ECOLOGICAL FACTORS AFFECTING ORGANOPHOSPH ATE TOXICITY TO IAR-VAE OP CULICIDAE- (DIPTER-A) THESIS FOR THE DEGREE OF M. S. hflCHIG‘AN. STATE UNIVER SITY ROBERT FRIEDRICH WILHELM SCHRDDER 1966 THF‘S‘S ‘ i ~m~h~mm VII" 1 LI [BRA RM Mic} ;3n‘~t3tc Un varsity ( ABSTRACT ECOLOGICAL FACTORS AFFECTING ORGANOPHOSPHATE TOXICITY TO LARVAE OF CULICIDAE (DIPTERA) by Robert Friedrich Wilhelm Schroder The most recent techniques and methods of rearing gages aegypti L. are described. An acetone suspension of fenthion (0,0-Dimethyl O-(4-(methylthio)-m-toly) phosphorothioate) was compared as to efficacy against fourth instar A. aegypti and Culex resturans Theobald, in various treatment combinations of pH, temperature, organic substance, and water type. DDT was used as a standard. Analysis of variance was calculated for each species, using a digital computer, correlating the effect of fenthion to the ecological factors and DDT. Results indicated that the various ecological factors, broken down into subgroups, were generally significant in relation to the fenthion check. In the course of investigation, a mermithid nematode was found parasitizing Aedes stimulans Walker. ECOLOGICAL FACTORS AFFECTING ORGANOPHOSPHATE TOXICITY TO LARVAE OF CULICIDAE (DIPTERA) By Robert Friedrich Wilhelm Schroder A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Entomology 1966 ACKNOWLEDGEMENTS The author wishes to express gratitude and apprecia- tion to Dr. R. A. Hoopingarner for his patience, guidance and counsel during the course of this study, to Chemagro Corporation for making this research possible, and a special tribute to my wife for her inspiration. ii TABLE OF CONTENTS PAGE INTRODUCTION ------------------------------------------ 1 REVIEW OF LITERATURE ---------------------------------- 3 Methods of Measuring Toxicity of Insecticides ------- 5 Intrinsic and Extrinsic Factors Affecting Insect Toxicity __________________________________________ 8 MATERIAL AND METHODS ---------------------------------- 15 Testing Methods ..................................... 17 Factorial Experiments ............................... 13 Preparation of Mortality Data for CDC 5600 Computer - 22 RESULTS OF HEARING ME HODS AND TOXICITY TESTS --------- 24 Results of Preliminary Experiments ------------------ 24 Classification of Culex Pasturans and Aedes Stimulans ----------------------------------------- 26 Effects of Ecological Factors ----------------------- 27 Treatment I. Acidic, hard water and diatomaceous earth ------------------------------ 30 Treatment II. Acidic, hard water, and organic wheat germ ------------------------------ 30 Treatment III. Acidic, soft water (distilled) and diatomaceous earth -------------------------- 33 Treatment IV. Acidic, soft water, and organic wheat germ -------------------------------------- 34 Treatment V. Basic, hard water, and diatomaceous earth ------------------------------ 34 iii PAGE Treatment VI. Basic, hard water and organic wheat germ -------------------------------------- 35 Treatment VII. Basic, soft water, and diatomaceous earth ------------------------------ 35 Treatment VIII. Basic, soft water, and organic wheat germ ------------------------------ 36 Treatment IX. Fenthion versus DDT ---------------- 36 Effects of Ecological Factors on Mortality of g; Resturans -------------------------------------- 36 Treatment I --------------------------------------- 59 Treatment II ______________________________________ 39 Treatment III ..................................... 39 ITeatment IV ...................................... 39 Treatment V --------------------------------------- 59 Treatment VI ______________________________________ 59 Treatment VII ..................................... 39 Treatment VIII .................................... 59 Treatment IX -------------------------------------- 39 DISCUSSION OF REARING METHODS AND TOXICITY TESTS ------ 41 Effects of Temperature ------------------------------ 43 Effects of PH _______________________________________ 44 Effects of Water Type ------------------------------- 45 Effects of Organic Matter --------------------------- 45 SUMMARY ----------------------------------------------- 47 LITERATURE CITED -------------------------------------- 43 .iv LIST OF TABLES PAGE I. Basic Design of Treatments for Mortality of Aedes Aegypti Treated with Fenthion ------------------- . 20 II. Mortality of the Rockefeller Strain of A. Aegypti Larvae Treated with Fenthion in Glass and Paper Containers -------------------------------------- . 29 III. Analysis of Variance of Treatments in Table I for Aedes Aegypti ----------------------------------- 31 IV. Analysis of the Chemical Content of Water from Tire Ruts --------------------------------------- 37 V. Analysis of Variance of Treatments in Table I for g. Resturans -------------—-------é ---------- 38 LIST OF FIGURES PAGE I. Dosage-Probit Mortality Line for Different Strains of A; Aegypti Treated with Fenthion . ----- 28 II. Toxicity Tests Performed at 16°C, ZAOC, and 30°C, - for A; Aegypti (Notre Dame) --------------------- 29 III. Dosage—Probit Mortality Curve for Q; Resturans Tested at 24°C. --------------------------------- 36a vi INTRODUCTION The physical and biological components of the environ- ment in which mosquito larvae are controlled with larvicide are complex. They vary from one location to another, even in a limited area, and do not remain constant in time.‘ The types of breeding sources harboring mosquito larvae are diverse too, and no single remedy can be found or recommended for effective control under all circumstances. Mosquito control efforts have also become increasingly difficult due to the development of insecticide-resistant populations. Since improper control can arise from all these factors, it is important to determine the correct cause for the opera- tional failures. It was the purpose of this investigation to determine the factors causing the failures. There was suspect of not just insecticide resistance, but ecological factors in the mosquito larvae habitat that affect toxicity tests. Temperature, organic and chemical content, and pH were selected for evaluation as possible factors affecting the mortality rates of larvae tested with fenthion. Toxicity tests performed on mosquito larvae showed variations in larval mortality to a given concentration of fenthion. Some of the possible causes for this are due to improper bioassay techniques and variations in sex, nutrition, instar and day-to-day fluctuations in the composi- tion of water in which the larvae were tested. To achieve the high degree of uniformity necessary for toxicity tests a standardization of all bioassay techniques, size, age and stage of nutrition of the mosquito larvae was followed throughout the experiments. REVIEW OF LITERATURE The earliest work investigated on rearing procedures for mosquitos was that of Boyd in 1926. He reared larvae of Anopheles quadrimaculatus say and_A. crucians at a temperature of 70° to 80°F. Marble chips were added to correct the pH, and Fleischmann's yeast served as the larval food. The first large-scale rearing of A._guadrimaculatus and A. crucians was performed by Boyd and Cain (1932). Larval food consisted of hay infusion and Fleischmann's yeast. Additional descriptions of rearing techniques were made by Brennan and Harwood, (1953); Busvine, (1957); Jones, (1957); Glover, (1931); Greenwald et 31, (1948); Lesson, (1932); Liles EE.§A3 (1960); MacGregor, (1924); Pefle.aE.e1; (1946); Repass, (1952); Trembley, (1944); and wray, (1946). In the literature as indicated by Busvine (1957), A; aegypti has been the most convenient insect in bioassay tests because it can be easily reared in the laboratory. The work of Granett and Powers (1937) and Johnson (1937), apparently were the first attempts at rearing and maintaining .A. aegypti. Their improvements were a constant temperature cabinet for rearing larvae and an improved larval diet of Fleischmann's yeast and blood albumen. Similar work was con- ducted by Johnson, (1947); Casanges gt_al., (1949); to improve rearing methods for A. aegypti used in bioassays of insecticides. The necessity of low oxygen concentration for the hatch- ing of Aedes mosquitos was shown in 1941 by Gjullin, Hegarty 4. and Bollen, and confirmed by Burgess (1959). He indicated that for quantity rearing of A. ae ti, the immersion of eggs in distilled water containing less than 2 p.p.m. of dissolved oxygen was very efficient in giving better hatches. Containers which exposed little surface of deoxygenated water to the air were most efficient for hatching eggs. Methods of maintaining egg production in laboratory colonies of mosquitoes have been observed in many of the works previously mentioned. It usually consists of making some source of blood available to the females at regular intervals. According to Trembley (1955), various small animals such as mice, guinea pigs, rabbits, and hamsters were held quietly while the mosquitoes engorged blood. To avoid this bothersome task of using live hosts, Lea gt 31. (1955) and Knierim gt El- (1955), used other proteinaceous sources, supplied on gauze- covered cotton pads. One of the sources used was a mixture of nine parts citrated beef blood and one part honey. Mos— quitoes in the laboratory of Liles 23 51. (1960), have been reared on a diet of citrated hemolyzed beef blood containing 10 percent sucrose. The addition of sucrose acts as a phagos- timulant if the blood is frozen. Blood lacking whole red cells is not engorged unless sweetened. The method of Liles gtlgl. (1960), was based only on work with the Ohio State strain of .50 aegypti. Evidence shown by Porter gt a1. (1961), suggests that adult female A. aegypti require blood meals and a honey- water solution while the males will survive on the honey—water alone. Porter and his co-workers designed a feeder out of a three inch high, 10 dram plastic vial which prevented eva- poration of the honey solution and was impervious to egg deposition. Many methods have been used in rearing A. aegypti in the laboratory. Recent work of Burgess (1959), and Klassen (1965), give a simplified method of rearing A. aegypti. Eggs used by Burgess came from a colony of mosquitos maintained on a 10% honey solution and allowed to engorge regularly on a guinea pig. The eggs were laid on moist strips of filter paper and then conditioned. Conditioning was accomplished by keeping the eggs moist for three days and then storing them at 23° - 300 C. and 30-60% humidity until used. Eggs were hatched in contain- ers having as little surface of the deoxygenated water exposed to the air as possible. Klassen (1965), described hatching eggs in regular tap water and through careful feeding obtained uniformly and rapid- ly developing larvae. KlassenS larval diet consisted of 1 part wheat germ, 1 part brewer's yeast, and 1 part Kellogg's concentrate. METHODS OF MEASURING TOXICITY 0F INSECTICIDES Lewallen and co-workers (1959), and Busvine (1957), agree that mosquito larvae reared in the laboratory were more uniform in response to chemical tests than larvae collected in the field. The optimum test would be full-scale field trials; but such factors as nutrition, exposure to insecticides, fatigue, and the difficulty of providing adequate replication to make up for great variability in mosquitos would not always produce unambiguous results. Hoskins and Craig (1962), also agree that 6 the use of bioassay to measure susceptibility of insects in field trials often must be made under very primitive con- ditions. The large numbers of experimental insects needed was also found difficult to obtain at times. These scientists agree that field trials should be postponed until laboratory tests have been performed. Tests on field-collected larvae are questionable, since the above factors are known to in- fluence the results. Campbell £3 31. (1933), (1934) and Jones gt 31. (1933), apparently described the first convenient methods for using Culicine mosquito larvae as test insects in bioassays. The tests were made in 120 cc. Erlenmeyer flasks containing 100 cc. of distilled water. At least four replications of each material were made. The desired acetone solution were pipett- ed into the water containing the 4th. instar larvae. A control using acetone was included in each test. Deonier _e_t_ 21- (I946), Bushland (1951), Nolan (1950), and Pal (1952), were some of the first to make use of mosquito larvae for routine bioassay experiments. All these methods were similar and soon a standard technique of insect tests for resistance was condensed into a WHO Technical Report (Symes ,§t_§1., 1962). Early work of Abbott (1925), gave a method of computing the percent effectiveness of an insecticide when actual counts of living and dead organisms in both treated and untreated samples were available. It was obvious to him that the insects which died a natural death must be considered, and by subtracting the percent mortality in the check from the figure for those dead in the treated he calculated the effectiveness of the treatment. Busvine (1957), said that such a qualitive statement can be positive if by replication an indication of the standard error can be appended. The only sound method of quantitatively comparing insecticides is by a ratio of equal toxic doses (Busvine, 1957; Bliss, 1938; Hoskins and Craig, 1962). Hoskins and his co-workers (1952) pointed out that one of the ways of assaying insecticides to find their critical doses was by tests based on quantal response, or simply the all-or-nothing reaction. Basically, it is the method used to estimate the magnitude of the dose that is sufficient to produce death within a portion of the test pop- ulation. Data from the mortality tests, in the form of percent mortalities, is plotted against dosages resulting in a dis- tribution in the form of an asymetrical sigmoid curve. Such sigmoid curves are difficult to use for the interpolation of LD5O's not actually tested (Hoskins gt 31., 1962). Therefore, the straight line regression curve was devised such that both variables were transformed into what is called a log-dosage- probit line (Bliss, 1934; Hoskins 23H§1., 1962; Busvine, 1957). Recent work of Busvine (1957), and Litchfield and Wil- coxon (1949), provide calculations and graphic methods for approximating the LDsO and the slope of the curve. These are obtained by plotting percent mortalities on logarithmic probability paper versus dosages. Once a minimum of three points is obtained, a straight line connecting the three points is fitted by eye. The critical dosages are interpolated with remarkable accuracy and methods are provided for detecting parallelism and the degree of heterogeneity of the lines. There are several factors that affect the interpolation of the regression line slope (Busvine, 1957; Synes gt g1., 1962; Bliss, 1935; Hoskins and Craig, 1962; WHO 1960; and Shepard, 1960). The slope is dependent on using the maximum number of specimens per test, as well as in the variance in the population of insects tested. The precision and validity of the test methods and data also determine regression line accuracy. INTRINSIC AND EXTRINSIC FACTORS AFFECTING INSECT TOXICITY TESTS. Many investigations have concerned factors that affect theresults of insect toxicity tests. Shephard, (1960); Bus- vine (1957); Hoskins and Craig, (1962); and Thomas, (1965); have categorized these factors into: (i) phySical state of insecticides in solution, (ii) intrinsic factors, and (iii) the state of the environment, or extrinsic factors. Busvine (195W; and Hoskins (1962), agree that physical and chemical factors of insecticides inevitably influence the susceptibility of insects to these poisons. In bioassay ex- periments, larvae are generally exposed to toxicants in the form of colloidal suspensions, which consist of the insecticide dissolved in a water miscible solvent (e.g. acetone) which is added to water. Hawkins (1956), indicated that the amount of water-soluble Solvent used affected the toxicity of DDT. However, Busvine (1957, p. 83), showed that the use of sol- vents such as acetone or alcohol does not give different results as long as they are used at the same rates. For some tests the quantity of solvent may be varied slightly for convenience in dosing. The quantity of certain insecticides (e.g. DDT) in colloidal suspensions declined over a period of time due to the deposition of particles on the test containers (Kruse, 1952). His data showed that the method of dispersion of the toxicant and the type of surface of the container strongly influence the amount of DDT remaining in suspension upon standing. Busvine (1957), Shepherd (1960), Hawkins (1956), Bowman gE.§1. (1958), and Curtis (1961) evaluated the use of various containers and concluded that sedimentation was more rapid in paper than in glass containers. Paper con- tainers are a good source of active ions which cancel the zeta potential of the suspension, allowing a huge propor- tion of DDT to be deposited on the walls. Other physical and chemical factors such as diameter increase in test containers, volume of water, exposure period, and nature of containers affect toxicity tests (Schmidt, 1958; WHO, 1960; Pal, 1952; Busvine, 1957; and Curtis, 1961). Intrinsic factors of mosquitos such as sex,age, size and specificity definitely interfere with and affect mortality counts in bioassays with insecticides. Busvine (1957, p. 12), very clearly states that specificity in insects creates the problem of a poison being effective against one species and 10 showing no effect against another species. The element of susceptibility levels of different insects occur along with the resistance factor. In recent years, the term resistance has been coined to describe strains of insects that have developed a tolerance to certain insecticides. (Lewallen, 1961; Lewallen and Nickolson, 1959; Mulla gt_§l., 1964; Phillips, 1940; Hoskins, 1959; Yates, 1950; Matsumura, 1963; and Brown, 1957). The mentioned names are only a partial review of resistance in mosquitos. Various aspects of resis- tance have been exhaustively covered in the past. Work by Forgash (1956), indicated that the male American cockroach, Periplaneta americana L., may have been more variable and susceptible to certain insecticides than the female. Lewallen (1965), attempted to determine if similar variability occurred in the susceptibility in larval stages of male Culex inornata, Q, bpharti, and Agdgg gigromaculig (Ludlow). He concluded that there was no difference between the sexes in either variability of response or in sensitivity to poisons. Differences in mortality that occurred between replicates were not due to a preponderance of one sex or the other, which might not be true for other species. Susceptibility levels vary greatly, due to differences in physiological age of the mosquito larvae (Busvine, 1957). In particular, there is a steady rise in resistance through- out the 4th instar. Late 3rd and early 4th instar larvae were specified, since resistance in pupae is much greater, and any measurement would be difficult if a large percent of 11 pupae developed during the test (Mitchener, 1953; Yates, 1950; and Thomas, 1965). Shephard (1960) and Busvine (1957), considered the in- direct effects of the environment in which an insect has been reared, and the direct effects during and after exposure to the insecticide separately. These workers have stressed the importance that temperature has on the action of insecticides. Since temperature affects the size of insects, therefore affecting their susceptibility, it is necessary to select a suitable rearing temperature. . The optimum water temperature for A. aegypti larvae are in the 770 - 840 F. range with time to pupation being five to seven days. Although more rapid growth occurs at 860 - 880 F., the adults that emerge are smaller than those reared at optimum temperatures (Fay, 1964). The temperature at which an insect is reared may also affect the type of lipids in the body (Busvine, 1958). Temperature is probably the one single factor that direct- ly affects insects during the period of exposure. Temperature variations also affect the action of the insecticide. At room temperature most insecticides are effective, so it is the extremes of temperature which are significant. Dustan (1947), Vinson and Kearns (1952), Hoffman and Lindquist (1949), Woodruff (1950), and Burgess and Sweetman (1949), found that DDT exhibits a negative temperature co- efficient, i.e., it is more lethal at lower than at higher temperatures. According to Guthrie (1959), DDT, pyrethrum, and lindane, gave better results at lower temperatures, and dieldrin and aldrin gave better results at higher tempera- tures. Temperatures during post treatment can influence the storage of insecticide, the toxifying processes, and elim— ination of the insecticide (Busvine, 1957). Nutrition definitely plays a role in the susceptibility of insects to insecticides. Hurlbut (1945), and his co- workers apparently were the first to study the nutritional factors affecting the larvicidal action of an insecticide on mosquitos. Larger amounts of DDT were required to con- trol larvae of Culex guinquefasciatus that lived in foul water. Results from his study indicated that the amount of DDT required to kill the culex mosquitos varies according to the media under test. It is stated vaguely that organic matter and pH should be considered in mosquito control work. Lewallen and Wilder (1963), did related work with ecological factors. They devised a method of preparing standard polluted water with steer manure. DDT, fenthion and other insecticides were compared as to the efficacy against fourth instar Culex pipiens quinquefasciatus Say in polluted and tap water. DDT and fenthion, based on LG comparisons, performed better in polluted water than in tap water. Phillips and Sevingle (194» made a study on the effect of diet on mosquito resistance to rotenone and nicotine. They indicated that resistance could be controlled to 13 a very large extent by artificial methods of rearing, es- pecially in the quality or quantity of food supplied. Nutrition is an important factor in the development of mosquitos and is a factor in the susceptibility to in- secticides as shown by David and Bracey (1946). Blood-fed 'A. aegypti were less susceptible to pyrethrin sprays then water or sugar-fed individuals. Thomas (1965), observed the ecological factor of tap water versus distilled water. Larvae of Culex pipiens fatigggg tested in distilled water gave higher L050 values than those tested in tap water. Effects of varying depths of water having identical surface areas on larval mortality using commercial malathion and parathion was conducted by Rai and Lewallen (1960). They concluded that a certain dosage was less effective against .9;.E; qginquefasciatus as the depth of water was increased. This was attributed to the homogeneous distribution of the insecticide throughout the water. Their work suggests that depth of water should be considered in effective larval con- trol. Weidhass, Gahan, and Ford (1955), made a study on the effect of temperature, aeration, pH, and the presence of soil on the toxicity of various insecticides to mosquito larvae. This study was developed to determine factors re- sponsible for the loss of effectiveness of insecticides applied in irrigation water. Temperature, aeration, and pH did not affect the loss in toxicity. The presence of 14 sand caused little loss in toxicity. Evaluations of fenthion against mosquito larvae have been performed by Jakob and Schoof (1963) , and Mulla _e_t_ 3;. (1964). Jakob and Schoof used the standard WHO larval test method to determine the LC for fenthion using larvae of 95 dieldrin-resistant A. qgadrimaculatus, dieldrin-DDT resistant .§;.£; quinquefasciatus, and DDT-resistant A. aegypti. The concentrations (p.p.m.): 0.01, 0.005, and 0.02 were the respective LC '5 for the mentioned larvae. 95 Mulla and his co-workers (1964), evaluated fenthion using the susceptible strain of Q. P. quinqgefasciatus. To determine toxicity, twenty five fourth instar larvae were placed in 100 ml tap water in wax paper cups. Desired con- centrations of insecticides were added and mortality was Observed after twenty-four hours. The LG for DDT was 50 0.035 p.p.m. The LC 's for Culex tarsalis and gnggromaculis, 50 tested with DDT was 0.30 and 0.65 p.p.m. respectively. The LC '3 for the above larvae treated with fenthion was 0.0056 50 and 0.0068 respectively. In all the tests conducted, fenthion was more effective as a mosquito larvicide. MATERIALS AND METHODS Strains of A. aegypti (Notre Dame and Rockefeller)were obtained from the University of Illinois, and larvae of Q. resturans were collected from tire ruts near East Lansing, Michigan. Any resistance by the Q. resturans mosquitos to- wards fenthion and DDT is unknown since no records of in- secticidal applications in this area are available. However the strain of_A._gggyp§i is susceptible to the mentioned insecticides. The procedures in handling, rearing, testing and ob- serving the mosquitos was kept as uniform as possible and any mortality in the controls nullified the experiment under progress. Adult A. aegypti were reared in a controlled tempera- ture room at 24° 0 $120 C and at a relative humidity of approxi- mately 40-50 percent. The colony was kept in a 2' by 2' screen cage and fed a 10 percent honey solution in a specially designed container (Porter, Kozuchi, and Kuck, 1961). A mixture of raw beef blood, sodium citrate, (20 grams/one- half gallon blood) and honey (10 ml/90 ml blood) was poured into a beaker covered with Parafilm which was then inverted on top of the cage, so that the females could pierce the membrane and engorge on the blood mixture. A simpler method was also used, whereby fresh, blood-soaked cotton was placed in a petri dish in a cage and renewed every two days. 15 16 A water filled 250 ml beaker lined with a paper towel was the site for oviposition. When sufficient eggs were deposited on the towel, it was removed and conditioned for three days. Conditioning was accomplished by placing the eggs on the paper towels on a water-soaked cotton pad and allowing them to dry naturally for three days. In order to hatch the eggs the paper strips were moisten- ed for a few hours and immersed in deoxygenated water. Containers which exposed little surface of deoxygenated water to the air were the most efficient in hatching eggs. Several hours later the first instar larvae were emptied into a large five gallon container half filled with tap water. Only a pinch of larval food consisting of 1 part wheat germ, 1 part brewer's yeast, and 1 part Kellogg's concentrate was added. As the larvae progressed, more food was added at regular intervals until they reached the third and fourth instar. Tests with insecticides were performed when the larvae reached the late third and early fourth instars. g. resturans collected in the field were scooped into a large container for transfer to the laboratory. The tem- perature was recorded and a 1000 m1 sample of the water was taken for later chemical analysis. Attempts to colonize this species failed, due to it's failure to reproduce in captivity. Other species were obtained by collecting third and fourth instar larvae from natural breeding places and their life cycle was completed in the laboratory. When the adults emerged, abnormal thrashings were observed in 17 the abdomen. Upon examination of the specimens, large mermithid nematodes were found. Since this parasitization would naturally interfere with insecticidal tests, bio- assays with these species were discontinued. TESTING METHODS. Technical samples of 84 percent fenthion and 72.2 percent DDT were made up as weight/volume solutions in acetone. A 1.0 percent and 0.1 percent stock solution were made for fermhion and DDT respectively. Preliminary tests were conducted to determine the L050 for the larvae before undertaking more complicated experi- ments. Basically the test was conducted with 100 ml of disstilled water per crystallizing dish containing 25 late third and early fourth instar larvae which had been trans- ferred from the rearing dish by a special scoop designed by the author. This scoop was a necessary tool in transferring the larvae, since care must be taken not to transfer excess water to the crystallizing dishes. A control was run with each group of treated larvae. Mortality in the controls was consistently nil and any control showing mortality nullified the test. Contamination was reduced to a minimum by rinsing the ancillary equipment in alkali and acid baths before wash- ing. The desired concentrations of insecticides were pipetted into crystallizing dishes, each concentration being replicated three times. Larvae treated with fenthion were held at 2400 for 24 hours and they received no food during this period. Those treated with DDT were held for 48 hours before mortality 18 counts began. Mortality counts included larvae found dead or moribund. The larvae which responded normally when probed were counted as alive. Average percent mortalities were plotted on log-probit graph paper as percent mortality versus dosage in p.p.m. At least three points were used in obtaining a straight line which was fitted to the points by eye. Once the LC5O was determined for the species of mosquitos, further investiga- tions were conducted to analyze ecological factors that might contribute to their mortality when treated with fen- thion. FACTORIAL EXPERIMENTS. The same procedures for rear- ing, handling, testing, and obServing larval mortality in the L050 tests were used in the factorial experiments. The 2x2x2 factorial design was the best method that could be used to evaluate the various ecological factors that affect mortality of larvae treated with fenthion and the DDT standard. (Snedecor, 1957). .0 Experiments were set up so that the three factors; pH, organic matter, and hard versus soft water were sub-divided into two levels, resulting in eight treatment combinations. The pH factor of the test solution was set at two extremes from the mean by addition of HCl and NaOH to give solutions of pH 5.0 and pH 9.0 respectively. The hard versus soft water factor requires tap water having a high mineral con- tent and distilled water which is mineral free. Several substances were considered for the organic factor, but due to l9 contamination only diatomaceous earth, low in organic con- tent, and wheat germ, high in organic content, were used in the experiments. For clarity Table I, shows the design of all the factorial arrangements of treatments for the experiments at various temperatures. This design was also used in the experiment assaying the ecological factors affecting the mortality of the swamp mosquitos, Q, resturans at 1900. In Table I treatments 1, 3, 5 and 7 have three replica- tions and a control all of which contain 1 ml of organic substance A (diatomaceous earth). The crystallizing dishes in rows 2, 4, 6 and 8 contained 1 ml of emulsified wheat germ. Then the first two treatments were filled with 100 m1 of tap water adjusted to pH 5. Treatments 3 and 4 were filled with distilled water adjusted to pH 9, and the fifth and sixth ones were filled with 100 ml of tap water adjusted to pH 9. Distilled water at pH 9 was poured into the cry- stallizing dishes of treatments 8 and 7. The ninth and tenth treatments contained 100 ml of distilled water. After the 100 ml water solutions were poured into the crystallizing dishes, twenty-five late third and early fourth instar A; aegypti larvae were transferred into each of the forty test dishes. The L050 dosages for A. aegypti and g; resturans were the insecticide concentrations used in these experiments. The dosage of 0.0037 p.p.m. of fenthion was pipetted into each crystallizing dish of the first nine treatments and 20 .Htheoo tam psospmohp hog mm>ama Opeswmos mm Um: soapmoaamoa Scam nom>hma mQMMSQmos Noaso so psosflhmme MOM Sago mm: HN pemspmohe "maoz I I I hope: msmsm tam soanpcom .Hx I I I thmtsmpm Sm . N I I I Moono coaepemm .NH I I I I Show among pmom m .HHH> I I I I enema msomomEOQMHQ pmom m .HH> I I I I Show pews; them a .H> I I I I spasm macmomsoumflm them m .> I I I I Show smog; pmom m .>H I I I I enema mSOoomEOPmHQ pmow m .HHH I I I I Show pews; them m .HH I I I I Spend mdomomEOPmHQ them m .H been honesz HHH HH H peopsoo omemwho make me sopasz have; Honpsoc mQOHmeHHQom mesmEpmohB coenpsoh spas emanate Hpmhmo< moped mo hpflamphoz how mpcospmohe mo swflmmm oammm H mqm<9 21 0.0007 p.p.m. of DDT was pipetted into the dishes of treat- ment 10. The same volume of acetone was pipetted into the control of each treatment. Thus there were three replica- tions and one control per treatment. All dishes were capped with perforated plastic covers to reduce evaporation. The forty test dishes were held at 24°C for 24 hours and the DDT at 48 hours before mortality counts were made. The experiment was repeated at two temperatures (16°C and 3000) and with the species collected in the field. The field specimens of Q, resturans were reared in the five gallon containers until they reached the fourth instar stage for testing. Twenty-five specimens were trans- ferred to the individual test dishes containing the treated solutions of water. One extra treatment, number eleven, was added which consisted of the water collected from the tire ruts in the field. It was tested with .0037 p.p.m. fenthion. To understand the significance of the eleventh treat- ment compared to the other tests, it was necessary to analyze the soluble and insoluble material in the swamp water. In the laboratory, chemical analyses were performed on the samples of water collected to determine the pH, percent organic matter, and percent insoluble salts. Five hundred m1 of the swamp water was poured into a flask immediately after returning from the field. The pH was recorded and the water was evaporated by heating over an open flame until approximately thirty ml. remained. This residue was transferred to an extracting flask. Diethyl ether 22 and redistilled hexane were used for the extraction. Twenty ml of ether was added to the water and the flask was shaken vigorously. The mixture was allowed to separate, the water was removed, and the ether layer was added to a tared beaker. The process was repeated and the total ether extract was evaporated on a steam bath until only the residue remained. The residue and beaker were oven-dried and weighed the next day to determine the percent of ether solubles present. The water removed from the ether extraction was poured into another extracting flask, and mixed vigorously with redis- tilled hexane as in the above process. The percent of organic material and insoluble salts in the water was cal- culated. PREPARATION OF MORTALITY DATA FOR CDC 3600 COMPUTER. Data from the bioassays of A. aegypti and g. resturans were arranged in chart form for ease of preparation of the card deck which was fed into the 3600 computer. The two-way analysis of variance was the best routine for the factorial experiments undergoing analysis. Input cards were prepared as described by Kiel, Kenworthy, and Ruble (1963). FARCEP, or Factorial with Replicates, was the experimental design used throughout the program. The first analysis of variance (AOV) was programmed from A. aegypti larval mortality data of the three temp- erature factorial experiments. Twelve variables were involved, consisting of the eight treatments in each experi- ment, temperature, replications, fenthion check, and DDT 23 standard. Data cards provide data for the AOV and there was a card for all the variables associated with each ob- servation, including location within the experimental design. A given variable occupied the same column on all cards. Once the program.was completed and all input cards were completed the deck was verified on the verifer (056) for correct punching and order. This problem deck was a collec- tion of cards containing the same variables for all points and all of the collections taken together was also called a deck. In this case a batch processing of three analyses was done for the same job. The F values were recorded in chart form and the significance of the individual values were based on the theoretical 5% and 1% points of F for convenient combinations of the degrees of freedom (Snedecor, 1957). A second AOV was programmed from Q. gesturans larval mortality data. There are eleven variables in this experi- ment: eight treatments, fenthion check, DDT standard, and replications. The input cards were punched, verified, and processed by the CDC 3600 computer. Again, the F values were recorded in a chart and the significance was determined as above. Data collected from the analysis of variance computer data sheet was studied and evaluated to determine if eco- logical factors affect the mortality of larvae treated with fenthion. RESULTS OF REARING METHODS AND TOXICITY TESTS RESULTS OF PRELIMINARY EXPERIMENTS. New techniques and methods were developed for rearing and testing A; aegypti larvae. .A; aegypti eggs that were moistened and immersed in deoxygenated distilled water showed a distinct increase in hatch rates. First instar larvae that were transferred to large five gallon jugs and fed properly, developed uniformly through the fourth instar at room temperature (24°C. 2°C.). Adults fed on a 10 percent honey solution survived 2-3 months. Females fed on fresh citrated beef blood at two day intervals were highly fertile in egg production. Preliminary tests to determine LC5O's of fenthion for the Rockefeller strain of A;_aegXEti larvae, were made in pyrex crystallizing dishes and parafin coated paper cups. Tests performed in paper cups showed variations in mortality rates. When a parallel test was made using glass crystalliz- ing dishes, a significant difference in mortality was noted between the two tests. In Table II, the tests indicate that 50% of the larvae died in the glass containers and only 5% died in the paper containers. Table II. Mortality of the Rockefeller Strain of A; Aegypti Larvae Treated with Fenthion in Glass and Paper Con- tainers. Container p.p.m. Number Tested % Mortality , (Approx.) glass disHes 0.0052 76 50% paper cups 0.0052 76 5 % 2L 25 From the previous table, it can be concluded that glass containers are better for testing fenthion. Once the problem of test containers was solved, tests with fenthion against the Rockefeller strain of A. aegypti larvae were conducted to determine the LC50° The log-probit graph in Figure I, represents the normal response of the Rockefeller strain to fenthion. The L050 determined from the line was 0.0052 p.p.m. A comparison using the Rockefeller strain was made to determine the effect of tap water versus distilled water on the LCSO of fenthion at room temperature. There was no significant difference between the LC50 with tap water and with distilled water. Wheat germ was the organic substance selected from a group of organic materials, to check the L050 for the Rocke- feller strain of aedes larvae tested with fenthion in a highly organic test solution. Conflicting results occurred with rabbit pellets placed in the test solution. Tests per- formed with Gas-Liquid Chromatography on the material showed definite chlorinated hydrocarbon contamination. Rabbit pellets were eliminated as a test material for the organic effect on the L050 of fenthion. Consistency in later con- firmation tests of organic matter indicated that wheat germ was an excellent test material and free from contamination. Preliminary tests were conducted to determine the L050 of fenthion at 20°C. The tables were prepared giving mortality rates at various concentrations and the LC50 value was 26 obtained for the late third and early fourth instar larvae of the Rockefeller strain of A. aegypti. Figure I, illus- trates the dosage-mortality regression lines obtained from the data. The L050 value was 0.0025 p.p.m. Repeated tests showed the L05O for 20°C. was lower than the LC5O obtained at 24°C. CLASSIFICATION OF CULEX RESTURANS, AAH)AEDES STIMULANS. Specimens were collected June 24, 1965 from shaded, water filled ruts south of East Lansing, Michigan. The larvae were members of the family Culicidae in the order Diptera, and classified as the species resturans, according to Ross (19A?)- Other larval specimens were collected in vernal ponds in the central Michigan area and their life cycle was com- pleted in the laboratory. The adult females were keyed down and classified as g. stimulans according to Ross (1947). The characteristics that separated this species from other re- lated ones was the identification of three epimeral bristles on the mesopleurae. There was not sufficient time to perform any toxicity tests on the late fourth instar of A. stimulans. In a short time, pupation occurred and adults emerged. Abnormal move- ments were noticed in abdomens of approximately 80% of the females and 40% of the males. Further investigation showed that the adult mosquitos were parasitized by a mermithid nemotode. Dr. H. E. Welch of the Research Institute of the Canada Department of Agriculture, Belleville, Ontario 27 identified the nematodes as belonging to_genus Hydromeris sp. Surveys of this parasite were made in the lower peninsula of Michigan during the spring of 1965. Parasitized mosquitos were found in the Southeastern section of the state. (un- published data). EFFECTS OF ECOLOGICAL FACTORS. When the Rockefeller strain of A. aegypti reared in the laboratory died out, a colony of the Notre Dame strain of A. aegypti was started. Thisstrain was the basic one used in all the rearing experi- ments except the one analysis on C. resturans. The L05O value was derived from the dosage—mortality curve in Figure 1. Compared to the L050 of 0.0052 p.p.m. for the Rockefeller strain, the LC5O of the Notre Dame strain was 0.0035 p.p.m. Apparently there was a difference in the response of the two strains to fenthion. The three ecological factors under investigation, pH, water type, and organic constituency, were broken down into subfactors: acidic (pH5) and basic (pH9), hard and soft water (tap versus distilled), and diatomaceous earth and organic wheat germ respectively. All possible combinations of the subfactors resulted in eighttreatment combinations. (Table l). The eight treatments were tested with fenthion at three temperatures: 16°C,, 2400., and 3000. First observations of the mortality rates in the experi- ments at the various temperatures indicated that DDT at a concentration of 0.0007 p.p.m. in acetone has a negative temperature coefficient in relation to fenthion, at 0.0035 p.p.m. 28 .soaspsom spa: acetate apmwmoa .m mo msflwhpm pectomMHm Mom mafia hpflamphoz panonmIowmmom .E.m.m ma soapmspsooaoo oOOON pm CHMMPW hmfiflmMmMOOK o . .OOdm pm EHmhpm MOHHoMoxoom IIIII .oo¢m pm cashew mama mapoz soo. moo. .H enemas moo. om Om 04 on ow on om om sqtqoad up 51119110“ 29 am mm 1. ZOHmBzmm Om D hpflamphoz Omaho>< ma P . .Aosmm ohpozv HpmNmo< {fl not .ooom est .oosm ..ooma an costoetom memos spaoawos ma III 3 NH .m mesmHm OH 1 Toots no em hOOOm 30 (Figure 2). The concentrations of DDT and fenthion were selected as experimental dosages because they covered the wide range required in testing mortality rates in these experiments. Mortality data from the three experiments were tabulat- _ ed and since all experiments were conducted as uniformly as possible, the batch processing of these three analyses was done for the same job. A program was developed using the analysis of variance with the FARCEP design. The F statistic values from the computer analysis were computed on the basis of a fixed effects model, comparing fenthion to the various treatment combinations and DDT (Table 1). The significance of all of the F values was judged at the 5% and 1% levels (Snedecor, 1947 p. 246-249). 0 - TREATMENT I. ACIDIC, HARD WATER, AND DIATOMACEOUS EARTH. Treatment I conducted at 160, 24°, and 30°C., was significant at the 5% and 1% levels. The low mortality at 16°C may be attributed to the absorption action of the diatomaceous earth and the slow action of fenthion at low temperature. At room temperature the mortality was near the fenthion check, indicating increased mortality toxicity of fenthion in acidic hard water mixed with diatomaceous earth. Reduced mortality was observed at 30°C., signifying possible evaporation at high temperatures and absorption by the earth material. TREATMENT II. ACIDIC, HARD WATER, AND ORGANIC WHEAT GERM.I This treatment conducted at various temperatures was significant at 16°C. (F value 42.6) and only slightly 31 Table III Analysis of Variance of Treatments in Table I for Aedes Aegypti Source of 2 Treatments variance 8X2 df X F—value I. 16 C. 565.8 2 282.9 242.5 ** 24 C. 220.5 1 220.5 189.0 ** 30 C. 100.0 2 50.0 42.8 ** Error 14.0 12 1.7 Total 900.3 17 II. 16 C. 289.0 2 144.5 42.6 ** 24 C. 14.2 1 14.2 4.2 * 30 C. 18.1 2 9.1 2.7 . Error 40.7 12 3.4 Total 362.0 17 III. 16 C. 152.4 2 76.2 40.4 ** 24 C. 24.5 1 24.5 13.0 ** 30 C. 57.3 2 28.7 15.2 ** Error 22.7 12 1.9 Total 256.9 17 IV. 16 C. 42.3 2 21.2 9.8 ** 24 C. 174.2 1 174.2 80.4 ** 30 C. 331.4 2 165.7 76.5 ** Error 26.0 12 2.2 Total 574.0 17 V. 16 C. 133.0 2 66.5 79.8 ** 24 C. 410.9 1 410.9 493.1 ** 30 C. 324.1 2 162.1 194.5 ** Error 10.0 12 0.8 Total 878.0 17 VI. 16 C. 193.0 2 96.5 96.5 ** 24 0. 93-4 1 93-4 93-h ** 30 C. 694.1 2 347.1 3A7.1 ** Error 12.0 12 1.0 Total 992.5 17 32 Table III (continued) Source of 2 2 Treatments variance SX df X F-value VII. 16 C. 234.1 2 117.1 105.4** 24 C. 288.0 1 288.0 259.2** 30 C. 342.3 2 171.2 l54.1** Error 13.3 12 1.1 Total 877.8 17 VIII. 16 C. 13.0 2 6.5 1.2 24 C. 107.6 1 107.6 19.2** 30 C. 230.1 2 115.1 20.5** Error 67.3 12 5.6 . Total 418.0 17 IX. 16 C. 49.3 2 24.7 16.4** 24 C. 46.7 1 46.7 31.1** 30 C. 140.4 2 70.2 46.8** Error 18.0 12 1.5 Total 254.5 17 NOTE: All figures in AOV rounded off to nearest tenths., F-values checked for significance at 5% and 1% levels from Table 10. 5. 3, p. 246-249 (Snedecor, 1957). 33 significant (F value 4.2) at 2400. The treatment at 1600. showed a mortality higher than the fenthion check, probably indicating that the highly organic wheat germ absorbed a per- centage of fenthion, and both were consumed then by the Agdgg larvae. In the slightly significant test (F value 4.2) at 24°C. the presence of wheat germ increased mortality over the check. The test conducted at 30°C. was not significant, indicating mortality of the treatment was the same as the check. The presence of a highly organic substance such as wheat germ increased the larval mortality. A review of the first two treatments and comparison to the effectiveness of fenthion, indicated that the presence of diatomaceous earth greatly reduces the mortality rate of .A. aegypti larvae at 1600. The presence of high organic wheat germ in the 16°C. and 24°C. tests increased larval mortality. Acid conditions may have also attributed to the increased mortality. TREATMENT III. ACIDIC, SOFT (DISTILLED) WATER, AND DIATOMACEOUS EARTH. The F values: 40.4, 13.0, and 15.2, definitely showed that the treatments performed at 16°, 24°, and 30°C. were significant, respectively. These combinations of factors (pH5, soft, diatomaceious earth) were most impor- tant at 1600. Mortality was higher than the fenthion check, possibly signifying that soft water (distilled) is the factor of concern, since in Treatment I the earth material in hard water reduced mortality. The performed tests of Treatment III indicate that the mortality is relatively stable, compared 34 to the direct increase of mortality with increased temp- eratures of the fenthion check. TREATMENT IV; ACIDIC, SOFT WATER, AND ORGANIC WHEAT GERM. According to the 5% and 1% level (Snedecor 1957), the fourth treatment performed at 16°, 24°, and 30°C. was signi- ficant. The F value (9.8) for 16°C. was least important of the three tests. The mortality rates at the various temp- eratures were fairly constant throughout the tests, point- ing out that the treatment is important in its relation to the checks. Since the mortality of the larvae increased directly with the temperature of the fenthion check, it can be concluded that the combination of acidic, soft water with wheat germ definitely affects the larval mortality. An analysis of the last two treatments promoted several Observations. The fourth treatment was very important in that the larval mortality remained constant as the fenthion check larval mortality increased with the temperature. This also was evident in the third treatment. Distilled water was important in the third treatment since the diatomaceous earth and acid conditions were the same in the first and third treatments. TREATMENT V. BASIC, HARD WATER, AND DIATOMACEOUS EARTH. The F values} 79.8, 493.1, and 194.5 for 160, 24c, and 30°C. are all highly significant. Mortality at 16°C. was lower than the check, but not as low as in the first treatment where the same existed under acid conditions. It is evident that basic conditions at pH 9.0 affect the mortality of 35 .A- aegypti larvae. Tests at 24°C. showed reduced mortality to the check and to treatment number I. The basic condition (pH 9.0) and the absorption property of diatomaceous earth accounted for the reduced mortality. This same phenomenon occurred in the 3000. test, but the mortality rate was greatly reduced. TREATMENT VI. BASIC, HARD WATER, AND ORGANIC WHEAT GERM. Experiments with this treatment were significant at the three temperatures, especially at 30°C. Mortality in the larval population was high, due to the highly organic wheat germ, under basic (pH 9) conditions. The 24°C. experi- ment showed decreased larval death compared to the fenthion check, but it was the same as the 160C. mortality. Larval mortality was relatively stable at 16°C. and 24°C. but ,A. aegypti larval mortality was nil at 30°C., suggesting that the basic (pH 9.0) solution reduced the toxicity of fenthion. TREATMENT VII. BASIC, SOFT WATER, AND DIATOMACEOUS EARTH. Here again all the tests were highly significant. A first observation at 1600. showed that the reduced mortality compared to the check and to the similar acidic test in treatment three. It can be assumed that the dif- ference was due to the pH 9.0 in this treatment. The high- 1y important value of this experiment at 24°C. seemed to be due to the presence of the base (pH 9.0). This is evident since mortalities were similar in treatments III and VII, and the only difference between them was the pH 9.0. 36 At 300C. mortality was nil, suggesting that the highly absorptive nature of diatomaceous earth in basic solution contributed to the low number of dead larvae. TREATMENT VIII. BASIC, SOFT WATER, ORGANIC WHEAT GERM. The treatment combination conducted at 16°C. was not sig- nificant at 5% and 1% levels. The tests conducted at 24°C. and 3000. were significant since the mortality in both cases was less than the check. This is attributed to the highly organic wheat germ and pH 9.0 of the distilled water. A comparison with the similar fourth treatment, suggested that the only different factor which could account for the difference in mortality was the basic pH 9.0. TREATMENT IX. FENTHION VERSUS DDT. Tests conducted at the various temperatures comparing fenthion and DDT in distilled water showed significant difference. At 16°C., fenthion was less effective than DDT; at 24°C. and 300C. fenthion was more effective than DDT. The negative temp- erature coefficient of DDT was apparent (Figure 2). EFFECTS OF ECOLOGICAL FACTORS ON MORTALITY 0F.§-.E§§: TURANS. The wild specimens of C. resturans were reared in their own water habitat until they reached the early fourth instar. The specimens were tested to determine the LC50 for fenthion and for DDT, the values of which were derived from the dosage—mortality curve in Figure 3. LC5O values for fenthion and DDT were 0.0035 p.p.m. and 0.0015, respectively. The same breakdown of the ecological factors affecting 36a .ooem pm ewpmoe manhdpmmm xmado tom o>a50 hpflawphoz pfiQOHmuommmom No.0 Ho.o eoo. moo. - b i p Pppbhp - L moo. .m mnswam o\ om on 04 on om on om SQIqOJd u: Aqrteqaow om 37 A. aegypti mortality was considered in this experiment. The eight treatment combinations were tested at 1700., which was the temperature of the native habitat water. The first eight treatments and DDT were compared to the fenthion check. One extra treatment was added consisting of the water collected from the tire ruts in the field. The final analysis consisted of comparing the larvae in their original habitat water with fenthion (Table 1). An analysis was made to determine the percent of soluble versus insoluble organic material in the tire rut‘water (Table IV). The data from the water analysis were necessary in understanding the significance of the water habitat and the fenthion check. Table IV. Analysis of the Chemical Content of Water From Tire Ruts. % Residue in Chemical Content Organic Matter in Grams H20 Ether residue 0.0503 0.01 Hexane residue 0.5194 0.1 An analysis of variance was programmed from the larval mortality data of Q;_resturans. The FARCEP design was in- corporated into the program. The F statistic values from the computer analysis were tabulated comparing the signifi- cance of fenthion with the various treatments and DDT (Table 5). The significance of the F values was determined at the 5% and L% levels (Snedecor l9h6 p. 2h6-2A9). TREATMENT I. The combination of factors} pH5, hard water, and diatomaceous earth at 170C. was significant at 38 TABLE V Analysis of Variance of Treatments in Table I for C. Resturans Source of 2 2 Treatments variance SX df X F—value 1. A 16.7 1 16.7 25.0 ** Error 2.7 4 0.7 Total (after 19.2 5 II. A 1.2 1 1.2 1.0 Error 16.7 4 4.2 Total 20.8 5 III. A 10.7 1 10.7 8.0 * Error 5.3 h 1-3 Total 16.0 5 IV. A 140.2 1 140.2 27.1 ** Error 20.7 A 5.2 Total 160.9 5 V. A 4.2 l h.2 3.1 Error 5-3 A 1-3 Total 9.5 5 VI. A 20.2 1 20.2 9.3 * Error 8.7 A 2.2 Total 28.9 5 VII. A 6.0 1 6.0 1.6 Error 15.3 A 3.8 Total 21.3 5 VIII. A 501.2 1 501.2 275.0 ** Error 7.3 A 1.8 Total 511-5 5 IX. A 32.7 1 32.7 7.8 * Error 16.? 4 h-2 Total A9.A 5 F values were checked for significance at 5% and 1% level. 39 thef% and 1% level. There was no mortality in the treat- ment compared to the fenthion check. TREATMENT II. The second treatment was not significant because the mortalities were the same. TREATMENT III. Acidic (pH 5.0), soft water, and dia- tomaceous earth were significant at the 5% level. Reduced mortalities in treatments I and II signify the absorption of fenthion by diatomaceous earth. TREATMENT IV. A combination of} acidic (pH 5), soft water, and organic wheat germ was highly significant. The larval death rate of Q. resturans was very high in relation to the check, suggesting that the highly organic wheat germ used as a food for larvae, absorbed the insecticide. TREATMENT V. The mortality in the treatment (basic, hard water, diatomaceous earth) was equivalent to the fen- thion check, so the relationship was not significant. TREATMENT VI. At the 5% level the combination of factors: basic, hard water, and wheat germ was significant. Increased mortality over the check apparently meant, as in treatment 2, that the presence of highly organic wheat germ increased larval deaths. TREATMENT VII. Not significant. TREATMENT VIII. This treatment was extremely important in that the mortality was much higher, than in the check. Again, it was due to the presence of highly organic wheat germ. TREATMENT IX. The relationship of mortality between 40 fenthion and DDT was only significant at the 5% level. DDT, in this particular experiment, was more effective in killing Q. resturans larvae at 17°C. It is very evident from this investigation that wheat germ, which is highly organic, increases mortality, probably because the wheat germ and absorbed fenthion is ingested as a larvel food. Another observation can be drawn from the experiments. Diatomaceous earth has a high attraction or absorptive property for insecticides. This absorption blocks the contact between the insecticide and mosquito larvae. Nearly 100% mortality in the larval water habitat, as compared to the fenthion check, indicated a high organic content in the water. Table A shows evidence of a relative- ly high organic content in the water. DISCUSSION OF HEARING METHODS AND TOXICITY TESTS Toxicity studies of this nature required the best tech- niques and methods that could be developed for the mass rear- ing of strictly standardized mosquito larvae. Many methods have been utilized in rearing mosquitos. The importance of standardizing age, size, temperature, food, and humidity have been stressed by Trembley, 1955; Busvine, 1957; Burgess, 1959; Mulla, 1961; and Thomas, 1965. The immersion of mosquito eggs in deoxygenated distilled water definitely increased the hatch rate of A. aegypti. This is in line with the findings of Burgess (1959) and Gjullin gt, 21. (l9h1). Egg production was maintained by making fresh citrated beef blood and 10% honey available to the adult female mosquito. This was suggested from the work of Knierim gt. 31. (1955), and Li1es (1960). It should be stress- ed that the use of five gallon rearing jars proved much more effective in standardizing the larvae, than the photographic tray rearing method used by many workers. Another factor which cannot be stressed enough is the type of containers used in toxicity tests. Results of this study definitely indicate that glass crystallizing dishes are better than paper cups. This is contradictory to the containers used by Mulla in all his toxicity tests. It is for this reason that little consideration has been given to his work on susceptability of various larval instars, 41 A2 evaluations of new insecticides, and resistance in mosquitos. For larvicide determinations, the standard WHO technique (1960) was used. Mortality counts were based on the quantal response, which according to Hoskins and Craig (1962), is simply the method to estimate the magnitude of a dose that is enough to produce death. In agreement with Lewallen gt. El. (1959) and Hoskins (1962), the best tests would be field tests, but many ex- trinsic and intrinsic factors of great variability would not always give unambiguous results. Field trials should be reserved until detailed laboratory tests have been com- pleted. In line with Thomas (1965), evidence was given that hard and soft water as individual factors are not signifi- cantly different. The presence of the Hydromeris gp. nematode which killed a large percent of A. stimulans could prove to be avery effective biological control method. In habitats where this parasite occurs, the tolerance of the mosquitos to insecticides is most likely to be greatly reduced. There are many factors that can cause variation in toxicity experiments. Factors such as density of population, volume, surface area, and depth of test solution have been described by Busvine (1957), Curtis (1961), Kruse (1962), and Rai (1960). Ecological factors, being the most important in evalua- tion of insecticides in the field, were studied in depth. It was highly important to understand the factors and A3 combination of factors that affect the toxicity of fen- thion. Once an understanding of the ecological factors was acquired, a better knowledge of the action of the insecticide in field tests could be achieved. The various factors investigated were temperature variations, pH 5 and 9, low and high organic material, and hard versus soft water. The significance of each of these factors can be attributed in many ways in relation to the fenthion check. EFFECT OF TEMPERATURE. In Figure 2, the toxicity of fenthion at 1600. is lower than DDT, and as the temperature at 1600. increased to 300C. fenthion was more toxic than DDT which indicated that DDT has a negative temperature coe- fficient. At the low temperature, the presence of acidic tap water mixed with diatomaceous clay definitely reduced the toxicity of fenthion. The presence of wheat germ in all the tests conducted at 1600. showed an increased mortality over the fenthion check. This would indicate that high organic content of wheat germ absorbs fenthion, and then the wheat germ was ingested by the larvae. Diatomaceous earth reduced the mortality in all the tests except one treatment with dis- tilled water. At room temperature (24°C.), mortality rates of the various treatments were relatively higher than at 16°C. This indicates that fenthion toxicity increases as the temperature increases. At 30°C., the response of A. aegypti larvae in all the treatments was lower than the high mortality rate of the fenthion check. High temperatures apparently breed bacteria more rapidly under certain conditions of pH, and organic matter. Thus, the toxicity of fenthion is reduced in the high organic solutions, while the interactions of diatomaceous earth, pH, and water type cause similar reduction in mortality. EFFECTS OF PH. The effects of pH are more difficult to understand. Acidic conditions of diatomaceous earth at 160C. seemed to be a part of the factors that greatly reduc- ed the larval mortality. Temperatures at 30°C. showed re— duced mortality due to the basic (pH 9.0) condition. The acidity of the treatments increased mortality over the. treatments under basic conditions which generally hydrolyze organphosphates. This relation of pH to high temperatures may be indicative of bacterial growth which indirectly affects the toxicity of fenthion. It is difficult to understand the significance of pH, but the evidence supported the importance of pH in fenthion toxicity tests. EFFECTS OF WATER TYPE. Soft versus hard water as in- dividual factors showed no significant differences in the toxicity of fenthion. Specifically, the treatment where soft water affects mortality was in the combination of acidic distilled water mixed with diatomaceous earth. In this combination, mortality was higher than the check, indicating #5 that distilled water was the factor, since in a similar treatment the only difference was hard water which greatly reduced mortality. At room temperature, treatments that showed a diff- erence in water type indicated that hard water caused higher mortality, than the similar soft water treatment. This gave sufficient evidence that water type is an important factor in the fenthion toxicity test. EFFECTS OF ORGANIC MATTER. The breakdown of the organic factor into two sub-factors: diatomaceous earth (low in organic material) and wheat germ (highly organic) proved to be highly significant in many of the treatment tests at various temperatures. A very evident situation was the reduced mortality of larvae at 16°C., in the presence of diatomaceous earth in acidic tap water. This reduction of mortality was evidently due to the high attraction of fenthion to the material, which in turn blocked the desired contact with the A. aegypti larvae. Supporting evidence for the effectiveness of wheat germ was obtained in treatments at 160C. All the treatments that contained wheat germ had a mortality higher than the fenthion check. Wheat germ apparently stabilized the larval mortality, as the tempera- ture and fenthion check increased. Lastly, results from the treatments of_g. resturans, at 170C. showed several important factors that affected fenthion toxicity. DDT was more effective at 1700. which was the temperature of the water habitat of the field larvae. A6 Fenthion killed approximately 10% of the larvae compared to 32% mortality with DDT. Treatments that contained wheat germ gave significantly increased mortality over the fenthion check. The other treatments containing diatomaceous earth showed reduced mortality compared to the check. The mosquito larvae collected from the field significantly verified the findings of organic affects on toxicity of fen- thion conducted on laboratory specimens. This is in line with the work of Lewallen gt_§l. (1963), indicating that DDT and fenthion consistently performed better in highly organic (polluted) water than in tap water. The limitations of laboratory testing must be considered when evaluating the results. SUMMARY The effects of various ecological factors were studied on larvae of A. aegypti and g. resturans reared under strict standardized conditions. Alliests on these larvae were per- formed in glass crystallizing dishes, and all variables were kept as uniform as possible throughout the experiment. Re- sults from the experiments showed that temperature, hard and soft water, pH, and organic content are very important in all the tests, and at 300C. it increased the basic action- catalyzed hydrolysis of organophosphorous insecticide. Dia— tomaceous earth which is low in organic matter, decreased mortality, while highly organic wheat germ significantly in- creased mortality. Water types were important at room temp- erature where hard water had higher mortality than the similar soft water treatments. Tests on field collected 9. resturans larvae were questionable since factors such as prior exposure to insecticide and variation in nutrition could have markedly influenced results. Mermithid nematodes of genus Hydromeris sp. were found parasitizing field populations of A. stimulans, killing nearly 90% of the mosquitos. A7 LITERATURE CITED Abbot, W. S. 1925. A method of computing effectiveness of an insecticide. J. Econ. Entomol. 18:265-269. Bliss, C.I., 1934. The method of probits. Science 79: 409- thO . . 1935. The calculation of the dosage-mortality curve. Am. Appl. Biol., 22: 134-167. . 1938. The determination of the dosage-mortality curve for small numbers. Quart. J. Pharm. and Pharmacol. 11: 192~2l6. Bowman, M. C., Acree, F., Schmidt, C. H., and Beroza, M. 1958. Fate of DDT in larvicide suspensions. J. Econ. Entomol. 52: 1038—1042. Boyd, M. F., 1926. A note on the rearing of Anopheline larvae. Bull. Ent. Res. 16 (4):308. , , and Cain, T. L. Jr. 1932. 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Econ. Entomol. 43:944-945. IIIIIIIIIIIIIIII ERSITY LIBRARIES IIII)(I(I)))TIII))I lllllllllll IIHII) 169 5319