TOXICITY OF FOUR INSECTICIDES TO LARVAE AND ADULTS OF EGYPTIAN ALFALFA WEEVIL, HYPERA BRUNNEIPENNIS (BOHEMAN).

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Authors Suhaibani, Ali Mohammad.

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University Microfilms International 300 N. ZEEB RD.. ANN ARBOR, Ml 48106

1318791 SUHAIBAWI, ALI MOHAMMAD TOXICITY OF FOUR INSECTICIDES TO LARVAE AND ADULTS OF EGYPTIAN ALFALFA WEEVIL# HYPERA BRUNNEIPENNIS (BOHEMAN). THE UNIVERSITY OF ARIZONA, M.S.# 1902

University Microfilms International 300 N. ZEEB RD., ANN ARBOR, Ml 48106

TOXICITY OF FOUR INSECTICIDES TO LARVAE AND ADULTS

OF EGYPTIAN ALFALFA WEEVIL, HYPERA BRUNNEIPENNIS (BOHEMAN)

by

Ali Mohammad Suhaibani

A Thesis Submitted to the Faculty of the

DEPARTMENT OF ENTOMOLOGY

In Partial Fulfillment of the Requirements For the Degree of

MASTER OF SCIENCE

In the Graduate College

THE UNIVERSITY OF ARIZONA

198 2 STATEMENT BY AUTHOR

This thesis has been submitted in partial fulfillment of re­ quirements for an advanced degree at The University of Arizona and is deposited in The University Library to be made available to borrowers under rules of the Library.

Brief quotations from this thesis are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the department or the Dean of the Graduate College when in his judgment the proposed use of the material is in the in­ terests of scholarship. However, in all other instances, permission has to be obtained from the author.

SIGNED: /Vi M- Sahjuba^U.

APPROVAL BY THESIS DIRECTOR

This thesis has been approved on the date shown below:

/tr2-— GE U. WARE late d Head of Entomology ACKNOWLEDGMENTS

I wish to express my appreciation to my major professor, Dr.

George W. Ware for his understanding, his suggestions, his patience

and his guidance throughout the duration of my graduate program and

the writing of my thesis.

Gratitude is also to Dr. L. A. Crowder and R. T. Ruber who

served on my graduate committee and for their help, suggestions and

criticism.

My very special thanks go to Norm A. Buck and Betty J.

Estesen for their assistance during the laboratory work and help in

acquiring the initial test insects.

To my wife, Medhaiwi and my daughter, Nahlah for their sup­

port throughout my research study, I owe the most special debt of

gratitude.

Finally, I am grateful to my government for its generous financial support during the period of my study.

iii TABLE OF CONTENTS

Page

LIST OF TABLES

LIST OF ILLUSTRATIONS vi

ABSTRACT vii

LITERATURE REVIEW. 1

Adult Stage 2 Egg Stage '. 3 Larval Stage 3 Pupal Stage 4 Feeding Habits 4

MATERIALS AND METHODS 12

Larval Testing 12 Stock Solutions and Dilution Procedures 12 Dish Treatment 14 Larval Selection 14 Treatment of Adult Weevils ...... 15

RESULTS 17

Determination of the LC50 and LC95 for Larvae 17 Determination of LD50 and LD95 for Adults 20

DISCUSSION 23

SUMMARY AND CONCLUSIONS 27

APPENDIX A: LC50S AND LC95S DATA FOR LARVAE AND LDcqS AND LD95s DATA FOR ADULTS 28

LITERATURE CITED 31

iv LIST OF TABLES

Table Page 2 1. LC50S and LC95S (yg/cm ) with upper and lower limits for four insecticides on Egyptian alfalfa weevil larvae (Hypera brunneip ennis) 18

2. LD50S and LD95S (yg/g) with upper and lower limits for four insecticides on Egyptian alfalfa weevil adults (Hypera brunne ip ennis) 21

v LIST OF ILLUSTRATIONS

Figure Page 2 1. LC5Q~LCg5 slopes (pg/cm ) for four insecticides on Egyptian alfalfa weevil larvae (Hypera brunneipennis).... 19

2. LD5Q-LD95 slopes (ug/g) for four insecticides on Egyptian alfalfa weevil adults (Hvpera brunneipennis) 22

vi ABSTRACT

Toxicity of four insecticides, methomyl (Lannat^®, Nudriri4*),

carbofuran (Furadar^l , methidathion (Supracid^®) and phosmet (Imidan^)

to fourth instar larvae and adults of Egyptian alfalfa weevil was in­

vestigated. Laboratory tests were conducted on weevils collected from

two alfalfa fields which were not treated that year with insecticides.

The methods of exposure were by direct contact of weevil larvae to uniform insecticide deposits in glass petri dishes, and by treating adults topically with acetone solutions of insecticides using a preci­ sion microapplicator. 2 The LC^QS, LCgj-s (yg/cm ) and their upper and lower 95 percent confidence limits were computed for larvae. Carbofuran was the most toxic insecticide, requiring an average of 0.01 yg/cm 2 to kill 50 per­ cent of the tested larvae. This compound was followed by methidathion, with the LC^q of 0.04, then methomyl, 0.23, and finally phosmet with the 2 LC^Q of 0.58 yg/cm . The LD^QS, LD^s (yg/g) and their upper and lower

95 percent confidence limits were also determined for adult weevils.

The sequence of LD^QS obtained was 2.37 yg/g for carbofuran, 3.40 for methidathion, 5.87 for phosmet and 8.93 yg/g for methomyl.

vii LITERATURE REVIEW

On April 11, 1939 an insect was discovered feeding on alfalfa

(Medicago sativa L.} near Yuma, Arizona, by L. P. Wehrle and T.

Hendrixon. At first, this insect was believed to be the alfalfa weevil, Hypera postica (Gyll.), but when specimens were submitted to the Bureau of Entomology and Plant Quarantine U.S. Department of

Agriculture, they were determined to be the Egyptian alfalfa weevil,

Hypera brunneipennis (Boh.), by L. L. Buchanan (Wehrle, 1940). A year later a small infestation was found at the University of Arizona Date

Experimental Farm near Tempe, Maricopa County, Arizona (McDuffie,

1941).

The native habitat of this weevil has not been definitely de­ termined, but it is known to occur in North Africa, particularly in

Egypt, and in Ethiopia. Because of the dearth of parasites in these regions, Dietrick and Van den Bosch (1953) reasoned that these regions might not be the native habitat of this species.

The exact mode of entry of H. brunneipennis into the United

States is not known. The most likely route of entry seems to be through the importation of date palm stock from Egypt to Yuma, Arizona, since it is known that large numbers of the adult weevile aestivate in protec­ ted places at the base of date palm offshoots (McDuffie, 1941).

H. brunneipennis is very similar to its more widely known rela­ tive, H. postica (Gyll.) or alfalfa weevil, which has been an

1 2 economically important weevil pest for many years (McDuffie, 1941).

The two species are very much alike, but H. brunneipennis is larger with a proportionately broader prothorax, is flatter onside view, has a more even and not so pronounced color, has slightly more erect setae, and the elytra are broader, and not so parallel in front as in II. postica (Michelbacher and Leighly, 1940). Also, in behavior, H. postica spins most of its cocoons in the surface litter while H. brunneipennis spins most of its cocoons on alfalfa stems (Wehrle,

1940).

Adult Stage

The adult of H. brunneipennis is a small snout beetle. The newly emerged weevil is bronze in color but within a short while it becomes brown (McDuffie, 1941). In Southern Arizona, the adults of

H. brunneipennis can be found in infested alfalfa from October through

June (Huber, 1982). New adults feed on leaves of the nearest avail­ able leguminous host and most adults migrate from fields in search of places for aestivation, where they remain inactive throughout the summer and early fall months. The heaviest feeding of adults occurs at night when the temperature is moderate. For aestivation, the adults choose protected locations in which there is adequate moisture.

During aestivation, adults do not feed under outdoor or indoor condi­ tions. Also, there is no development of the sex organs throughout aestivation and the adults migrate back to the fields before attaining complete sexual maturity. Oviposition begins late in December reaching its peak during February in Yuma, Arizona, and lasts for approximately 3

3 to 4 months from late December to mid-April (Wehrle, 1940). Some H. brunneipennis adults may live for 17 months and through two summers by re-entering diapause (Madubunyi and Koehler, 1977). Diapause can be terminated in the alfalfa weevil by using the synthetic juvenile gona­ dotropic hormone CIO, 11-epoxyfarnesenic acid methyl ester). This can be very helpful for continuous rearing in the laboratory of insects that diapause, and for potential control of insects, since termination or prevention of diapause in an insect exposes it to the consequences of a hostile environment and its stresses (Bowers and Blickenstaff,

1966). Adults are capable of flight to diapause sites and their dis­ persal is dependent upon wind direction (Sherburne et al., 1970).

Err Stage

The newly deposited egg of H. brunneipennis is shiny yellow but in about a week it becomes dark. The egg is oval, and averages 0.62 mm in length and 0.40 mm in width. The eggs are deposited in clusters in small, dry, easily punctured stems of alfalfa and the average cluster contains 14 eggs (Wehrle, 1940).

Larval Stage

H. brunneipennis has four larval instars. Larvae of each stage vary greatly in body size and there is an overlapping between succes­ sive stages.

The body color of first-stage larvae varies from yellowish to light gray and the head is black. The body color of second-stage larvae is gray and the head is yellow but soon becomes dark. 4

The head of third-stage larvae is mostly black and the body is light green with a visible white stripe down the middle of the back.

The head capsule of fourth-stage larvae is almost entirely brown but

with visible darkness near the edges. The body is light green with a

broad white stripe in the middle of the back paralleled by smaller

stripes on each side. Larvae of H. brunneipennis are similar to com­

parable stages of H. postica with no distinguishing characteristics

(Wehrle, 1940).

Pupal Stage

The pupal stage is passed in thin, white, netlike cocoons,

usually oval in shape. Cocoons are attached to the growing leaves of

the host plant and some of them to dead leaves and stems above the

ground. The newly formed pupa is usually light green but gradually takes the structure and light-bronze color of the adult. The exo- skeleton of the newly formed adult is soft and thin, and in a few days

it hardens and the adult is able to emerge from the cocoon. The pupal period extends to about 15 days during early March, but gradually shortens to 6 to 7 days during April. Adults remain in cocoons 2 to 5 days before emerging but most of them emerge in 2 to 3 days (Wehrle,

1940).

Feeding Habits

First and second larval stages feed on the tender portions of host plants, usually on buds or stem tips. Eighty percent of the fol­

iage consumed is by third and fourth larval stages. Adults of both 5 sexes consume more foliage at night tfian during the day (Okiwelu,

1977).

Adult feeding on the stem epidermis and the conducting tissues beneath it causes excessive water loss, disrupts translocation, and results in cessation of growth. Also, under field conditions, reduc­ tion of yield and quality caused by adult feeding would be more severe since drying makes the plant brittle and many leaves are lost during the harvest operations (Allen et al., 1959). The effect of adult feeding on alfalfa quality depends to some extent on the growth stage and growth rate of the plant. A smaller number of adults causes some­ what greater damage to growing alfalfa than does a larger number of adults feeding on mature alfalfa (Mathur and Pienkowski, 1967).

Of several methods of sampling to measure weevil populations, the sequential stem method was the best and provided the most accurate estimate. The population can be estimated as percent of stems in­ fested, times the number of larvae per infested stem, times the number of stems per square foot. This provides an estimate within 90% of the actual population. Using the sweep net, eight to nine sweeps with a

15-inch net were required to produce the same estimate (Blickenstaff and Huggans, 1969).

The economic threshold of H. brunneipennis infestation in alfalfa is 15 to 20 larvae per sweep and control measures must be applied to prevent the population from reaching the economic injury level (Cothran and Summers, 1972). However, the sweep net method of sampling is incapable of providing acceptable predictive information as to the maximum population level. At the same time, population 6

curves based on this kind of sampling fail to describe accurately the

actual population pattern, and the greater degree of error falls early

in the developmental period. In contrast, much more accurate curves are

derived from absolute sampling techniques (square-foot) in which stem

tips are dissected and all larvae are counted (Cothran and Summers,

1972). Correlations of larval numbers and first cutting hay yields dis­

closed that every increase of one larva per sweep above 22 larvae at the

larval peak was associated with a yield reduction of 9.2 pounds (Koeh-

ler and Rosenthall, 1972).

There are many parasites that are effective in the control of jl. brunneipennis (Boh.). Some of these parasitize the adults such as

Microctonus aethiops (Nees) (Stehr and Casagrande, 1971), Campogaster

exigua (Meigen), and Hyaolmyodes triangulifera (Ioew) (Brunson and

Coles, 1968). Others parasitize larvae, for example, Bathyplectes

curculionis (Thomson), Dibrachoides druso (Walker) (Brunson and Coles,

1968) and Bathyplectes corvina Thoms. (Fisher et al., 1961). In ad­

dition, some parasitize the eggs, i.e., Patasson luna (Girault) and

Peridesmia discus (Walker) (Brunson and Coles, 1968). Control from

these parasites is sometimes not very effective either because of hyper-

parasitism, i.e., Dibrachys cabus (Walker) attacks the alfalfa weevil

parasites of Bathyplectes spp. (Thomson) (Day, 1969), or because of the

defense reaction of H. brunneipennis by encapsulating the eggs of

Bathyplectes curculionis with blood cells (Van den Bosch and Marble,

1971).

Two other sources of biological control are from predators i.e., Solenopsis invicta Buren, the red fire ant, which is very 7

effective in controlling weevil larvae (Morrill, 1978), and the fungal

pathogens, i.e., Entomophthora phytonomi, (Puttier et al., 1978), and

Beauveria bassiana (Hedlund and Pass, 1968). In the latter case a 98

to 100% relative humidity is required for a high rate of infection to

occur.

Selecting resistant varieties of the host plant helps in manag-

ing the pest species and decreasing its damage. Alfalfa varieties dif­

fer significantly in susceptibility to oviposition by the alfalfa weevil.

The phenotypic and genotypic correlations between stem diameter and

oviposition supported previous observations that stem diameter is

associated with oviposition preference. For example, low oviposition

was associated with small stem diameter, a decrease in stem pith or an

increase in stem solidness (Busbice et al., 1968).

One of the physical methods used in controlling alfalfa weevil

is thermal treatment by flaming. Alfalfa weevil larvae and pupae can

be controlled by burning the alfalfa after cutting. The egg and adult

stages are also vulnerable if control measures are properly timed and

applied. In addition to reducing weevil populations, the growth of

certain weed pests is effectively retarded in flamed areas (Dorsey and

Stevens, 1969). Experimental physical control methods are X-irradiation

of adults (Burgess and Bennett, 1966) and gamma irradiation of larvae

(Burgess and Bennett, 1971). Neither method has been promising.

The carbamate insecticide methomyl (Lannate^ or

(S-methyl N-[(methyl carbamoyl)] thioacetimidate) was registered for

agricultural use in 19.70 on a limited number of vegetable crops (Dupont,

1970). In recent years the number of crops for which methomyl is 8

registered has increased to include many vegetables, cotton, alfalfa,

soybeans and citrus. Methomyl is toxic to insects both by direct con­

tact and ingestion of the insecticide (Leitch and Pease, 1973).

The other carbamate insecticide used in this research was

carbofuran (Furadail®>, (2,3-dihydro-2,2-dimethyl-7-benzofuranyl

methylcarbamate). Carbofuran is a broad-spectrum insecticide-

nematicide which has demonstrated excellent efficacy on a variety of

crops. The first registered use for carbofuran in the U.S. was on corn

in 1968 (Cook, 1973). In recent years, formulations of carbofuran have

been registered for use in controlling corn rootworm, nematodes and

wireworms on sugarcane, and alfalfa weevil on alfalfa.

Carbofuran, a methylcarbamate, is a readily reversible cholin-

esterase inhibitor capable of causing toxic effects as the result of

ingestion or inhalation (Cook, 1973). Field studies done by Fahey et

al. (1970) showed that the carbofuran residues were not found on green

alfalfa 14 days after treatment and that dehydration of alfalfa re- 14 duced the carbofuran residues by 65%. Ivie and Dorough (1968) used C

labeled carbofuran and demonstrated that 3-hydroxy carbofuran glucur-

onide was the major carbamate residue excreted in the milk of treated

cows.

The organophosphate methidathion (Supracide or GS-13005)

(S-2,3-dihydro-5-methoxy-2-oxo-l,3,4-thiadiazol-3-ylmethyl 0,0-dimethyl

phosphorodithioate) was introduced in 1964, as a broad-spectrum contact

and stomach poison insecticide and acaricide. The main areas of 9

application are against chewing and sucking pests in citrus, alfalfa,

cotton, cereals and vegetables. Depending upon crop species, rate of application, and weather, effective control of pests ranges from one to three weeks (Eberle and Suter, 1976). Methidathion acts as.an in­

hibitor of cholinesterase, and its clinical effects are typical of

anticholinesterase compounds (Grob et al., 1965). Polan and Chandler

(1971) found that the degradation of methidathion to carbon dioxide

occurred readily in lactating cows and no accumulation of the compound or other metabolites was indicated. The main routes for the excretion of metabolites of methidathion if applied orally to rats are urine and

expired air (Dupuis et al., 1971). On green alfalfa, methidathion residues persisted for 21 days after treatment and the dehydration of

alfalfa reduced the methidathion residues by 50-60% (Fahey et al.,

1970). The residues of this compound can be determined by using

electron-capture gas chromatography or thin layer chromatography as used by Eberle et al. (1967).

The other organophosphorus insecticide used in this study was

phosmet (Imidan® or Prolati®) (0,0-dimethyl S-phthalimidomethyl phos- phorodithioate). This compound is registered for use on a variety of crops and it is active against a wide range of insects such as alfalfa weevil, boll weevil, codling moth, oriental fruit moth, and many others (Batchelder et al., 1967). Miller et al. (1969) found that, after 21 days following treatment, phosmet residues on alfalfa were less than the sensitivity of the analytical method (0.6 ppm) used for residue determination. Also, phosmet is found to be absorbed 10 by treated leaves but not translocated in the plant (Menn and McBain,

1964).

Previous studies on control of both H. postica and H. brunneipennis, indicated no perceptible difference in susceptibility to insecticldal sprays and any material effective against one was just as effective against the other (Koehler, 1971). Organochlorine in­ secticides such as DDT, toxaphene, aldrin, and chlordane were used in the past to control Egyptian alfalfa weevil in Arizona (Russell and

Barnes, 1951), but these weevils were able to develop resistance to some of these insecticides (Alder and Blickenstaff, 1964). Methomyl, carbofuran, phosmet, and methidathion are now recommended for con­ trolling this pest. Organophosphate and carbamate insecticides are less persistant in the environment and occasionally more selective than organochlorine insecticides. Carbofuran is excellent both in terms of initial larval kill and persistence of activity for a limited period of time (Koehler, 1971). Summers et al. (1971) also found that carbofuran gave better control of the alfalfa weevil and was longer- lasting than phosmet and methidathion. Summers (1975) stated that control of the Egyptian alfalfa weevil remained adequate for 14 days post-treatment, and larval populations continued to decline in the methidathion and carbofuran plots in spite of an increasing number in the untreated check. Also, he mentioned that methomyl was signifi­ cantly better at 0.6 kg Al/ha than at 0.3 kg Al/ha, and the population was close to the economic threshold level 7 days post-treatment. Re­ garding the formations used, Pass (1966) found that sprayable formu­ lations of carbofuran and methidathion were much better than the 11 granular formulations used to control alfalfa weevil larvae. Summers and Cothran (1972) found that effective weevil control could be obtained with carbofuran applied at 1.12 kg Al/ha as early as 80 days prior to cutting.

Insecticides should be applied under ideal spraying and weather conditions to insure effective control. For example, methidathion is much more effective against the alfalfa weevil when it is not rainy and the temperature is between 85-90°F (29.4-32.2°C) (Armbrust and Wilson,

1970). Also, methidathion is more effective than carbofuran against the weevils when applied late in the spring in warmer temperature

(Summers and Cothran, 1969). Methidathion and phosmet did not give satisfactory control when applied at 50°F (10°C) or less (Dorsey, 1966).

Van Meter and Pass (1970), found that the most effective insec­ ticide for 3-day-old adult weevils was carbofuran, but against 14-day- old adults carbofuran required slightly higher dosages to obtain a 50% kill of this age. This might be related to the increase of lipids in the adults. Carbofuran and phosmet, when applied early in the season, had little or no effect on the weevil parasite (Bathyplectus curculionis) (Davis, 1970).

The objective of this study was to examine the effectiveness of four different insecticides; carbofuran, methidathion, phosmet and methomyl against larvae and adults of Egyptian alfalfa weevil by deter­

mining the LC^Q and LD^Q respectively for each insecticide. Addition­ ally, the most effective insecticide against this pest was to be determined in order that recommendations for field control of this weevil could be formulated. MATERIALS AND METHODS

Larval Testing

Egyptian alfalfa weevil larvae used in this study were collected from untreated alfalfa fields on the Lone Butte Ranch of Gila River

Farm, located on the Gila River Indian Reservation, Maricopa County,

Arizona, from February 27 to March 22, 1981, using sweep nets. The larvae were kept in brown paper bags with fresh alfalfa and transferred to the laboratory inside an ice chest. In the laboratory, the larvae were held in the refrigerator at about 12°C until the time of treatment.

The following insecticides were used: Methomyl (S-methyl N-

[(methyl carbamoyl)oxy] thioacetimidate), recrystallized, (100%) provided by E. I. DuPont de Nemours & Company; methidathion (S—2, 3- dihydro-5-methoxy-2-oxo-l,3,4-thiadiazol-3-ylmethy1 0,0-dimethyl phos- phorodithioate), (97.2%), provided by the CIBA-Geigy Corporation; carbofuran (2,3-dihydro-2,2-dimethyl-7-benzofuranyl methylcarbamate)

(98%), provided the FMC Agricultural Chemical Group; and phosmet (0,0- dimethyl S-phthalimidomethyl phosphorodithioate), (99.8%), provided by the Stauffer Chemical Co.

Stock Solutions and Dilution Procedures

The stock solution of each insecticide was prepared by weighing

30 mg of the technical insecticide using a Mettler H6T, analytical balance. The insecticide was placed in a 25 ml volumetric flask and

12 I

13

dissolved in technical acetone. The stock solution then contained 1200

yg/ml. The preferred dilutions were prepared by diluting with acetone

and both stock and dilutions were kept in the refrigerator at 5°C.

Petri dishes were washed with a laboratory detergent (Liqui-

Nox®>, rinsed with tap water, then distilled water, finally with dis­

tilled acetone, and then allowed to dry. Rinsing in acetone reduced

surface tension and permitted more uniform coverage with the insecti­

cide dilutions.

Five dilutions of each insecticide were selected besides the

controls which were treated only with acetone. The dilutions were pre­

pared as follows:

1:10 1.2 yg/ml

-1:100 12 yg/ml -I"Li;12 1 yg/ml

-1:50 24 yg/ml

-1:25 48 yg/ml 1200 iJg/ml — •1.5:25 72 yg/ml

•2:25 96 yg/ml

-2.5:25 120 yg/ml

-3:25 144 yg/ml

Range-finding experiments were conducted to determine those

dilutions that would give 10 to 90% mortality. This insured good

distribution for log-probit analysis. Dish Treatment

Glass petri dishes consisting of a top and bottom were used in this experiment. One milliliter of the insecticidal solution was ap­ plied to the inside of the inverted top plate and another milliliter of solution applied similarly to the bottom plate. Immediately after applying the insecticidal solution to the plates they were continually rotated while tilting, allowing the solution to cover the entire sur­ face. The sidewall of the bottom was treated during rotation but not the sidewall of the top, since it was not accessible to the larvae.

The solution was kept moving across the plates until the acetone eva­ porated leaving a fairly uniform film of the insecticide. When the top plate was placed over the bottom, the insects had access only to treated surfaces and thus the specified amount of insecticide. The procedures used were provided by Dr. R. H. Langille (personal communica­ tion, 1981).

Larval Selection

For each insecticide, ten fourth-instar larvae were placed in each dish for each replication, starting with the lowest concentration, the control, which was treated only with acetone, and advancing up the concentrations. Four replicates of each concentration for all the insecticides tested were made. The time was recorded when one-half of the dishes contained larvae. Mortality readings of the larvae were made

4 hours after that time. Percent mortality was determined for each insecticide to include not only the number of dead but also the moribund larvae. Moribund larvae were those that could not move forward on their own when touched several times with a probe.

Treatment of Adult Weevils

Adults used in this study were collected from an alfalfa field on the farm of C. B. Shiflet, 2 miles southeast of Coolidge, Pinal

County, Arizona, using a sweep net or picking them by hand from alfalfa crowns. The adults were held in an ice cream cylinder used

as a cage, with the cardboard top removed and replaced with mosquito net. Fresh alfalfa was placed in the cage for adult feeding. The in­ sects were transferred to the laboratory for treating.

All dosage-mortality tests of the adults involved topical ap­

plication of the four recommended insecticides dissolved in technical

acetone. Topical applications were made with a model M IS CO micro- applicator equipped with a 0.25 cc syringe and a 27 gauge needle, one for each insecticide to avoid contamination. Needles were blunted and polished, and the tips were bent at an angle of about 80°

(Reynolds, 1962). Calibration of syringes was accomplished by meas­ uring the weight of a volume of mercury from the syringes at a known temperature, and converting this to yl/dose.

All dilutions were prepared as described earlier and held at

5°C in a refrigerator. The number of dilutions were five for each tested insecticide, and were applied in increasing concentration starting with the controls. For each concentration there were two replicates with 10 adults per replicate, for a total of 120 insect per test. The volume used for all topical tests was 1.0 yl per 16

' adult, applied to the ventral surface of the thorax. After treating,

the adults were held in petri dishes at room temperature which ranged

from 73° to 78°F (22.8° to 25.6°C). Mortality counts were made 24 hours

after treatment.

An adult was considered dead if it laid on its back with its

legs folded and unreactive when touched with a probe. Twenty adults

were weighed from each treatment to provide the average adult weight

for computation. RESULTS

Determination of the LCqp and LC95 for Larvae

The and for the larvae were determined as concen- 2 tration/area (yg/cm ). The larvae in this case were dosed while they

were crawling directly on the film of insecticide deposited uniformally

on the inner top and bottom surfaces of petri dishes. Readings of

larval mortality were made after 4 hours of exposure to the toxicant

and the data were recorded. Data were analyzed using a computerized

probit analysis program and subsequent calculations were made to convert 2 LC^QS and yg/cm . The average and LC^,. with upper and

lower 95 percent confidence limits for each insecticide are presented

in Table 1. The dosage-mortality curves are presented on log-probit

scales in Figure 1. 2 Carbofuran had the lowest LC5Q, 0.01 yg/cm and 0.04 2 yg/cm , indicating it as the most toxic of the compounds tested.

Methidathion was the second most toxic, with an of 0.04 and an 9 LCg^ 0.09 yg/cm . The third was methomyl, with an LC^Q of 0.23 and 2 LCg^ 0.58 yg/cm . Phosmet was the least toxic with an LC^q of 0.58 2 and an of 2 yg/cm . All of the slopes were relatively steep,

suggesting that larvae were susceptible to the action of each and that

resistance is not present nor will it become quickly established

(Hoskins and Gordon, 1956).

17 2 Table 1. LC-^s and LCgcs (yg/cm ) with upper and lower limits for four insecticides on Egyptian alfalfa weevil larvae (Hypera brunneipennis) (Average of four replications).

Lower Limit Upper Limit Lower Limit Upper Limit Insecticide 95% CL 95% CL 95% CL 95% CL Slope LC50 LC95

Carbofuran 0.01 0.01 0.02 0.03 0.04 0.05 2.75

Methidathion 0.04 0.04 0.05 0.08 0.09 0.11 4.71

Methomyl 0.18 0.23 0.26 0.48 0.58 0.87 4.13

Phosmet 0.47 0.58 0.69 1.50 2.0 3.69 3.06 Figure 1. Lc -Lc , slopes (~g/crn 2 ) for four insecticides on Egyptian alfalfa weevil larvae50 9~Hypera brunneipennis) Average of four replications). 95~------~------Jr------r------~---

~ ~ ~ ~ ~

:so~~r------~~------?-----~L---~------a / 1 I ~ / I / ~ / I I / I / E / I I / I I / I / ~ I &: / I I / •w I I / =1o I I / ..L .------~------~------L------.a·- 0 ~ L

1.------~

·01 .os ·1 .s 1 2 3 log Dosage (ugtcm2) 20

Determination of LD50 and LDqs for Adults

Mortality counts of adult weevils were made 24 hours after treat­ ment. The data were also analyzed using the same probit analysis as used for the larvae. The average and (yg/g) of two replications of each insecticide, and their upper and lower 95 percent confidence limits, are provided in Table 2. The LD^Q-LD^^ slopes of each insecticide are presented in Figure 2. The of carbofuran was 2.37 yg/g, methida- thion was 3.40, phosmet was 5.87, and methomyl, with the lowest activ­ ity, was 8.93 yg/g. Table 2. LD50S and LD95S (yg/g) with upper and lower limits for four insecticides on Egyptian alfalfa weevil adults (Hypera brunneipennis) (Average of two replications).

Lower Limit Upper Limit Lower Limit Upper Limit Insecticide 95% CL 95% CL 95% CL 95% CL Slope LD50 LD95

Carbofuran 2.02 2.37 2.72 4.88 6.02 8.50 4.03

Methidathion 3.02 3.40 3.76 5.88 6.91 8.07 5.34

Methomyl 8.34 8.93 9.61 12.78 14.59 18.42 7.75

Phosmet 5.20 5.87 g.62 10.93 13-45 19.13 4-60 Figure 2. LD -LD slopes (~g/g) for four insecticides on Egyptian alfalfa weevil 5 95r-______a_d_u2_t_s __ (_~_YP_e_r_a~brru 7n_n_e_i_p_en_n_i_s7)~(A_v_e_r_a_g_e_o_f__ t_w_o_r_e_p_l_i_ca_t_i_o_n_s_) ______

.. .a·- .....0 'r------

0.1~1------:::------10 100 N Log Dosage (ugfg) N DISCUSSION

Two organophosphorus (methidathion and phpsmet) and two carbam­ ate (carbofuran and methomyl) insecticides were selected to evaluate their toxicity to Egyptian alfalfa weevil larvae and adults. Field experience has demonstrated that two of these insecticides, carbofuran and methidathion, are very effective against these insects even at low dosages (0.3 kg Al/ha). The other two insecticides, phosmet and meth­ omyl, have given satisfactory results when applied at higher dosages,

1.12 and 0.54 kg Al/ha respectively (Moore et al., 1978).

In this study, carbofuran was the most effective insecticide against both the larvae and adults (Tables 1 and 2). Koehler (1971) and Summers et al. (1971) obtained similar results using carbofuran for controlling this pest in the field.

Methidathion was the next most effective compound. Similar results were obtained by Summers et al. (1971), Summers (1975), and

Armbrust and Wilson (1970) with methidathion against field populations of the alfalfa weevil. Summers et al. (1971) stated that phosmet was as effective as carbofuran against the weevil, but did not measure- up to carbofuran in immediate or residual protection. At the same time, Summers (.1975) found that carbofuran and methidathion gave adequate control of the larval population of Egyptian alfalfa weevil for at least 14 days.

Methomyl and phosmet were the two other compounds evaluated against larvae and adults of Egyptian alfalfa weevil. These two

23 insecticides gave promising results when used at the higher dosages, but neither was as effective as carbofuran or methidathion. Summers (1975) found that methomyl was significantly better at 0.6 than at 0.3 kg AI/ ha, and that the weevil population approached the economic threshold level seven days after treatment. At the same time, Armbrust and

Gyrisco (1965) obtained a poor reduction of the weevil population when phosmet was applied at 0.6 kg Al/ha. This would suggest that phosmet and methomyl should be used at higher dosages in order to give satis­ factory control of weevil populations. Methomyl was more effective than phosmet against the larvae (Table 1), but the opposite occurred with adults (Table 2).

The log-dosage probit (ld-p) response curve is the key factor in interpreting insecticide toxicity studies. A plot of log-dosage versus probits gives the ld-p line, which, for a homogeneous group is approximately straight over the mid-mortality range, usually at least from 20-80% (Hoskins, 1960). The important properties of the ld-p line are its slope and the LC or LD values extrapolated from it. The slope of the ld-p line is a measure of the variation in response to an insecticide (Hoskins, 1960). A steep slope indicates that the test population is homogeneous in response to the toxicant. A flat slope indicates a broad range of response within the test population, or in other words, a heterogeneous population. In this study, the LC^Q-

LCg^ slopes of the ld-p lines for the four insecticides tested on the larvae (Fig. 1) are somewhat similar, but the methidathion and methomyl slopes are somewhat steeper than carbofuran and phosmet slopes. This could suggest that methidathion and methomyl are more specific in their site of action. Carbofuran and phosmet, because of their shallower slopes, are less specific and may attack more than one enzyme. Also, the more the ld-p line slope shifts to the right, the less its toxicity. Phosmet, with its ld-p line slope far to the right, requires the highest dosages (Table 1) to give the same percent mor­ tality (50%) as the other three insecticides.

Similar results were obtained with the adult weevils (Fig. 2), except that methomyl shifted to the far right, indicating that it required the highest dosages (Table 2) to give the same percent mor­ tality (50%) as the other insecticides.

Toxicity of the four insecticides used in this study to the larvae and adults of H. brunneipennis is difficult to compare because of the methods used. With the adults, a known dose is administered to each insect, and the toxicity, based on a weight-to-weight basis, is readily determined. With the larvae, the dose is variable, depending on the activity of the insects: the greater the distance crawled the greater the exposure, and thus the greater the dose ac­ quired. The latter method depends on and assumes a uniform amount of activity on the part of the larvae. This, of course, is unlikely to happen for several reasons, such as the degree of hunger motivating the search for food; irritation of the insecticide to the larvae, resulting in their attempt to escape; and, time remaining until the next molt, which may reduce or increase the activity, and the dose.

Thus in the case of larval testing, the toxicities are only relative and could result in an entirely different sequence of toxicity were the insecticides applied topically in known doses as with the adults. SUMMARY AND CONCLUSIONS

Of the four insecticides tested, carbofuran and methidathion

were more effective than methomyl and phosmet against larvae and adults

of Egyptian alfalfa weevil. Higher dosages of all of these insecticides

were required for the adult weevils than with the larvae, but, because

the toxicity studies were conducted using two different methods of

testing, it is difficult to make a very accurate comparison. Methomyl

was more effective against the larvae than adults while the opposite

was found with phosmet. It is concluded that carbofuran and methida­

thion were the most effective insecticides tested against larvae and

adults of Egyptian alfalfa weevil and that significantly higher dosages

of methomyl and phosmet were required. Since carbofuran and methida­

thion have short residual lives and since they are effective at very

low dosages, they are two of the best if not the best, insecticides

to use for control of both larvae and adults of Egyptian alfalfa weevil.

27 APPENDIX A

s AND LC95s DATA FOR LARVAE AND

5Qs AND LDg5s DATA FOR ADULTS

28 S 2 Table A.1. LC5ns and LC_,. (yg/cm ) with upper and lower limits for four insecticides on Egyptian alfalfa weevil larvae (Hypera brunneipennis).

Lower Limit Upper Limit Lower Limit Upper Limit Insecticide Rep. 95% CL 95% CL 95% CL 95% CL Slope LC50 LC95

Carbofuran 1 0.01 0.02 0.02 0.03 0.04 0.05 4.62 2 0.01 0.01 0.02 0.02 0.03 0.03 3.30 3 0.01 0.01 0.01 0.03 0.04 0.06 2.75 4 0.01 0.01 0.01 0.03 0.03 0.05 3.30

Methidathion 1 0.05 0.05 0.06 0.09 0.10 0.13 5.67 2 0.04 0.04 0.05 0.06 0.07 0.09 6.84 3 0.04 0.04 0.05 0.08 0.09 0.12 4.91 4 0.04 0.04 0.05 0.07 0.08 0.11 5.55

Methomyl 1 0.17 0.23 0.27 0.52 0.65 1.01 3.69 2 0.19 0.24 0.27 0.45 0.55 0.79 4.49 3 0.19 0.22 0.26 0.47 0.59 0.89 3.88 4 0.18 0.22 0.25 0.46 0.53 0.77 4.18

Phosmet 1 0.33 0.47 0.59 1.63 2.40 5.71 2.31 2 0.47 0.59 0.71 1.59 2.20 3.91 2.90 3 0.55 0.64 0.74 1.32 1.60 2.27 4.12 4 0.51 0.62 0.72 0.44 1.90 2.86 3.46 Table A.2. kD5_s and LD_5s (yg/gi) with upper and lower limits for four insecticides on Egyptian alralfa weevil adults (Hypera brunneipennis).

Lower Limit Upper Limit Lower Limit Upper Limit Insecticide Rep. 95% CL 95% CL 95% CL 95% CL Slope LD50 LD95

Carbofuran 1 1.95 2.31 2.67 4.99 6.24 9.04 3.81 2 2.09 2.43 2.76 4.77 5.80 7.96 4.35 5- Methidathion 1 3.16 3.54 3.90 5.99 7.02 9.14 5.53 2 2.87 3.26 3.63 5.76 6.80 8.99 5.15

Methomyl 1 8.29 8.91 9.62 12.97 14.93 19.22 7.34 2 8.38 8.95 9.60 12.59 14.25 17.62 8.15

Phosmet 1 5.09 5.79 6.56 11.18 13.97 20.53 4.31 2 5.31 5.95 6.67 10.67 12.92 17.72 4.89 LITERATURE CITED

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