$ X matings at irra- from first T 9 ) diation* mating mating 2nd 3rd 4th 5 th Comments Pupae irradiated at 30,000 R 33-28 D-8 5 1 t 23-18 D-13 18-13 D-25 6 13-9 D-25 40 38 6th mating, D-18 D-ll D-14 9-4 D-24 4-1 D-19 5 11 (liven as hours between irradiation and emergence; thus, higher numbers indicate younger larvae and pupae, but older adults. 11 D =: female died, L ~ female lost (including escape, accidental death, or discard), without ovipositing; age (days) at time of death or loss shown by associated numbers, t indicates that the female died or was lost when less than 6 days old and thus could not have ovi- posited, refers to comments. '" Ttalic figures indicate that the next 1 or more egg batches from the same female were corroborative; i.e., sterility was within a few percentage points of the first batch from the indicated mating; or, if the first batch was 100% sterile, so were ajl subsequent batches. 11 This female was given additional blood meals and/or matings, but no eggs (or no additional eggs) were laid.

Table 6.--N $ x N? matings (controls) : Eggs in first egg batches when each normal, untreated male (N #) was matctl with untreated females (N 9 ).

Sterile eggs (%) in first batches6 No. first- First Subsequent (N $ X N 9 ) batch eggs (X<5 X matings Age from first X9) (hr)« mating mating 2nd 3rd 4th 5th Comments Controls for larvae irradiated at 5000 R 145-130 132 130-119 102 Controls for pupae irradiated at 5000 R 56-48 116 2 48-40 0 D-21 30-24 0 L-8 27-24 25 40 124 0 22-18 160 11 0 D-14 0 D-31 t D-8 6th mating, 0% Controls for adults irradiated at 5000 R 1-25-5 0 D-8 88 3 122 100 119 1 2 1 t 0 Controls for pupae irradiated at 10,000 R 56-48 17 100 50-46 149 0 Controls for adults irradiated at 10,000 R 0 0 0*c ^Batches 2-5: 142, 133, 93, 65 eggs; 1%, 0%, 3%, 37% sterile 120 0 117 0 0 D-13 Controls for larvae irradiated at 15,000 R 67-63 •Batches 2-6: 137, 101, 83, 66, 46 eggs; 4%, 140 I* 0%, 1%, 0%, 20% sterile 56* 0 1 *Estimated figure 88 10 846 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA LVol. 60, No. 4 Table 6.--N3 X N$ matings (controls) : Eggs in first egg batches when each normal, untreated male (N# ) was mated with untreated females (N5). (Continued.) Sterile eggs (%) in first batches11 No. first- First Subsequent (N $ X N 2 ) batch eggs (N# X matings Age from first N9) - (hr)a mating mating 2nd 3rd 4th 5th Comments Controls for pupae irradiated at 15,000 R 53-50 120 0 50-46 162 4 46-42 0 D-9 0 2 195 4 42-37 123 0 33-30 158 2 30-26 113 8 7 26-22 183 I 195 1 22-17 174 1 135 1 17-11 243 100* *Female remated: batch 2: 181 eeers. V/< sterile Controls for adults irradiated at 15,000 R 36 Controls for pupae irradiated at 20,000 R 44-40 0 t 0 0 0 1 40-34 176 2 16* 9 *Batch 2: 0% sterile 0 L-21 16-11 182 0 13* 20** 1 2 *Batch 2: 0% sterile. **Batch 2: 1% sterile 11-7 120 2 12* *Batch 2: 0% sterile 7-3 0 t 0 2 Controls for pupae irradiated at 30,000 R 23-18 0 t 0 18-13 169 4 13-9 133 3 34 2* 0 *Batch 2: 39 eggs, 100% sterile 71 0 133 0 4-1 124 2 t 2 t 65* *Batch 2: 11% sterile

a Age when corresponding test group of treated flies was irradiated, given as hours between irradiation and emergence; thus, higher num- bers indicate younger larvae and pupae, but older adults. b D = female died, L = female lost (including escape, accidental death, or discard), without ovipositing; age (days) at time of death or loss shown by associated numbers, t indicates that the female died or was lost when less than 6 days old and thus could not have ovi- posited. * refers to comments. c Italic figures indicate that the next 1 or more egg batches from the same female were corroborative; i.e., sterility was within a few percentage points of the first batch from the indicated mating; or, if the first batch was 100% sterile, so were all subsequent batches.

ACKNOWLEDGMENTS Preliminary investigations of insect transmission of bluetongue virus in sheep. Amer. J. Vet. Res. 24: I thank my wife, Ann Harbour Jones, for her 1195-1200. assistance. She not only helped me organize the data, Jefferson, M. E. 1960. Irradiated males eliminate screw- but then wrote this paper in its entirety, with a mini- worm flies. Nucleonics 18: 74-76. mum of advice from me. Although the organization Jones, R. H. 1961. Observations on the larval habitats and style are hers, frequent consultation insured that of some North American species of Culicoides (Dip- the interpretation of the data is mine. tera: Ceratopogonidae). Ann. Entomol. Soc. Amer. I also thank my former assistant, Norma S. Allan, 54: 702-10. without whose painstaking care these experiments 1966. Culicoidcs biting midges, p. 115-25. In C. N. Smith [ed.] Insect Colonization and Mass Production. would have been impossible. For his many helpful Academic Press, New York and London. remarks about this manuscript, which greatly clari- LaChance, L. E., C. H. Schmidt, and R. C. Bushland. fied our thinking and, we hope, our presentation of 1967. Radiation-induced sterilization for insect con- the results, I am grateful to Dr. Leo E. LaChance, trol. In W. W. Kilgore and R. L. Doutt [ed.] Bio- Insect Geneticist with this Division. logical, Physical, and Advanced Chemical Methods of Pest Control. Academic Press, Inc., New York. REFERENCES CITED (In press.) Curtis, L. C. 1962. A gynandromorph in Culicoides. Smith, C. N. 1963. Prospects for vector control through Mosquito News 22: 53. sterilization procedures. Bull. World Health Organ. Foster, N. M., R. H. Jones, and B. R. McCrory. 1963. 29, Suppl.: 99-106.