TECHNIQUES AND RESULTS OF APPLYING THIOUREA TO SYNTHETIC AND GRASS SILAGE MEDIA FOR THE CONTROL OF STOMOXYS CALCITRANS LINNAEUS by HARRY B . EINBURGH AN ABSTRACT Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Entomology Year 1956 ,,,»——-—+» ’ Approved :z1:l~if%:4_i_\q l HARRY B. WEINBURGH ABSTRACT This research was initiated because of the need for a compound with low mammalian toxicity, yet exhibiting insecti- cidal properties, to be used for control of stable fly popula- tions in silage. Since previous workers had shown that thio- urea was toxic to house fly and flesh fly larvae, its effec- tiveness as a larvicide to the stable fly was investigated un- der laboratory conditions. Standard procedures were modified and refined for a method of continuous rearing of the stable fly exposed to a varying laboratory environment. The size of the adult rearing cages proved to be not necessarily a limiting factor: large cages (3 x 3 x 3 feet) or small cages (15 x 15 x 24 inches) were equally satisfactory. The adult food, beef blood, was preserved with freshly prepared sodium citrate solution, but could not be retained for more than ten days when stored at 50° Fahrenheit. Techniques were developed for the evaluation of thiourea as a stable fly larvicide. The test media were composed of CSMA media, oat hulls and distilled water. Counts of emerged adults were facilitated by use of funnels coated with "tangle- foot" and inverted in the test Jars. The maintenance of con- stant temperature and humidity proved to be critical for ob- taining reliable results. I The effect of thiourea on the development of the stable fly was determined when mixed with or when sprinkled on the 2 HARRY B. WEINBURGH ABSTRACT synthetic media, and when mixed with grass silage. The median lethal dosages were determined to be 63, 13 and 12 parts per million, respectively. Thiourea sprinkled on the synthetic media was apparently more effective as a toxicant than when mixed with the synthetic media. The data also suggested that thiourea, when mixed with grass silage, produced sufficient mortality to stable flies to warrant future investigations of its toxic properties under field conditions. TECHNIQUES AND RESULTS OF APPLYING THIOUREA TO SYNTHETIC AND GRASS SILAGE MEDIA FOR THE CONTROL OF STOMOXYS CALCITRANS LINNAEUS by HARRY B . WEINBURGH A THESIS I Submitted to Michigan State Universiti‘ ~‘ in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Entomology 1956 ACKNOWLEDGMENTS The author wishes to express his sincere thanks and ap- preciation to those who have been helpful in planning and en- couraging this work. It would not have been possible to secure adequate treatment of this research had it not been for their personal interest. To Professor Ray Hutson, the writer wishes to express his deep appreciation for the guidance and council freely given in all stages of this investigation. His advice, backed by his long experience in Entomology, was invaluable. Particularly helpful were Drs. Herman L. King, Robert G. Haines and Gordon E. Guyer, who freely gave their guidance, criticism and constant encouragement in the conduct of this study, and Dr. Don w. Hayne, Zoology Department, who generously assisted in the statistical analysis of the data. The author also extends thanks to Dr. Phillip Granett, Department of Entomology, Rutgers University, New Brunswick, New Jersey, who furnished pupae of Stomoxys calcitrans Linnaeus, Mr. w. S. van Scoik, American Cyanamid Company, who furnished the technical thiourea and the bulletin on this material, and Mr. William Van Alstine, Okemos, who furnished the beef blood necessary to rear the adult flies. The writer is especially indebted to his family for their interest, patience and encouragement during the study. 11 TABLE OF CONTENTS INTRODUCTION . . . . . OBJECTIVES . . . . . MATERIALS USED . . REVIEW OF LITERATURE . PROCEDURE EXPERIMENTAL RESULTS . DISCUSSION . . . . SUMMARY AND CONCLUSIONS LITERATURE CITED . iii 13 21 36 142 1414 INTRODUCTION The stable fly, Stomoxys calcitrans Linnaeus, has been a pest of domestic animals and man, causing irritation, discom— fort and serious economic loss on occasions. The stable fly lays its eggs in moist, rotting vegetation. Under laboratory conditions the eggs hatch in 48 hours. The larvae develop by feeding on the decaying matter and usually pupation starts on the ninth day. The adults start emerging on the fourteenth day, mate after four days, and begin egg-lay- ing on the twenty-first day. Both sexes subsist primarily on mammalian blood. The optimum rearing temperatures range be- tween 800 and 85° Fahrenheit. The recent increase in the use of silage on farms has pro- vided this insect with an excellent growth medium for the im- mature forms. Since silage is used as food for beef and dairy cattle, the use of chlorinated hydrocarbon and organic phosphate insecticides, which have produced excellent control of stable flies, is prohibitive because of their toxic properties. Be- cause of potential toxicity problems the United States Food and Drug Administration has banned all chemicals in milk. Consequently, entomologists and farmers have expressed the desire to have made available a compound which would be ‘non-inJurious to warm-blooded animals and still destructive to insect life. Previous research by other workers had 1 2 produced evidence that at least one compound, known chemically as thiourea, exhibits these desirable qualities. This material has been shown to be toxic to the larvae of the house fly, Musga_ domestica Linnaeus, and the flesh fly, Lucilia sericata Meigen, is practically non-toxic to mammals, and is partially soluble in water. Since earlier investigators indicated the possible values of thiourea as a larvicide, experiments were conducted to determine the effect of thiourea on the immature forms of Stomoxys calcitrans. OBJECTIVES The major objectives in this study were as follows: (1): To design a method of continuous rearing of the stable fly, Stomoxys calcitrans, under variable laboratory con- ditions; and to further design techniques necessary for evalu— ating the effect of thiourea on stable fly populations. (2): To determine the effect of various concentrations of thiourea on the immature forms of the stable fly, Stomoxys calcitrans, when (a): mixed with synthetic media, (b): mixed with grass silage, and (c): sprinkled on synthetic media. f-l- i—' _.—.__-—_—_— MATERIALS USED 1. Thiourea (Thiocarbamide) Thiourea is a white crystalline solid, stable at normal room temperatures, with a chemical structure as follows: 038 NH2 Thiourea, on heating, isomerizes to ammonia thiocyanate, with which it forms an eutectic mixture. The technical compound has a melting point between 1700 and 1800 Centigrade. Decomposi- tion takes place at 2000 Centigrade; H28, 082 and NH3 are evolved and guanidine thiocyanate remains. The solubility in water ranges from three grams per 100 grams of solution at minus five degrees Centigrade to 100 grams per 100 grams of solution at 1000 Centigrade. The apparent density of the chemical is fifty pounds per cubic foot. The thiourea was obtained from the Agricultural Chemicals Division, American Cyanamid Company, 30 Rockefeller Plaza, New York 20, New York. 2. CSMA Standard Fly Larval Medium The CSMA (Chemical Specialties Manufacturing Association) standard fly larval medium is composed of alfalfa meal, dried brewers grains and soft wheat bran. The media were procured from the Ralston-Purina Company of St. Louis, Missouri. 4 3. Cat Hulls The oat hulls, which were mixed with the CSMA media, are known commercially as "Full O'Pep" Poultry Litter. They were purchased from the New Century Company, 3939 South Union Avenue, Chicago 9, Illinois. 4. Beef Blood The beef blood was obtained from Van Alstine Custom Meats, 5416 North Okemos Road, Okemos, Michigan. 5. "Tanglefoot" The "tanglefoot'l was prepared by mixing two pounds of rosin with one quart of hot castor oil. 6. Battery Jars The battery Jars were the standard type used in many kinds of biological experimentation, having dimensions of six inches in diameter and ten inches in height. 7. Sodium citrate The crystalline sodium citrate, used to preserve the beef blood, has a molecular weight of 29A and the following formula: Na206H507°2H20. REVIEW OF LITERATURE Woodbury (1943) stated that the stable fly, Stomoxys cal- citrans, can be reared by methods similar to those employed for the house fly, Musca domestica, except that the adults are fed mammalian blood. Campau, Baker and Morrison (1953) reared stable flies by using house fly media for the larvae. McGregor and Driess (1955) modified earlier procedures to make rearing simpler. The larval medium was prepared by mixing one part by volume of CSMA media with five parts of wood shav- ings and slightly moistening with water. Various techniques have been employed to obtain oviposi- tion. Champlain, Fisk and Dowdy (1954) used a water-saturated sponge about one inch thick, fitted horizontally in the bottom of a rectangular box of Plexiglass. The container was then placed away from the source of light within the cage. After sufficient oviposition, the eggs were easily recovered by :flushing from the sponge with water. McGregor and Driess (1955) used a one-inch ball of damp cotton batten wrapped in :3 black cloth which, after moistening with a few drops of five Iper cent ammonia, was placed in a saucer or Petri-dish in the (oviposition cage. Female flies were attracted by the ammonia Aand therefore eggs were laid on the dark surface, which facili- ‘tated the collecting of eggs. Mold has been a very serious problem in larval media. Cnnamplain, Fisk and Dowdy (1954) added sand to the surface of 6 5%.. 3.... ml! 7 the medium to prevent mold formation. Other workers have sug— gested maintaining a moist media to inhibit mold development. Many inorganic and organic insecticides have been used to kill house fly larvae. Hadjinicolaou and Hansens (1953) deter- .mined that extremely small amounts of aldrin, dieldrin and chlordane controlled house flies when added to the larval media. Field tests showed that these materials alone could not be exs pected to result in complete fly control because of the diffi- culty in finding all breeding places. Tests of these materials on turkey manure and a large manure pit produced excellent con- trol with the first application, but each subsequent applica- tion indicated decreasing effectiveness. Cunningham and Eden (1955) determined the LDSO concentra- tions for endrin, aldrin, dieldrin, chlordane and DDT in the larval media to third instar house fly larvae. Endrin was the most toxic. Standifer (1955) investigated 25 formulations of fifteen chlorinated hydrocarbon and organic phosphate insecticides to determine their respective toxicities to third instar house fly larvae. Aldrin emulsion was the most toxic of the chemicals tested; EPN-300 wettable powder and diazinon emulsion were Inearly as effective; while BHCa emulsion and BHC wettable pow- der were the least effective. McCauley, Grainger, Lindquist and Fay (1955) tested seven chlorinated hydrocarbon insecti- cides, DDT, methoxychlor, toxaphene, BHC (95 per cent same ——‘I—3Benzene'hexachloride 8 isomer), chlordane, dieldrin and aldrin as larvicides in spray applications on the surface of breeding media containing non- resistant house fly larvae. Chlordane produced superior con- trol of all instars, whereas BHC, aldrin, toxaphene and dieldrin were most effective in media containing early instar larvae. very low concentrations of endrin, heptachlor, lindane and parathion appeared to be highly effective as house fly larvi— cides, based on field tests by Sampson (1956). Greater concen- trations of other insecticides were required to produce equiva- lent toxicity. In an attempt to find a material non-injurious to mammal- ian life yet toxic to fly larvae, various research workers have experimented with thiourea as a fly larvicide. Hoskins, Blox— ham and van Ess (1940) tested thiourea as a larvicide for the flesh fly, Lucilia sericata. A concentration of four-tenths per cent by weight when added to the synthetic diet resulted in complete control, while a concentration of two-tenths per cent produced 85 per cent control. Green (1946) applied a coarse spray of technical thiourea at the rate of 33 grams per gallon of water per 100 square feet of manure. This type of application resulted in complete control of fly larvae for two weeks. McGovran and Piquett (1945) determined the LD5Oa of thiourea to third instar house fly larvae to be 81 parts per million. Contrary to the findings of Sampson (1956), these o is the dosage required to kill 50 per cent of the population of test organisms. .flgfii mums! i 9 workers found DDT and thiourea to be about equally toxic to fly larvae. Konecky and Mitlin (1955) found that a concentration of 5,000 parts per million of thiourea resulted in complete in- hibition of house fly larvae. The mammalian toxicity of thiourea has been investigated, using rabbits, rats, dogs, guinea pigs and humans as test or- ganisms. Flinn and Geary (1940) fed thiourea in small amounts to rabbits, dogs and rats and observed no ill effects. Groups of rabbits given nine grams of thiourea per kilogram of body weight by means of a stomach pump, behaved in the same manner as animals in the control group. In a continuation of this experiment, 50 per cent of the rabbits died after receiving ten grams of thiourea per kilogram of body weight. Mortality increased to 100 per cent when 11 grams of thiourea per kilo— gram of body weight were administered. Symptoms of toxicity were observed during periods not exceeding 36 hours in these tests. The deaths were probably due to drastic changes of ionic concentrations in the blood stream, rather than direct toxic action by thiourea. Rabbits were fed an aqueous solution containing 25 milligrams of thiourea per kilogram of body weight each day except Sunday for 12 weeks. Increase in weights of test animals did not differ significantly from that of the con— trols. In subsequent tests, rats were fed thiourea in drinking water at the rate of 60 milligrams per kilogram of body weight for nearly 12 weeks. The weight gain was more gradual in the exposed group when compared with the control group, but the 10 percentage gain at the end of the test was the same for the two groups. Autopsies on the rats and rabbits receiving low doses of thiourea revealed no lesions in any of the tissues examined. Hartzell (1940) administered thiourea to guinea pigs at the rate of 45 milligrams per kilogram of body weight each day during a period of six weeks. No evidence of toxic action was observed. Rats that ingested concentrations up to 49 milli— grams of thiourea per kilogram of body weight for periods of 22 to 28 days exhibited no symptoms of poisoning and made gains comparable to the controls. Kennedy (1942) found that 200 milligrams of thiourea in the diet daily for ten days resulted in abnormal thyroid glands in rats. MacKenzie and MacKenzie (1943) also determined that relatively high dosages of thiourea caused enlargement of the thyroid gland. Purves and Griesbach (1946, 1947) observed tumors of the thyroid gland in rats given 0.25 per cent thiourea in their drinking water for 12 months or more. There was a tendency for such tumors to become malignant after 20 months. Since no abnormal growth changes were found in tissues other than the thyroid gland after long periods of feeding, it was concluded that thiourea had no direct carcinogenic action. Flinn and Geary (1940) fed dachshund puppies a diet con- taining 25 milligrams of thiourea per kilogram of body weight for ten weeks. The dogs evidenced no injurious effects; they were lively and gained weight in a normal manner. Hartzell 11 (1940) fed thiourea to three young males of a litter of five dogs, while the other male and the female served as controls. Thiourea was administered in a ten per cent solution by stomach pump at a dosage equivalent to one gram of thiourea per kilo- gram of body weight. Every animal increased in weight almost two-fold during the period of the test and no toxic manifesta- tions were evidenced at any time. Hartzell (1940) also reported that in human feeding trials, 6.8 grams of thiourea were consumed during a period of two days with no discomfort experienced by the subject. Studies on the metabolism of thiourea, in man by Medes (1937) and in rabbits by Blood and Lewis (1943), indicated that the compound was excreted in urine within 48 hours, probably in unchanged form. Positive tests for thiourea were obtained in urine collected one hour after ingestion. Schulman and Keating (1956) injected thiourea labeled with radioisotopic sulfur in rats and recovered 98 per cent of the radioactivity in the urine within 48 hours. The major portion of the thio- urea was excreted unchanged; only 12 per cent of the radioac- tivity was associated with secondary sulfur products. There was a 55-fold concentration of radioactivity in the thyroid as compared with other tissues of the animals. I Thiourea structurally resembles urea, therefore it is probable that the degradation scheme may be similar. The end products, although not definitely known, are theoretically pre- sumed to be ammonia and sulfur-containing acids. An anonymous 12 report (1956) indicated that high temperatures were required to initiate hydrolysis. Therefore, since no data to the con- trary has been produced, the rate of degradation is assumed to be slow in manure and silage. PROCEDURE Wild stable flies were collected at the Michigan State University experimental dairy barn in an attempt to establish a laboratory strain; but, due to some inexplainable reason, the flies did not survive. Consequently, Stomoxys calcitrans pu— pae were obtained from the Department of Entomology, Rutgers University, New Brunswick, New Jersey. After some refinement in rearing technique, a stock colony was adequately established. The rearing procedure to initiate and maintain a stock colony, briefly, was as follows: All experimentation during the winter and spring was con- ducted under constant environmental conditions (85° Fahrenheit temperature and ten per cent relative humidity). However, the research during the summer and fall was conducted under ex- tremely variable environmental conditions (600 to 85° Fahren- heit temperatures and 20 to 95 per cent relative humidities). The adults were provided fresh beef blood as a daily diet. To prevent coagulation, 64 milliliters of a 0.17 molar sodium citrate solution were added to each liter of blood. Blood was obtained at weekly intervals from a local slaughter house and stored at 50°;t 5° Fahrenheit. Waxed paper sundae cups were used as feeding receptacles within each cage to eliminate ex- tra glass washing. The blood was poured into the cup, after which absorbent cotton was pressed into the liquid with a 13 14 stirring rod. Cellucotton was preferred over absorbent cotton since the former rapidly absorbed the blood. Used cups were discarded daily and replaced with freshly prepared feeding con- tainers. A rapid method for obtaining a known amount of Stomoxys calcitrans eggs was necessary to expedite the testing proce— dure. The female flies would deposit eggs in fermenting lar- val medium, but it was difficult to separate the eggs from the medium for the tests. The method of McGregor and Driess (1955) was tried but did not work under the conditions in the labora- tory. The ammonia-moistened cotton quickly dried due to the low humidity and probably was no longer attractive to the fe- males. During the course of colony maintenance, it was observed that the female flies would deposit many eggs in the feeding containers, particularly when the blood level decreased. Al- though washing the eggs from the blood-saturated cotton was very time-consuming, it proved to be the most satisfactory manner in which to secure an abundant supply of eggs for test- ing procedures. Contrarily, eggs for colony regeneration were easily ob- tained. Containers of fermenting larval substrata were placed in the breeding cages, and subsequently the females deposited eggs in the medium. The cups were removed each day and the contents added to battery jars containing additional media. 15 The larval medium was prepared by mixing 180 grams of CSMA media, 20 grams of wood shavings and 500 milliliters of distilled water. The wood shavings varied in weight and water absorption capacity, depending upon the kind of wood. While this variation did not greatly affect rearing of the stock colony, serious inconsistencies occurred in the testing pro- cedures. Consequently, oat hulls were substituted for wood shavings. However, 50 grams of hulls were necessary to manu— facture a medium consistent with the other method. The vessels stocked with egg-seeded media were covered with muslin and dated. If mold formed in the media, it was inhibited by stirring, and additional water was added if neces- sary. The eggs hatched in one to two days and the larvae be- gan feeding actively until the eighth day. Pupation began on approximately the ninth day. The adults began emerging five days later in the jars and were released in wire screen cages (15 x 15 x 24 inches), closed at one end with muslin and with a muslin sleeve fitted to the opposite end (Figure 1). When the colony had increased in size to the point that the females were producing a total of several thousand eggs each day, tests with thiourea were started. The test medium was prepared by mixing thoroughly 90 grams of CSMA media and 25 grams of oat hulls. Known amounts of thiourea were dissolved in 300 milliliters of distilled water, added to the dry ingredients and again mixed. W"$f.r' M‘- v nus. ”'1' ' A \ .I. 3.1150337 n m "‘1' .l ‘ O I‘ - -. l.‘ A‘-‘< . .. O” ‘l 3“ . " ’f 2&1. -I .. q . -- .3:- Figure l. R \ ’. “ \Tw A11 .- 7.x. Adult rearing cage. 16 17 The test eggs were washed from the blood—soaked cotton, placed in a Petri-dish and gathered in a medicine dropper. One hundred eggs were counted by use of a stereoscopic microscope and deposited on filter paper. The eggs were transferred from the filter paper to the surface of the test media at the rate of 100 eggs per vessel. The experimental technique consisted of four replications for each concentration of thiourea and equivalent controls. All containers were covered with muslin, labeled for concentra- tion and date, and retained until ample time for emergence elapsed. Mold usually developed in the media within the first three days, but agitation generally inhibited the mold. If stirring was not successful, 50 milliliters of distilled water xvere added to aid in prevention of mold growth. Subsequent to pupation and before adult emergence, a :ihort-stemmed six-inch funnel (Figure 2), smeared with “tangle- fNoot" on the outside, was inverted in each test jar to trap tune adults (Figure 3). Daily emergence counts were simplified by this technique. A second series of experiments were designed to test the effect of various concentrations of thiourea in a grass silage fluadium. The concentrations of thiourea were dissolved in 50 In1;lliliters of distilled water, thoroughly mixed with the si- lage, and 100 eggs were introduced. Counts of adult flies were made similarly by the method previously described. Figure 2. Inverted funnel coated with "tanglefoot." 18 Figure 3. Covered test jar containing inverted funnel. ‘1 20 In a final series of tests, an attempt was made to deter- mine whether any significant change in inhibition of larval development occurred when the thiourea was combined with eggs or when sprinkled on previously-established larvae. Therefore, six-day—old larvae were added to the media prior to application of various concentrations of thiourea on the basis that under natural conditions this situation might normally exist. The medium was prepared in the usual manner for testing except that the thiourea was omitted. The thiourea was first dissolved in 50 milliliters of distilled water and then sprinkled on the snirface of the media by means of a clothes-sprinkling device IEitted to an Erlenmeyer flask. Counts of adult emergence were eugain made by use of the short-stemmed funnels smeared with '“tanglefoot.” The experimental data were analyzed by following the pro- cuedures exactly as outlined by Finney (1947, 1952) in chapters Ikbur and six, except that an adjustment was necessary in Table 11[ because of negative values obtained from two of the concen- tusations. A second cycle for fitness was computed in Table \EEI. -“<— ‘— EXPERIMENTAL RESULTS The results in this investigation pertain to the follow— ing series of experiments: A. The determination of the effect of various concentra- tions of thiourea on stable fly larvae when mixed with synthetic media. B. The determination of the effect of various concentra— tions of thiourea on stable fly larvae when mixed with grass silage media. C. The determination of the effect of various concentra— txions of thiourea on stable fly larvae when sprinkled on syn- thetic media . A. Thiourea Mixed With Synthetic°Media Thiourea solutions ranging in concentrations from 250 to 3M3,000 parts per million (three per cent by wet weight) resulted if! complete inhibition of larval development when mixed with Synathetic media (Table I). Response to lower concentrations ‘Of‘ 12.5 to 100 parts per million indicated a gradient of larval Inortality (Table I). Two additional tests for determining the ILIESC,were conducted, but due to extreme changes in temperature 8116! humidity, results were invalidated. Quantity of thiourea to produce an LD5O was determined to be approximately 63 parts FNEJ? znillion when mixed with the growth substratum. The statis- tiJcail analysis of the data is shown in Table II and Figure 4. 21 22 B. Thiourea Mixed With Grass Silage Media Solutions of thiourea mixed with grass silage media pro- duced complete inhibition of larval development at concentra- tions greater than 75 parts per million, while concentrations of less than 50 parts per million resulted in a reduction gradient of larval survival (Table III). The statistical treatment of the data is provided in Table IV and Figure 5. C. Thiourea Sprinkled 9§_Synthetic Media When concentrations of thiourea were sprinkled on infested larval substrata, a gradient of mortality resulted (Table V). 'Fhe'probit analysis of the data is given in Tables VI and VII anud accompanying Figures 6 and 7. The sprinkling of various (noncentrations of thiourea on the synthetic media resulted in 21 much lower median lethal dose (LD50) when compared with that cflotained by mixing equivalent concentrations of thiourea with tune synthetic media. .I r...» 23 KEY TO SYMBOLS USED IN STATISTICAL ANALYSIS Conc. ppm. . Slope of line . Concentration of thiourea ex— pressed in parts per million . Natural mortality . Empirical probit . Number of insects tested 100 r/n . p = (p'-C)/(l-C): ”Abbott's formula" . Number of insects killed . Weighting factor . Natural logarithm of concen- tration . Working probit . Expected probit 24 TABLE I EFFECT OF VARIOUS CONCENTRATIONS OF THIOUREA ON THE DEVELOPMENT OF THE STABLE FLY, STOMOXYS CALCITRANS, WHEN MIXED WITH SYNTHETIC MEDIAa Conc. Repli- adults emerged Total Mean Difference cations A B C D from control ppm. % 30,000 4 0 20,000 4 0 10,000 4 0 5,000 8 0 2,500 4 0 1,250 4 o 625 4 0 312 4 0 100 4 4 8 17 13 42 10.5 -77.3 50 4 59 68 36 33 196 49.0 4.2 25 4 30 62 45 27 162 40.5 -12.7 12.5 4 42 22 52 19 135 33.8 -27.2 o 12 143 132 147 135 557 46.4 aMean length of test: 20 days I.“v.‘u . I'D-*0. ' .1" msama.: saefi.fl\mmsm.s II I. l' m.mumo..moam56m ago .eaa mm H omen ksmea.a +.mamfi.o u m wma.mn AmvmnBSUm H20 mm H x moaapcm m u wuomoq .IIIIII oHo.sH Ramsa.a +.mHmH.o u msa.om 063.:H mmm.ma Asmom.:-xvsmea.a + :Hm:.m n w www.mmfl.m mom.sms.a mmm.mm:.a smn.mmw.m mos.flss.a sem.:mz.fi oMuxvn.+.m u w m cm hxscm mxsnm $54 u p 336 um smog um oso.omm omm.mmm -- mm.Hs -- -- -- em mmm oo: .. o msm.m cam.» om.m mm.m sm.m m:.m m om mom oo: mm.m m.ma mam. mum. Hm.m ma.o no.3 sa.: AH- om mmm cos mm.m mm msa.m mmm.m HH.m mo.a mw.: om.: mm- am now 00: Hm.m om mmm.msm mas.oam mm.m mm.eo mm.m mm.m ms om mmm oo: om.: cod has x3: h as w .m.m Q .Q n c x .EQQ mwmmmv .smmp .thHMWHKZH.HHDHEMHEmn Mme ZO mo Bommmm HH mqmde PROBIT 0F KILL 6.0. 5.0_ 4.0_ J l J I 2.0 3.0 u o 5.0 Looe (ppm.) IFigure 4. Effect of various concentrations of thiourea on the development of Stomoxys calcitrans when mixed with synthetic media. 27 TABLE III EFFECT OF VARIOUS CONCENTRATIONS OF THIOUREA ON THE DEVELOPMENT OF THE STABLE FLY, STOMOXYS CALCITRANS, WHEN MIXED WITH GRASS SILAGE—mm- Total .33 Cone . Repli- adults emerged Total Mean Difference [ 3 cations A B C D from control 1 ppm' % I: i 100 4 0 j t f 75 4 0 . ‘ 50 4 0 3 2 0 5 l .3 -94 .0 25 4 3 l3 4 0 20 5 .0 -76 .4 O 4 13 22 19 31 85 21 . 3 aLength of test: 28 days ("N O 2 .EQQ MH n OmQA ma u x woflapcm mswmm.m m.m u mo..mopmsqm ago 0m:H.H\O00m.N u x 000.0 u nmvmpmsdw ago . on mam.sw msm.ms mme.m0 NOH.mw:.ofl 0mo.mmm.0 mam.mm0.m H0o.s0m.oa mom.mmm.0 Hmo.sme.m x0mza.a + onoa.m Aom0w.m:xv0mza.a + mmmm.0 n % mhzcm hxzcm mxzcm 0m:H.H u n mmmm.0 u.m om0s.m u.m som.mm0.a 00:.H0m -- mm.o0m -- -- -- ms mam cos x- o Hma.mo0 mmmqmmm 0os.m .mmqmmm as.m He.m 0e mm owm co: mm.m mm :zm.:0m mms.0mm mmm.0 ma.0m mm.0 mm.0 so am mam oo: Hm.m om m0m.mom mm0.mmH 000.0 m0.:: mm.0 ona 60H no: 007 mm.: ms eso.mea 300.00a m0m.e sw.mm om.» 99H ooH co: so: 00.: ooa has as: a 3: % .m.m Q .Q n c K .EQQ Amwumw .ocoo “mmmpumzmlmapwemweKEmv «Ham: muaqu mmamw meHz mmtz 2mm: . mzqmeHoqao m>XOZOEm .wqm mamaem mme mo ezmzmoqmsmn axe zo ammsone mo WZOHaamazmozoo WDOHmas as aomamm >H mqm<9 PROBIT 0F KILL 29 7.0.. 5.0- L I _L l 3.0 4.0 5.0 6.0 Loce (ppm ) Figure 5. Effect of various concentrations of thiourea on the development of Stomoxys calcitrans when mixed with grass siIage. ON THE DEVELOPMENT OF THE STABLE FLY, STOMOXYS CALCITRANS, WHEN SPRINKLED ON SYNTHETIC MEDIA (TeSES 1 & 2)3 TABLE V EFFECT OF VARIOUS CONCENTRATIONS OF THIOUREA 3O Total Adfilts C°“°' 323335. 123323 A—i-emeié 9% Tom “ea“ 31333333333321 ppm. # # #1 # # #’ #* % 6,400 6 330 0 3,200 6 330 0 1,600 6 330 0 800 6 330 0 400 9 480 1 3 3 7 0.8 -97.4 200 9 480 6 6 2 14 1.6 -93.8 100 3 150 2 1 1 4 1.7 -93.4 92 3 150 19 3 2 24 8.0 -68.4 50 3 150 4 3 5 12 4.0 -84.2 42 ' 3 150 21 17 18 56 18.7 —26.2 25 3 150 19 11 22 52 14.0 -24.9 0 9 480 105 110 103 318 25.3 8Mean length of test: 23 days 31 .800 m n 0094 0:.0 u mo..amnmsvw «no .800 N woafipcw m n K woaapcm H00m0.o H000H.H u H:>.0 u szmnMSUm H39 .800 0H woaapcm . . : H00.:m 00000.0 000: \:0H0 - x 0H0.Hm 00H.00 000.00H x0s:m.i. 000:.: u 0 0000 000.000.0 000.0mm.0 0H0.HH:30 0:84 A 00000; 2000:. + 000:: u o .00 . S. 00. 00 .0. 0. 33.11.03... + 3...... .. a 3 .00 a 0.2.0 .. .0...“ 00. apaaanmnopa m .mpdsaa mocmofigcoo 000:. n n H0m:.0 ".0 0000.: u.m 00:.msm.a 000.He0 -- 00.0Hm -- u- -- 0: H0 004 I- 0 :em.mm0 000.00: H0.0 00.00H 00.0 HH.0 0.00 00 00a 00H H0.m 00 000.000 000.00H 00.0 00.00 00.0 H0.0 0.00 00 0:H 00H 00.: 00H 000.000 000.:0m :0.0 00.00 00.0 as.0 0.00 00 0:H 00H 00.0 000 H00.00H 000.::H :0.» 0a.:m 00.0 00.» 0.00 00 0:H 00H 00.0 00: 0H0.Hm 000.:H NH.0 00.0 0H.0 00a 00a 00a 00a 00.0 000 03: x3: 0 3: M .m.m 0 .0 a c x .800 AOJROV .0800 AH 0009 - mHmwumn Ema zo «WMDOHmB mo mZOHB mo Bommmm H> mqm 00 000000 HH> mqm 000<0 PROBIT OF KILL 35 "i' 6.0 L4“? 5.0- 1 3.0 4.0 5.0 L0Ge (ppm ) Figure 7. Effect of various concentrations of thiourea on the development of Stomoxys calcitrans when sprinkled on synthetic media (test 2). DISCUSSION OF TECHNIQUES AND RESULTS Great difficulties were encountered in establishing a laboratory stock colony of stable flies. Wild stable flies were collected at the Michigan State University experimental dairy barn and deposited in a Jar containing grass silage. The breeding chamber was placed in a constant temperature room maintained at 80° Fahrenheit. The eggs hatched and larval de- velopment continued in a normal manner. After emergence, adults were transferred to a codling moth cage that had been modified by replacing the door with a cloth sleeve, and cit- rated beef blood was provided as food. However, the flies perished without producing eggs. This procedure was repeated with additional wild flies, but these also were unable to re- produce. Reasons for death were not definitely known, but probable factors included contamination of breeding cage and presence of toxic substances in the adult food supply. Therefore, because of lack of time, arrangements were made with the Department of Entomology, Rutgers University, New Brunswick, New Jersey, through the courtesy of Dr. Phillip Granett, to provide stable fly pupae at intervals so that a stock colony could be initiated and maintained. Considerable difficulty was experienced in rearing consecutive generations. Persistent loss of adult flies may have been due to failure 36 37 to eliminate completely pesticide contamination of cages as well as possible toxic substances in the food. In addition to cage contamination hazards, it was believed that small cages provided inadequate breeding space, and as a consequence, insufficient mating and egg deposition would oc- cur. To avoid the above-mentioned sources of error, a cubical cage three feet in each dimension was constructed of non- pesticide-contaminated materials. However, subsequent modifi— cation of rearing procedure indicated conclusively that stable flies could be satisfactorily maintained in either a large cu- bical cage (3 x 3 x 3 feet) or in a smaller rectangular cage (15 x 15 x 24 inches). Since mortality of adult flies continued to occur after the contamination problem had been eliminated, attention was directed to the adult food supply. In the original procedure an excess quantity of 0.17 molar solution of sodium citrate prepared with tap water had been stored at 50° Fahrenheit and used until coagulation or spoilage occurred. However, it was believed that under the above system of constituting and main- taining an adult food supply it was quite likely that certain toxic elements evolved to cause prolonged mortality within the stock colony. To eliminate suspected toxic effects from adult food re- tained under these conditions, a fresh 0.17 molar solution of sodium citrate prepared with distilled water was added to each new quantity of blood. As a further precaution citrated blood 38 was never retained more than seven days. With these changes in procedure, mortality numbers decreased and the stock colony progressed sufficiently. The experimental details were next undertaken. The larval substrata for the initial tests were prepared by mixing CSMA media and wood shavings with distilled water. However, since extreme variations in composition of the wood shavings existed, a relatively non-porous substance with a constant texture was sought as an alternate. Oat hulls have been utilized as a component of stable fly media by other investigators. There- fore, after preliminary experiments indicated that oat hulls would suffice for this project, they were substituted for wood shavings. The system for making daily counts proved quite efficient. Glass funnels, despite a coating of "tanglefoot," were easily manipulated by using the shortened stems as handles. When flies emerged, the majority would fly out of the funnels and subsequently become affixed in the "tanglefoot.” The trapped flies were removed each day with a pair of forceps and indi— vidually counted. The few flies that escaped the trap were counted without difficulty. Therefore, since the total tabu- lation was essentially accurate, the method was considered to be reliable. The first tests of thiourea mixed with synthetic media indicated that the LD5O for stable fly larvae was approximately 63 parts per million. Efforts to reproduce this data failed 39 because of extreme variability in laboratory conditions. Be- fore subsequent tests could be conducted, and because of space requirements, both stock and test colonies were transferred to a room with inadequate temperature and humidity controls. As a consequence, considerable time was lost in readjusting the colonies to the changeable conditions. During the course of developing experimental techniques, observations were made of the influence of temperature and humidity on the consistency of test results. It became appar- ent that both factors were critical in effecting good duplica- tion. As an example, natural mortality of adults increased when room temperatures ranged more that 50 from a mean of 800 Fahrenheit. Humidity appeared less critical than temperature, but radical changes in air moisture were indirectly related to variation in testing procedure. In addition to temperature and humidity factors, mold for- mation in the media was an interfering factor. Prevention of mold growth was accomplished either by agitation of media or by replenishment of moisture content. Additional distilled water was necessary only when mold formation rapidly increased. Mold development was more readily observed at low humidity values than at the Optimum range. Usually the appearance of mold would occur within the first 100 hours. The reasons for this phenomenon were not definitely known but it was assumed that newly-hatched larvae were unable to maintain sufficient agitation of media to inhibit mold growth during this interval. 40 However, as larvae matured and feeding activity increased, mold formation was noticeably absent. In a second series of tests thiourea, when mixed with grass silage, produced an LDBO of 12 parts per million with stable fly larvae. However, results were not too conclusive ‘F“T as time permitted only one test, and additional complications ? occurred with this series. Predacious mites were discovered at the termination of the experiment among the surviving adults, ; ,4 and it was unknown to what extent this condition may have af- g pg fected the outcome of the test. Despite difficulties encountered in this series, evidence of a trend of apparent toxic effect by thiourea to stable fly larvae in grass silage is present in the results. If subse- quent research substantiates these findings, thiourea may have definite practical application and commercial usage. Should thiourea prove to be effective and safe for use in grass silage as a toxicant to stable fly populations, the mat- ter of application would be problematical. Therefore, in a final series of investigations, an attempt was made to deter- mine whether thiourea would produce similar toxic reactions if various concentrations were sprinkled on synthetic media. Results from duplicate tests in this series indicated the possibility that thiourea increased mortality of stable flies when distributed in the above-described manner. The data sug- gested that thiourea sprinkled on media was more effective as a toxicant to stable flies than when mixed with the media. It 41 was not definitely determined that such was the case, but it is reasonable to assume that a greater degree of contact action was responsible for the increased mortality. The mode of tox- icity by thiourea to stable flies is also unknown; however, it is probable that toxicological symptoms could result from either direct contact or by metabolic processes following ingestion. Although the results in each series of tests were not definitely conclusive because of insufficient data, statisti- cal interpretations provided evidence that thiourea was toxic to stable fly populations in either grass silage or synthetic media under laboratory conditions. Field testing may yield different results, but it is logical that if application tech- niques are properly designed, similar responses are likely to occur . SUMMARY AND CONCLUSIONS This investigation can be summarized briefly by the fol- lowing statements: 1. Standard techniques for initiating and maintaining a stock colony of Stomoxys calcitrans were revised and refined i F to meet certain environmental conditions in the laboratory. - IL "T'VZZ‘ 2. Techniques were developed for evaluating thiourea as #fie a larvicide for the stable fly under laboratory conditions. 3. The effects of thiourea on the development of the stable fly were determined when various concentrations were mixed with or sprinkled on synthetic media, and when mixed with grass silage. The LDSO values were as follows: mixed with synthetic media, 63 parts per million; mixed with grass silage, 12 parts per million; sprinkled on synthetic media, 13 parts per million (mean value). 4. Counts of emerged flies were facilitated by use of funnels coated with "tanglefoot" and inverted in the test jars. The following conclusions were drawn from the results of this research: 1. For maximum colony sustenance, the adult food supply (beef blood) should be preserved with freshly prepared sodium citrate solution, and should not be retained more than ten days, if stored at 500 Fahrenheit. 42 43 2. Size of the rearing cage for adult flies is not neces- sarily a limiting factor. 3. Maintaining constant temperature and humidity condi- \u/ tions is necessary for obtaining reliable results in research of this nature. A. Thiourea sprinkled on media was more effective as a toxicant to stable flies than when mixed with the media. 174*” 5. The mode of toxicity of thiourea may have been initi— &F'T‘.T_ f ated either by direct contact action or by metabolic processes following ingestion. LITERATURE CITED Anonymous 1956 Thiourea Technical Bulletin. American Cyanamid Company. Blood, F. R. and H. B. Lewis 1943 The Metabolism of Sulfur. Jour. Biol. Chem. 139:413-420. Campau, R. A., G. J. Baker and F. D. M rrison 1953 Rearing the Stable Fly for Laboratory Tests. Jour. Econ. Ent. 46:524. Champlain, R. A., F. w. Fisk and A. C. Dowdy 1954 Some Improvements in Rearing Stable Flies. Jour. Econ. Ent. 47:940-941. Cunningham, H. B. and w. G. Eden 1955 Toxicity of Several Insecticides to House Fly Lar— vae. Jour. Econ. Ent. 48:109-110. Finne , D. J. 19 7 Probit Analysis, A Statistical Treatment of the Sig— moid Response Curve. Cambridge University Press, Cambridge, and Bentley House, London. 256 pp. Ibid. 1952 Probit Analysis, A Statistical Treatment of the Sig- moid5Response Curve. Cambridge University Press, Cambridge, and Bentley House, London. 318 pp. Flinn F. B. and J. M. Geary 1940 Feeding Tests with Thiourea. Contrib. Boyce Thomp- son Inst., 11:241-247. Green H. B. 1946 Research Points to Better Fly Control. Mississippi Farm Research, 19:3. Hadjincolaou, J. and E. J. Hansens 1953 Chlorinated Hydrocarbon Insecticides to Control House Fly Larvae. Jour. Econ. Ent. 46:34-37. Hartzell, A. 1940 Preliminary Feeding Tests with Thiourea. Contrib. Boyce Thompson Inst., 11:249-260. 44 45 Hoskins, w. M., H. A. Bloxham and M. w. Van Ess 1940 The Insecticidal Effects of Organic Compounds. Jour. Econ. Ent. 33:875—881. Kennedy, T. H. 1942 Thiourea as a Goitrogenic Substance. Nature, 150:233-234. Konecky, M. S. and N. Mitlin 1955 Chemical Impairment of Development in House Fly. Jour. Econ. Ent. 45:219—220. MacKenzie, C. G. and J. B. MacKenzie 1943 Effect of Sulfonamides and Thiourea on the Thyroid Gland and Metabolism. Endocrinology 32:185-209. McCauley, R. H. Jr., M. M. Grainger, D. A. Lindquist and R. w. Fay 1955 Laboratory Comparisons of Some Insecticides as Lar- vicides Against Non-resistant House Flies. Jour. Econ. Ent. 48:269-273. McGovran, E. R. and P. G. Piquett / 1945 Toxicity of Benzene Hexachloride to House Fly Larvae. Jour. Econ. Ent. 38:719. McGregor, w. S. and J. M. Driess 1955 Rearing the Stable Fly in the Laboratory. Jour. Econ. Ent. 48:327-328. Medes, G. 1937 Metabolism of Sulfur. VI. Oxidation in the Body of the Sulfur-containing Amino-acids and Some of Their Partially Oxidized Derivatives. Jour. Biol. Chem. 31:1330-1346. Purves, H. D. and w. E. Griesbach 1946 Studies on Experimental Goitre; Thyroid Carcinomata in Rats Treated with Thiourea. Brit. Jour. Exp. Path. 27:294-299. Ibid. 1947 Studies on Experimental Goitre; Thyroid Carcinomata in Rats Treated with Thiourea. Brit. Jour. Exp. Path. 28:46 Sampson, w. w. 1956 Insecticides Used to Control House Fly Larvae. Jour. Econ. Ent. 49:74-77. 46 Schulman, J. Jr. and R. P. Keating 1956 Studies on the Metabolism of Thiourea. I. Distribu- tion and Excretion in the Rat of Thiourea Labeled with Radioactive Sulfur. Jour. Biol. Chem. 183:215- 221. Standifer, L. N. 1955 Larvicides for Control of the House Fly. Jour. Econ. Ent. 48:731-733. Woodbury, E. N. 1943 Rearing Insects Affecting Man and Animals. Campbell, F. L. and F. R. Moulton, Editors. Laboratory Proce- dures in the Chemical Control of Insects, American Association for the Advancement of Science, Pub.20:75. Date Due Demco—293 R M'ClTl'llflfilLfljfllflilMlllllllliflflfllfllilalzlflfllTl'ES 8 242