AD-A093 171 INSECTS AFFECTING MAN AND ANIMALS RESEARCH LAB BAINE-ETC F/6 6/6EVALUATION OF INSECTICIDES, INSECT GROWTH REGULATORS, SKIN AND --ETCIUINOV 80 D L KLINE. R N ROBERTS N00014-79-F-0070

UNCLASSIFIED NL

11llllm7m

11111 1.00

11111-2 114 11.

MICROCOPY RESOLUTION TEST CHARTNATIONAL BUREAU Or STANDARDS- 1963-A

.T.)E O OF IS IERCA INSTIUCTIONREPORT DOcuME,:T-la,.. WW , EF,€ oR> COMP,,L..T-,NG VoRM_i /,V. REPO,,T NUMBER '' l:OV"T ACCESSON i." ECiPiENT', CATA,.OG NUMBERi

Annu<al Report No. 1 1,6-D-kO-1> '"/14. TITLE (and subtitle) EVALUATION OF INSECTICIDES, INSECT s. l1?EO, ~tT•'>E*' tfoVi

•- Annual - Sept. £ to;ROWTH REGULATORS, SKIN AND CLOTHING REPELLENTS, Oct. , 1980AM OTHER APPROACHES TO THE CONTROL OF COASTAL

r=4 SAND FLIES, CULICOIDES SPP. s. PERFORMING ORG. REPORT NUMSE!

7. AUTHOR(e) I. CONTRACT OR GRANT NUMBER(*)

Daniel L. Kline and R. H. Roberts1mmq P. 0. Box 14565 N00014-79-F-0070

Gainesville, FLorida 326049. PERFORMING ORGANIZATION NAME AND ADDRESS I. PROGRAM ELEMENT. PROJECT. TASKI,, AREAS WORK UNIT NUMlE RS

Insects Affecting Man and Animals Research A G U M

Laboratory NR 133-997

0 I. CONTROLLING OFFICE NAME AND ADODRFl,,, 12. REPORT DATE

g Office of Naval Research 01 November 1980

Code 443 13. NUMBER Of PAGES

S80 N. Quincy Street, Arlington, VA 222170< 14. MONITORING AGENCY NAME A ADDRESS(II different from Conrolfign Office) IS. SECURITY CLASS. (of #at #open)

Office of Naval ResearchCode 443 Unclassified800 N. Quincy Street Is. DECLASSIriICATION/DOWNGRADING

Arlington, VA 22217 SNEOULE

16. DISTRIBUTION STATEMENT (of this Report)

Approved for public release; distribution unlimited.

17. DISTRIBUTION STATEMENT (of the abettoct entered In Block 30, it different from Report)

Ill. SUPPLEMENTARY NOTES beeL1G9C

IS. KEY WORDS (Continue on 'ovaltie alde II neceeary and Identify by block numbAr)

Seasonal populaLion dynamics, Culicoide.s spp., treated screens, larvicides,repellents, adulticides.

20. 0A1ITRACT (Continue an revrae old* it neceeery and identify by block minber)

"opulation dynamics and control studies on CuTidoidez: sand flies were con-,p.. ducted at Parris Island, South Carolina, and Yankeetown, Florida.

C) Adult seasonal patterns were monitored by light traps. Four species, C.

fur ens Poey, C. hollensi Melander and Brues, C. mcllcus (Coquillett), and

ILJ C. missiosippicnis Hoffman are considered abundant. C. fzu 'r,' and C. mneteus

are present from mid-April through late October; C. ho.criris and C. mr.isois.ip-

- i- eflnss peak in the s erin and fall of the year. Larval habitat characterizatit ADD ,Io 1473 EDiTION OF I NOV st IS OSSOLETE

S/N 0102014601 C C I OcomaSECURITY CLASSIFICATION OF T74i$ PAGE (1ten D o Wk' e)

20. ABSTRACT (Continued)

-4tudies were initiated. Fluctuations in larval density were correlated withplant cover.

Candidate insecticides were evaluated as larvicides, residual applicationson household screens, and as aerosol adulticides. Based solely on toxicity toCuZicoides larvae, the decreasing order of effectiveness was chiorpyrifos,temephos, fenthion, malathion, naled, and propoxur. Based on knockdown capa-bility, toxicity, and longevity of the insecticides as a residual applicationon household screens, the decreasing order of effectiveness was propoxur,chlorpyrifos, malathion, and fenthinn. Seven insecticides were evaluated inour wind tunnel screening as aert;o[ adulticides. In order of decreasingtoxicity they were Decamethrln 9, permethrin, resmethrin, d-phenothrin, naled,malathion, and fenthion. na (7. / . 1d&4-'

Four commercial products (Avon's Skirf-So--Soft, Johnson's Baby Oil, Claubo,and mineral oil) were protective against sand fly bites. The mode of actionwas observed as trapping on oily skin rather than repelling attacking midges.

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OFFICE OF NAVAL RESEARCH

Contract No. NP 14-79-F-,070

Task No. NR 133-997

( ~ ~ I t MA ~!lLRFPT.NO. jAL 17 - -p 'i-I Evaluation of insecticides, insect growth regulators,

skin and clothing repellents, and other approaches

to the control of coastal sand flies, Culicoides sppe

by

/U Daniel L./Kline-m R. H.foberts /

Insects Affecting Man and Animals Research Laboratory

P. 0. Box 14565Gainesville, Florida 32604

I7) November 1989 ')

Reproduction in whole or in part is permitted forany purpose of the United States Government

Approved for public release; distribution unlimited

IIS ' S ~ .

SUMMARY

Population dynamics and control studies on Culicoide8 sand flies were conductedat Parris Island, South Carolina, and Yankeetown, Florida.

Adult seasonal patterns were monitored by modified New Jersey light traps.Three species (C. Juvens Poey, C. hollensis Melander and Brues, and C. melZeus(Coquillett) at Parris Island and 2 species (C. furens and C. mizssssippiensisHoffman) at Yankeetown are considered major pests. C. furens and C. meleushave several peaks from mid-April through late October. Both C. hollensis andC. m ssissippiensi. peak in the spring and fall of the year.

A detailed habitat characterization study has been initiated at Yankeetown.The relationship between larval population dynamics and factors such as timeof year, tidal dynamics, vegetative cover, and soil factors is being studied.Larval counts made from extraction of larvae from weekly samples taken betweenMay 28 and October 6 showed significantly greater numbers for Distichlis thanfor Juncus and Spartina vegetated areas.

Chemical studies with candidate insecticides include screening as larvicides,evaluating as residual applications on household screens, and laboratoryscreening as aerosol adulticides. Based solely on toxicity to Culicoideslarvae, the decreasing order of effectiveness of 6 insecticides was chlor-pyrifos, temephos, fenthion, malathion, naled, and propoxur.

Based on knockdown capability, high toxicity, and longevity of the insecticideas residual application on household screens, propoxur was the most effectivechemical evaluated. Next in effectiveness was chiorpyrifos, followed closelyby malathion. Fenthion was the least effective for all 3 factors, and demon-strated no practical use on window screens at any concentration tested.

Results of droplet size tests with malathion against field-collected C. rmi, ''c-oippiensas indicate that aerosols from standard ground ULV generators with avolume median diameter (VMD) of 8-10 jim should be highly efticlent for bitingmidge control. Seven insecticides were (valuated In our laboratory screeningprogram as aerosol adulticides. Ranked in order of decreasing toxicity theywere Decamethrin0, perme thrin, resmethrin, .- phvnothrin, naled, malathion, andfenthlon.

Four commercial products (Avon's Skin-So-Soft, Johnson's Baby Oil, Claubo, andmineral oil) were field-tested as skin applications against 6 species ofbiting midges. All were .ffective as protectants against bites, however, themode of action was observed to be trapping the midges on the oily skin surfacerather than repelling attacking midges. In the absence of a suitable repel-lent any of these commercial materials might serve as useful substitutes tomechanically prevent bites.

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I. INTRODUCTION

Because of the location of many military installations, particularly Navybases, on coastal tidal areas in the continental United States and theCaribbean, and the widespread distribution of Culicoide8 sand flies(biting midges) in these areas, the midges pose a serious threat to theefficiency of personnel stationed at these bases. Historically, controlof biting midges has been hampered by a lack of knowledge of theirpopulation dynamics and a lack of chemical products labelled for controlof either immatures or adults. Nevertheless, there has been no sustainedresearch effort to derive the data necessary for recommendations on in-secticidal control and personal protection. This report contains theresults of our first year's studies aimed at filling this informationvoid. Studies were conducted on basic population dynamics, chemicalcontrol, and personal protection.

II. POPULATION DYNAMICS

Population studies of Culicoides were conducted at two sites: ParrisIsland Marine Recruit Depot, South Carolina, on the Atlantic coast, andYankeetown, Florida, on the Gulf coast.

The Parris Island site is a good example of a type of salt marsh known aslow, or regularly flooded marshes. The characteristic feature of thismarsh is vast expanses of smooth cordgrass, 51]artina a,'Ucr-niflora Lois.,which grows in several height forms between mean sea level and mean hightide. Other characteristics of this site are high tidal amplitudes (>2.5m), vast dendritic creeks, many deep tidal. channels, and a very soft andsilty marsh substratum.

At Yankeetown are found salt marshes typical of the entire Florida Gulfcoast north of Tampa. The characteristic feature is broad expanses ofblack needle rush, eJioitus racmicr7"dz, Scheele grading into upland. Atthis site S. altlerniij'ra seldomly occurs in extensive stands, but isgenerally found fringing the edges of tidal creeks. Large patches offlistichZis eicata (Linnaeus Greene) are occasionally found at this site.Normally J. oemerwianus grows just above the normal high tide line, butthe Yankeetown marshes, like other northern Gulf coast marshes of Florida,have an unusual flooding pattern. From May through October the monthlymean sea level rises to more than a foot above the annual mean sea level;during this time even the dw,., and ,itiohlze] areas are flooded by eachhigh tide. During the rest of the year Ti ozo- and J'. ffohis{ remainunflooded by even most spring tides.

At both study sites, adult t'u4i,&.h'e seasonal iopulat ion patterns weremonitored with New Jersey light traps, modified by replacing the standarddelivery cone screen with 40-mesh brass screening. Data were obtained onspecies composition, seasonal icidence, and relative abundance. Thelight trap data (Tables I and 2) indit-ate that 3 species (C. fltrrcno Poey,

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L'. holleneis Melander and Brues, and C. melleuo (Coquillett) at ParrisIsland, and 2 species (C. furmns ; :d C. mtesissippiensis Hoffman) atYankeetown are abundant enough to be considered as major pests. At bothsites C. furens was present In trap collections from mid-April throughlate October with definite peaks in June, August, and late September -early October. 0. mollous has a similar seasonal pattern. Both C.hollensis and C. rn'szifipi vwi. are present in the spring and fall ofthe year with definite peaks in October through November, and Marchthrough mid-April.

At Yankeetown, larval population and habitat characterization studieswere initiated near the end of May. A study area, located within atypical section of the salt marshes, is subdivided into eighty-five 25-m 2

plots. Each plot was characterized according to major vegetative covertype, Spartiru, Juncus, Distichli, and various mixtures of these grasses.Previous studies have shown that C. furens and C. mississippiensisutilize these intertidal plant communities as breeding locations. How-ever, little is known about the relationship between the larval popula-tion dynamics of each of these ('ulioo1"des species, and other factors suchas time of year, tidal dynamics, and various soil characteristics. Suchinformation is very essential before control measures can be directedagainst immature stages. If larval distribution can he more definitivelydelimited, spatially and temporally, then larvicide treatments can berestricted to productive areas.

Sod samples (ca. 10 cm diam. X 8 cm deep) are taken weekly from eachmajor vegetative cover type by means of post hole diggers. Tie locationof each sample is determined through random selection of the plots inwhich the desired vegetation type is found. Larvae are extracted by ourrecently developed 2' agar technique (Kline et al. 1980). A portion oflarvae recovered from each vegetative type is reared for sex and speciesidentification.

The data (Table 3) collected between May 28 and October 6 show the meannumber of larvae recovered from sod samples was significantly greater for,'iste;d i[% areas (7.46 per sample) th~in for u i,-; and ,'irt Pia areas(5.64 and 4.75 per sample, respectively). Preliminary statisticalanalyses show that the factors DAY (week samples were taken), LOCATION(vegetative cover), and their interaction significantly explain ca. 30%of the variation in larval couits. We feel that a large portion of theremaining 70% variation mav be explaiimd by tidal dynamics aid soilcharacterist Ics.

The identification ,of larva, recovered from soil samples and held foremergence :,how.-; a trend of dec reas i ug ,. ,? nd Increasing 2. mie' *o-,-ip;i!'w'io; (Table 4) during this sai', rig period. This trend substantiateslight trap data (Tabile 2) which showed '. j'I .c' was more prominent Inlate spring throtigh the stimmer months, and ". ", e.' ., eduringlate fall and winter months.

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At Parris Island substrate sampling consisted of taking random samplesfrom Sp .'na vegetation at different locations throughout the base. Thenumber of samples taken during any week depended on the limited timeavailable for sampling by our biologist stationed at this site. The data(Table 5) varied too much from amj, to sample, and from week to week tomake many generalizations. It was noticed, however, that samples takenfrom underneath large oak tree trunks which had fallen into the marshconsistently produced large numbers of larvae with little variation. The8 samples taken during the last sampling week (July 24-30) is a goodexample of this. Much more substrate sampling needs to be done at thissite.

None of the larvae recovered were reared to adults, but 4 emergence trapswere placed in the Sp'azrti /, marsh and rotated weekly during the samplingperiod. Emergence trap data (Tabl, 6) showed the same seasonal trends asthe light traps (Table 1) with regard to C. f'urcns and C. hoglensis. NoC. melZeus were found in any of these collections. This is not sur-prising since past studies have shown that C. melieus prefers intertidalsandy beaches (Linley and Adams 1972; Kline and Axtell 1975). Much workneeds to be done at Parris Island to more definitively delineate Culicoideslarvae distribution and characterize the breeding habitats of the majorspeices.

111. CHMIiCAL CONTROl. STUDIES

Chemical studies have been divided Into 3 main areas with candidate in-secticides: screening as larvicides, evaluiation of residual applicationson househo d screens, and laboratory scre, ning as aerosol adulticides.

Larvicide Screening

Lirvae for these tests were obtained from field-colILcted substratesamples takun from known breeding areas at Yankeetown. Larvae wereexttracted from thet substrate by the aigar extraction method previouslycited. Only 3rd :tnd 4th Instar larvae were tised In these tests. Thelarvae were transferred with ani angled stainless steel probe into a50 ml g ass beakr containing 25 ml of untreated estuarine water. Thisbeaker was then decanted with a swirl ring ict ion into a 250 ml glassbeaker containing lo) ml of estuhr Inc water that is either untreated, ortreated at predetermined distriminatitg concent rations (ppm) of insecti-cide. Since the insecticides are formulated In acetone, the checks arealso conducted with acetone-water solutions. For each test, duplicatebeakers are treated at each concentrat ion level, thus exposing 20 larvaeat each level. Each te.:t is replic.ted at least 5 times.

At 24 hr postexposure mortality counts art, made. Larvae arc considereddead if, when touched with a probe, they Iack the ability to move intheir characteristic serpent ine mot ion, I.e., rapid flexing of the body.

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Since the larvae are found in sod, the effects of habitat sod placed inthe treatment water also were studied. The dose-response relationship isdetermined by probit analysis of the log-transformed mortality data.

During this past year, 6 insecticides were evaluated as larvicides:temephos, propoxur, fenthion, naled, malathion, and chlorpyrifos. Basedsolely on their toxicity (Table 7) to biting midge larvae, chlorpyrifosand temephos were an order of magnitude more effective than the otherchemicals, followed in order by fenthion, malathion, naled, and verydistantly by propoxur. Addition of habitat soil did not change the orderof effectiveness, but 3-4 times as much temephos and chlorpyrifos and ca.1.25 - 1.5 times as much fenthion, malathion, and naled were required tocause the same mortality as estuarine water alone. Habitat soil hadlittle or no effect on the killing effect of propoxur.

Evaluation of Treated Window Screens

The evaluations of residual applications of candidate insecticides wereconducted against C. mississippinesis using 2 different methods: a smallscreen method used previously by several investigators (Jamnback 1961,1963; Dukes and Axtell 1976), and a newly developed large screen method(Roberts and Kline 1980). Aluminum screening, 16 X 18-mesh, was used inboth methods.

For both methods, insecticides were tested in concentrations of 1, 3, 5,and 8% active ingredient (weight/volume). The insecticide solutionswere agitated thoroughly before the treatment period. Screens used inthese tests were treated by dipping in an acetone solution of technicalinsecticide or, in the case of emulsifiable concentrates, in a watersolution for about 10 seconds After treatment, the screens were hungbeneath ti caves of a building to dry. Control tests were conductedwith screens immersed in acetone only.

During the past year chlorpyrifos, fenthion, malath ion, and propoxur, allformulated in acetone with technical grade materi;al, were tested by thesmall screen method. For large screen tests chlorpyrifos and propoxurwere tested as emulsifiable concentrates, and as technical grade materialin acetone.

The small screen method uses a divided test chamber that consists of two500 ml paper cartons with the open tops abutting and taped together witha 9 cm diam. screen inserted in the middle. The bottom of one of thecontainers was replaced with transparent plastic to allow light to enter

and the bottom of tle opposite container was left Intawt to create partialdarkness during testlng. A hole ca. 2.5 cm in diameter Is made in thebottom of the other carton and closed with a stopper. Total darkness wascreated by covering the plIastic window with a lid.

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Adult biting midges used in these small screen tests were collected fromnatural populations by either inducing them with CO to enter a fieldtrailer and subsequently aspirating them off a window, or attractingthem into a CO -baited suction box trap immediately before testing.

2While in the box trap the biting midges had access to 10% sugar water.Approximately 30 midges were introduced into the chamber through thestoppered end. All tests consisted of at least 4 replicates, but as manyas 8 replicates were obtained for some chemical concentrations.

When the lid was removed the biting midges, attracted by light, quicklypassed through the treated screen. Lighting was standardized by placinga 25-watt fluorescent fixture ca. 8 cm in front of the test chambers.After passing through the treated screens, the midges were lightly anes-thetized with CO and transferred to holding cages which consist of a 215cc. paper carton with a fine mesh cloth as a top. Cotton pads soaked in10% sugar water solution were placed on top of the holding cages toprovide food and water for the midges during the holding period. Theholding cages were placed in large styrofoam chests, containing moistenedcotton pads to maintain a high humidity environment, and moribund anddead adults were counted at 0.5, 1, 2, 3, 4, and 24 hr postexposure.

After each test, the treated screens were removed from the test chamberand hung outside under the eaves of a building to age and weather. Thescreens were tested ca. every 7 days, depending on the availability ofadult biting midges, to determine the longevity of each treatment.

The large screen technique utilizes the biting midges attractancy to CO2and light to induce natural (field) populations of biting midges topass through a treated screen into a collecting cage. Essentially, thetrap is a box shell 30.5 cm (11 X D X W) except for one side which wasleft open for insertion of a large 950 cm2 -treated screen. On the sid,directly opposite the treated screen, a small hole is cut for attachmentof a collection cage. During testing the trap is oriented so that thescreen is nearly horizontal ca. 20-30 cm above ground level, and the col-lection cage is almost perpendicular with the ground. CO2 is released(ca. 100 ml/min) at ground level below the trap from a compressed gastank with a flow rate controlled by means of a single ntage regulator andmetered with a GilmontO compact flow meter. Four exposures of at least25 biting midges each are made for each treated screen. As with thesmall screen, counts of moribund or dead midges are made at 0.5, 1, 2, 3,4, and 24 hr. after exposure. After completion of the tests the boxesare left in the collection area for aging and weathering with screens inplace but in a vertical position. Effectiveness of the candidate insecti-cide is test.ed at various Interval.s after treatment, depending on theavailability of natural. populations of adult biting midges.

Criteria for effectiveness were knockdown capability (I hr mortalitycount), high toxicity (4 hr mortality count), and longevity of the in-secticide when weathered.

Based on these 3 criteria, propoxur (Tables 8-10) was the most effectivechemical tested. At 8% propoxur caused 87 - 100% knockdown, and 95-100%

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high toxicity for 35 days after treatment. After propoxur, the mostpromising chemical was chlorpyrifos, followed closely by malathion. At8% both were nearly equally effective, with malathion having a slightedge in knockdown capability, aud chlorpyrifos with an edge in longevityof high toxicity. Fenthion was the least effective for all three factors,and demonstrated no practical use on window screens at any concentrationtested.

In Table 9 the results of testing emulsifiable concentrate formulationsin water at 8% are presented for chlorpyrifos, propoxur, and malathion,as well as the 8% technical in acetone formulation for malathion. Basedon these data we have concluded that water-based formulations of emulsifi-able concentrates are not as effective as residual window screen treat-ments as technical in acetone formulations.

Future evaluations of candidate insecticides for residual screen treat-ments will be a 2-step testing program in which we plan to use the smallscreens for initial testing of new materials and the large screens forsecondary evaluation under more natural conditions. Secondary evaluationswill also include different formulations and methods of application.

Aerosol Adulticide Studies

Droplet-size studies.--In mosquito control, droplet size of an insecticideaerosol influences efficiency in killing adult mosquitoes (Mount et al.1975). To determine the effect of droplet size on insecticidal efficiencyon biting midges, laboratory wind tunnel tests were conducted with cagedadult female C. sr.p{'h'?in . A Berglund-Liu Monodisperse AerosolGenerator (Thermo Systems, Inc.,) was used to introduce uniform sizeddroplets into a wind tunnel for exposure of insect samples.

Droplet size was varied by a technique involving evaporation of a volatilesolvent (isopropanol) from a solution containing a nonvolatile insecti-cide (malathion) at different concentrations. Exposure time was variedto provide different dose levels.

A series of tests with field-collected C. miSi.Ssij1cnsis adult femaleswere conducted for 3 aerosol sizes (2.8, 4.8, and 8.2 vim diameters) ofmalathion at 6 dose levels. Mortality readings were made at 1, 2, and 24hr. after treatment.

Each treatment was replicated 4 times on different days. The results(Table 11) showed an increase in mortality (lower LD-50) with time aftertreatment due to a slow response to malathion. The smallest aerosol size(2.8 rim) was clearly less efficient than the 2 lnrger sizes (95% fiduciallimits do iot overlap at 2 and 24 hr posttreatment). The largest size(8.2 tim) produced the lowest ID-50 at each time, however, the differencebetween 4.8 and 8.2 yim was not large (957; fiducial limits overlap at eachreading). The leVel ing-off t ndency betwlV en 4.8 and 8.2 Jim indicatesthat the optim,|m size is not mth, if any, greater than 8.2 tim. Further

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tests with larger sizes will be necessary to confirm this. The resultsof these tests indicate that aerosols from standard ground ULV generatorswith a volume median diameter (VMD) of 8-10 pm should be highly efficientfor biting midge control.

Relative toxicity tests.--We feel. that compounds registered for groundULV application for mosquitoes, if used properly, can give some temporarylocal reductions in biting midges. However, insufficient studies havebeen conducted to provide adequate data on control by this method.Therefore, the evaluation of candidate chemicals against adult C: Zj'o e3in wind-tunnel tests is an essential p;.rt of our research program.

The wind tunnel system used was basically that described by Mount et al.(1976). Several changes were necessitated by the small size of the adultbiting midges. The major change was the substitution of the 16-meshgalvanized screen wire wind tunnel tube with 40-mesh brass screen. Alsothe fabricated atomizing nozzle was changed to a commercially-availablestandard stock nozzle (#12891 I/8JJ, Spraying Systems Co., Chicago, IL).The wind tunnel consists of a cylindrical tube 15.5 cm in diameter throughwhich a column of air is blown at a rate of 6.4 km/ha. Approximately 25adult female biting midges are confined in cardboard exposure cages, 8.6cm in diameter and 5.0 cm high, with 40-mesh brass screen ends. Thecages are placed in the center of the tube for exposure. One-fourth mlsolution of the desired concentration of the technical insecticide inacetone (wt A.I./volume diluent and expressod as % concentration) isatomized at 105.5 g/cm 2 into the upwind portion of the tube, and the in-sects are exposed momentarily as the aerosol passes through the cage. Weuse an automatic pipette for convenience and efficiency.

Following exposure, the insects are lightly anesthetized with carbondioxide and transferred to new 8.b X 5.0 cm cardboard holding cagescovered with fine mesh cloth screen tops. Cotton pads, soaked in a 10%sucrose solution, are placed on the cloth screens to sustain the insectsuntil all mortality counts are made. The holding cages are placed inlarge styrofoam chests, containing moistened cotton pads to maintain ahigh humidity environment. Knockdown is checked 1 hr after treatment,and mortality is recorded 24 hr after treatment. Checks are exposed tocontact sprays containing acetone only and handled in the same manner.

In our testing procedure candidate insecticides are first tested at con-centrations of 0.25% (wt/v). Then the concentration is successively re-duced by one-half until the 24-hr mortality falls below 50Z. Pivettingsof a serial dilution of a given Insecticide are made from low to highconcentrations without cleaning the wind tunnel. However, the tunnel iscleaned at the beginning and end of each test se'ies and before each newinsecticide is tested.

Since we do not have a laboratory colony of T" /z o '/,, field collectionsof adults are made with CO2,-bait,! suction traps. The biting midge's are

i-

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trausported to the laboratory and knocked down; ca. 25 are transferred toeach exposure cage within a cold room. Duplicate cages are used in eachtest and at least 3 replicates are made with each concentration of eachinsecticide. At least 4 discriminating concentrations of each insecti-cide are used in each replicate.

This test is not intended to yield results that can be compared closely;however, it does eliminate those insecticides that are not toxic enoughto warrant further testing, and it identifies the range of discriminatingconcentrations for those insecticides that are highly effective. Thisinformation is useful in the design of additional test programs.

The dosage/mortality data are used to determine the LC-50 and LC-90 ofeach compound. The dose-response relationship is determined by probitanalysis of the log-transformed mortality data.

During this past year, 4 synthetic pyrethroid (Decamethrin®, permethrin,resmethrin, and d-phenothrin), and 3 organophosphorous (naled, malathion,and fenthion) compounds were evaluated in our laboratory screeningprogram.

The 24 hr LC-50's and LC-90's and their respective 95K fiduci.,l limitsare pre.enIed in Table 12. The compounds are ranked in order of decreasingtoxicity (LC-90) to C,.,,', ::'.naz. The synthetic pyrethroids wereby far the most effective insecticides. Of the 3 organo-phosphorouscompounds tested, naled was slightly more effective than malathion.Fenthion was the least effective of all the insecticides tested.

Relatively quick knockdown Is a desirable, characteiistic of biting midgeadulticides. All the pyrethroids produced relatively quick knockdown(within I hr) at concentrations near that required for 24-hr mortality.Naled produced the bost knockdown among the 3 O-P compounds. Fenthiondemonstrated practical lv no knockdown within 1 hr, and malation showedonly moderate kiockdown.

IV. PERSONAL PROTECTION

Prior to ilitiation of the present contract, we (Schreck et at. 1979)had field tested the effectiveness of deet-treated net jackets against 4species of biting midges (' .'', .;, ' a, ' ;Z .

and C. hn,,)vz; Wirth and llIanton). We found that the jackets providedca. 98% protection against all SpcCies except C . ::'.. They gaveonly ca. 59% protect ion against this species.

During this past ycar 4 tommrelciaI products were-ff ield-to.ted as skinapplications against 6 species of biting midges. Tsts were designed toevaluate Avon's Skin-So-Soft, Johnson's Baby Oil, Claubo, and mineraloil using dcct as Lie standard.

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The test procedures were described by Schreck et al. (1979). The com-mercial formulations and mineral oil were applied at full strengthwhereas a deet standard was applied at a 12.5% or 25% ethanol solution.All applications were made in 1 ml aliquots and spread evenly over theforearm of a subject from wrist to elbow. Six subjects participated ineach of the studies.

All subjects wore a headnet and shirt with sleeves rolled to the elbow.Gloves were worn by the treated subjects, permitting exposure of onlythe forearm whereas exposure of the hands of the check subject allowedextra freedom for killing and counting biting midges. All wore longpants, usually military fatigues. Treated arms were continuously ex-posed to the natural population of midges. Tests were made in units of5 min each because of the fluctuating activity of the midges. Up to 215-min tests were made in 1 replication of a test series, depending onmidge activity and weather conditions at each location.

The purpose of the 5-min test was to establish an index of biting pressureby using the untreated check subject as an indicator of activity whilethe other subjects concurrently exposed treated arms to the midge popula-tion. At the end of 5 min, the number of bites occurring on each of thetest subjects and the check were recorded. The subjects (each separatedby 8 - 10 m) then changed positions, moving in a clockwise direction sothat no one remained at a location longer than 5 min. A differentsubject was used as a check in each replication of the study. Duringeach 5-min test, the check subject counted only those midges that werebiting the forearms and hands, and these were crushed so that they werenot counted more than once. Each material was paired directly with deetin each tes t series. Since it was not always possible to distinguishspecies when they were biting, collections were made by aspiratingmidges in the act of biting the untreated check subject during tests.Subsequent determination of species composition of the biting midgepopulation was then made.

Results of the study (Table 13) show Skin-So-Soft, Johnson Baby Oil,Claubo, and mineral oil are effective as protective applications for theprevention of bites from at least 6 species of the genus CuZico;,ide.Certainly, in the absence of a suitable repellent these commercialmaterials might serve as useful substitutes to mechanically preventbites. Observations made by all test subjects revealed that whereas themode of action of deet was to repel attacking midges, the other materialsmerely trapped them on the oily skin surface, thus preventing bites.

As a caution, iL must be noted that Skin-So-Soft is marketed not as askin app] l,:ation at full strength but as a bath vil at the rate of 1/2cap full in a bath tub of water. It would therefore bc prudent to in-vestigate what effect, if any, long-term skin applications might have onthe potenLil user of this as well as the other mat:erI.ls tested.

Ik -pow

-12-

REFERENCES CITII)

Dukes, J. C., and R. C. Axtell. 1976. Residual effectiveness of insecticide-treated screens for control of biting midges, Cidic-o dl f'urcrs (Poey)(Diptera: Ceratopogonidae) . Mosquito Ncws 36: 488-491.

Jamnback, 11. 1961. The effectiveness of chemically-treated screens inkilling annoying punkies, !'u1Z*,o:, 'ao tuc: . Econ. ntomo1.54: 578-580.

Jarnuback, It. 1963. Further observations of the effectiveness of chemically-treated sci'eens in killing bit ing midges, :'luio ,n: (Diptera:Ceratopogonidae). .1. Econ. Entomol. 56: 719-720.

Kline, D. L., and R. C. Axtell.. 1975. Cul, coidcr m, '7leur (Cq.) (Diptera:Ceratopogonidae): seasonal abundance and emergence from sandy inter-tidal habitats. Mosquito News 35: 328-334.

Kline, D. L., R. H1. Roberts, and D. A. Focks. 1980. Extraction of CuZicoides;LS~$S2>l, ;H offman larvae from salt marsh soil with a new agar

technique. Mosquito News (accepted for publication).

Linley, J. R., and G. H. Adams. 1971. Ecology and behavior of immatureu ?,) ; ~'I.:t:w (Cq.) (1)iptera:Cera ogonidae). Bull. Ent . Res.

62: 113-127.

Mount, G. A., N. W. Pierce, and K. F. BaIldwin. 1975. Droplet size of aerosolsdispersed by portable and truck-mounted generators. Mosquito News:35: 195-198.

Mount, G. A., N. W. Pierce, and K. F. Baldwin. 1976. A new wind-tunnel systemfor testing insecticidal aerosols agiinst mosquitoes and flies. MosquitoNews 36: 127-131.

Roberts, R. I1., and 1). L. Kline. 1980. A methed to evaluate insecticides ashousehold --creen treatments against ', I, spp. blting midges.Mosquito News: 40: 399-402.

Schreck, C. E., 1). Kline, and N. Smith. 1179. Protection afforded by theinsect repellent jacket against four species of biting midge (Diptera:

!'c/, ,it;'s;)M. Mosquito News 39: 739-742.

--m

-13-

BIBLIOGRAPHY OF PUBLICATIONS/MANUSCRIPTSPREPARED UNDER THIS CONTRACT

PUBLICATIONS

Roberts, R. H., and D. L. Kline. 1980. A method to evaluate insecticides ashousehold screen treatments against Cuiiooides spp. biting midges.Mosquito News 40: 399-402.

MANUSCRIPTS

Kline, D. L., and R. H. Roberts. 1980. Effectiveness of chlorpyrifos, fenthion,malathion, and propoxur as screen treatments for control of Culicoides

mississippiensis Hoffman (sent to J. Econ. Entomol. for review).

Kline, D. L., R. H. Roberts, and D. A. Focks. 1980. Extraction of Culiioidesmississippiensis Hoffman larvae from salt marsh soils with a new agartechnique. Mosquito News (accepted for publication).

Schreck, C. E., and D. L. Kline. 1980. Repellency determinations of fourcommercial products against sE: species of biting midges (Diptera:Ceratopogonidae: Culicoides). Mosquito News (accepted for publication).

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-19-

Table 5.--Recovery of Culicoides larvae from substrate samples taken fromSpartina habitats at Parris Island, South Carolina.

: Number Range of : Avg. no.Number pos. pos. : larvae per

Dates samples samples samples sample

March 19-25 6 6 1-200 65.3March/April 26-02 4 4 1- 9 0.8

April 03-09 26 4 1- 7 0.710-16 -- -- -- --

17-23 15 9 1- 18 2.724-30 12 8 1- 18 4.7

May 01-07 7 4 1- 2 0.908-14 9 6 1- 30 6.2

15-21 4 3 6- 19 8.022-28 8 6 2- 21 9.1

May/June 29-04 4 3 2- 5 2.3June 05-11 12 8 1- 7 2.0

12-18 -- -- --

19-25 -- -- -- --

June/July 26-02 4 3 1- 4 1.8July 03-09 8 4 1- 12 2.1

10-16 4 3 1- 7 3.517-23 4 3 5- 12 7.324-30 8 8 10- 75 43.4

a/ All samples taken from under fallen tree trunks.

-20-

Table 6.--Seasonal emergence pattern of Cuticcides spp. from Spartina habitatsat Parris Island, South Carolina.

Number collectedDates C. hollensis C. furens

March/April 26-02 15 0April 03-09 13 0

10-16 8 017-23 36 024-30 25 0

May 01-07 16 508-14 41 1015-21 33 722-28 23 54

May/June 29-04 -- -June 05-11 4 15

12-18 3 219-25 4 59

June/July 26-02 0 47July 03-09 0 23

10-16 0 9317-23 a 0 224-30=' 0 85

-21-

Table 7.--Toxicity of candidate chemicals as larvicides against CuZicoidesmississippiensis Hoffman.

Water only : Water/habitat soil

Chemical LC-50 LC-90 LC-50 LC-90

Chlorpyrifos .001 .002 .003 .007

Temephos .002 .006 .013 .025

Fenthion .011 .023 .014 .032

Malathion .121 .402 .198 .601

Naled .418 .826 .500 1.036

Propoxur 3.348 7.944 4.000 7.924

-22-

Table 8.--Knockdown effectiveness of small treated screens (technical inacetone) against Culicoides mississippiensis Hoffman atYankeetown, Florida.

Avg. % mortality-'-/ ( hr postexposure) at daysConc. : treated screens were weathered

Chemical: (%) : 1 : 7 14 : 21 : 28 35

Propoxur 1 92 66 69 16 4 33 93 98 82 41 10 25 100 100 93 76 64 648 100 98 100 96 87 90

Chlor-pyrifos 1 50 NT- 1 10 2 --

3 74 NT 33 32 7 --

5 88 NT 82 63 50 88 93 NT 90 74 64 48

Malathion 1 41 15 20 11 2 63 87 45 47 27 3 35 87 55 53 39 40 188 98 93 97 76 58 53

Fenthion 1 5 4 3 2 2 13 11 1 3 1 1 15 27 2 6 5 2 28 47 11 12 5 8 6

Checks 2 2 3 2 2 2

a/ Mortality data are corrected for control by Abbott's formula.

b/ Avg. of 4-6 replicates of ca. 25 flies each.

c/ .nsufficient natural populations for testing.

-23-

Table 9.--Toxic effectiveness of small treated screens (technical inacetone) against Culicoides mississippiensis Hoffman atYankeetown, Florida.

Avg. % mortalitya'b/(4 hr postexposure) at daysConc. treated screens were weathered

Chemical: (%) : 1 7 14 21 28 35

Propoxur 1 95 69 69 17 2 53 95 98 82 50 11 15 100 100 98 81 67 758 100 100 100 100 95 97

Chlor-pyrifos 1 83 NT-E 47 47 27 --

3 89 NT 80 62 36 --

5 99 NT 98 93 73 738 99 NT 100 99 97 97

Malathion 1 64 22 27 15 4 63 94 58 58 27 3 155 99 70 69 39 43 348 99 99 98 87 79 80

Fenthion 1 35 17 3 1 3 13 63 22 12 6 1 45 77 23 24 21 2 38 83 83 77 33 52 31

Checks 3 4 5 4 10 7

a/ Mortality data are corrected for control by Abbott's formula.

b/ Avg. of 4-6 replicates of ca. 25 flies each.

c/ Insufficient natural populations for testing.

-24-

Table lO.--Effectiveness of large treated (8% wt/vol) screens againstCulicoides mississippiensis Hoffman at Yankeetown, Florida.

Avg. % mortality (4 hr. postexposure) at: Formula-,/-- days treated screens were weathered-

Chemical lation-° 1 7 14 21. 35

Malathion t 95 92 88(est.) -- 77

EC 75 51 62 -- 76

Propoxur EC 74 52 29 --

Chlorpyrifos EC 99 89 72 66 49

a/ Avg. of 4-6 replicates of 25 flies each.

b/ t = Technical insecticide in acetone.EC = Emulsifiable concentrate; water-based formulation.

-25-

Table ll.--Effect of droplet size on dose units of malathion required for 50%mortality of caged Culicoiaes mississippiensis Hoffman exposed towind tunnel generated aerosols.

Aerosol : LD-50/ (95% fiducial limits)droplet 1 hr. 2 hr. 3 hr.(Gm) posttreatment posttreatment posttreatment

2.8 13.6 8.60 3.53(9.63 - 19.56) (7.55 - 9.84) (3.03 - 4.08)

4.8 9.80 4.93 1.86(8.48 - 11.43) (4.29 - 5.65) (1.07 - 2.69)

8.2 8.17 4.69 0.96(5.83 - 11.94) (4.01 - 5.46) (0.68 - 1.24)

a/ LD-50 is presented in arbitrary dose units (1 unit = 0.0066 pg technicalmalathion).

-26-

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-27-

Table 13.--Mean numbers of bites/min received from CuZicoides spp. in pairedtests with a deet standard as a 12.5 or 25% ethanol solution andthe candidate materials at full strength.

Yankee own . Ft. 14 rs Parris sland:__FM_ _ : FL- : SC_

Paring of : No. of : Mean : No. of : Mean : No. of : Meancandidates : repli- : no. of : repli- : no. of : repli- : no. ofand standard : cations : bites : cations : bites : cations : bites

Deet 6 0 3 1.46 3 .007

Skin-So-Soft 6 0.06 3 0.25 3 0

Deet 4 0 2 .13 3 .007

Johnson's Baby Oil 4 .01 2 2.0 3 .003

Deet 4 0 2 .75 3 .093

Claubo 4 .38 2 .25 3 0

Deet 2 .25 3 .003

Mineral oil 2 .38 3 0

Check (total of2 arms) 12.8 83.7 11.7

a/ Species identified in biting collections were 58% C. mississippiensis, 17%C. barbosai, 13% C. floridensis, and 12% C. furens.

b/ Species identified in biting collections were 94% C. barbosai 5% C. furens,and 1% C. floridensis.

c/ Species identified in biting collections were 80% C. hol ensis and 20%C. melleus.

d/ Deet tested at 25% of ETOH.

e/ Deet tested at 12.5% in ETOH.