A Bioassay for Detecting Compounds Which Stimulate or Deter Feeding by the Sweetclover Weevil

.\ bioassay employing sweetclover root disks impregnated with various plant extracts has been developed, The bioassay has been IIS{.'(! to demonstrate the distrihut ion of substances influencing feeding in fractionated water-methanol-chloroform extracts of Melilotus injesta Among' 19 Melilotus species screened by Manglitz and Gorz (196'1), 1\1. in[esta Guss. was the only species not fed upon to an appreciable extent by the adult sweetclover weevil, Sitona cylindricollis (Fahraeous). The weevil resistance of M. injesta was confinned by Gross and Stevenson (1964) and Radcliffe and Holdaway (19601). Since the resistance of 1\1. iniesta is manifested by a refusal of the adult weevils to feed extensively on this species. it appears that resistance may result from either a lack of attractiveness or the presence of deterrent substances. Thus, studies to determine the chemical na ture of this resistance should focus first on detectingsubstances which would stimulate or deter weevil feeding. One prerequisite for a study of the chemical nature of resistance is a suitable bioassay for selectingbiolog-ically active fractions in various plant extracts. This paper describes such a bioassay and g-ives results obtained from preliminary experiments using the bioassay. MATERIALS Ar.;n METHOlls.-One of the first requirements in the development of an effective bioassay is a suitable means of presenting the extracts or Iractions to the weevils for feeding. For a bioassay involvingthe sweetdover weevil, which feeds exclusively from the leaf margins. the requirements for the feeding medium are: (a) the medium must be thin enough to allow feeding on the edges but rig-id enough to support the weight of the weevils; (b) the consistency of the medium must permit chewing by I Coleoptera: ell rculionidae. e A cooperative investigation between the Nebraska Agric'ullul"ul Experiment Station. University of ,:\1ebraska, Lincoln, and the Entomology Research Division and Crops Research Division, Agr. Res, Serv .• r;SDA. Supported in part by ARS Grant no. 12-14-100llO27(33). Contribution no. 280 of the Department of Entomology, t.nlvcrsity of Nebraska. Published with approval of the Director a. Paper no. 2028. Journal Series, Nebraska Agrlcultura! Experiment Statinn, Accepted for publication April iii, 1967. :: Assistant Professor of Agronomy, Univcrslty of Nebraska; R('search Entomologist, Entomology Research Division, Agr. Res. Serv., USDA: Research Geneticist. Crops Research Division, Agr. Res. Serv .. USDA; and Professor of Agronomy, University of Ncbruska: respectively, Cuss. and M. officinalis L. Lam. leaves. Indications arc that substances responsible [or the resistance of M. it/· testa or the susceptibility of M. officinalis to feeding by adult sweetclover weevils, Sitona cylindricollis Fahraeus, reside in the water-methanol fraction. the weevil; (c) the medium must be relatively inert so as to prevent appreciable feeding on the untreated material and to avoid the presence of substances which alter the feeding response of the weevil to the active chemical components; and (d) except for changes caused by feeding. the material should remain constant in size and shape so that the extent of feeding can be readily determined. Many materials. including pith from the Japanese elder as used by Thorsteinson (1955), agar blocks, and disks of potato tuber, carrot, sweet potato roots, cabbage, and celery hearts were tested as media for the bioasay, but all were unsuitable with respect to one or more of the foregoing requirements. Disks cut from swectclover roots and subjected to certain pretreatments are the most satisfactory of the bioassay media thus far tested. First-year plants of field-grown Evergreen sweet clover (AI. alba Desr.) provided the roots used in preparing the bioassay disks. The plants were dug in November 1965, and fleshy branch roots from 12 to 15 mm in diam were selected. Sections approximately 20 mm long, cut from the central portion of these roots with a 9-mm cork borer. were sliced into disks 0.1 mm thick with a hand microtome. The disks were washed several times in distilled water, suspended in water (50 mlj300 disks) in a 250-ml flask, frozen, and lyophilized. To inactivate enzymes and remove alcohol and chloroform-soluble constituents, the lyophilized disks were refluxed for 4 hr with boiling 100% ethanol followed by 4 hr of extraction with chloroform and 'I hr with methanol in a Soxhlet thimble. After treatment. the disks were stored under methanol at -20°C until used. Prior to use in the bioassay, disks were attached to no. I stainless-steel insect pins cut to a length of 13 mm. The 5 disks used for each treatment were placed in a 50-mm watch glass containing 0.15 to I ml of plant extract which had been evaporated nearly to dryness under vacuum. Sufficient water was added to nonaqueous extracts or fractions so that the disks were still damp after the organic solvents had evaporated. Petri plates 15 mm deep and 95 or 145 .III.~I/.It 1967 AKESOl\; ET AL.: BIOASSAY WITH SWEETCLOVER WEEVIL 1083 ...--_..-_.._-_ ..---Table I.-Feeding preference of the sweetclover weevil nn sweetclover root disks treated with water-methanel or lhlnroform phases of a methanol-chloroform-water extract III sweetclover leaves. ,1 ";I!nt.'s for each test are averages of 5 disks. ""ah,," with different Ieuers differ significantly at the I % level arrording to Duncan's multiple range test. Calculations fur signihr;lIIfr. were made only within gTUUI:»i. " \Llte'-methanol (I: t ), 0.:> m!. "lI'ate,-methanol phase (0.:; ml) representing 0.125 g fresh plant material, ~ t.hloroform-rnethanol-water 0,::):]), 0.5 ml, 'Chloruf"rlll phase (0.25 ml ) representing 0.125 g fresh plant nnnrinl diluted with 0.2.1 ml or methanol and 0.05 ml water.