Report on a visit to the laboratory of Dr Raymond Noblet, Clemson University South Carolina 26/3/1991, and comments on the development of a new bioassay system sponsored by Abbott

world Health Organization - Onchocercrasis Control Prog ramm6 - Insecticide Research Unit Page 1 DE: C. Back IRU, Bouak6 A: Dr. D. Quill6v6r6 Chef VCU, Ouagadougou DATE:3o/3/1991 lA/11 151 Raymond Noblet, Clemson the development of a newSUJET:ReportonavisittothelaboratoryofDr.University, South Carolina, 26/3/1991, and comments on bioassay system sponsored by Abbott' REF:IRU/CB/91.26 ATT.: Directeur OCPDr. D.A.T. BaldrY, GenEve Chef WOA, Bamako Chef EOA, Kara Dr. Y. Bissan, AREM, Bamako Mr. L. Yam6ogo, Ouagadougou CC : Mr. M. Mr. M. Ocran, IRU Ouedraogo, IRU One of the major obstacles to the evaluation of B.t.H14 formulations as black fly larvicides is the difficulty to run large-scale laboratory bioassays. Black flies cannot yet be reared efficiently and current bioassay techniques imply systems installed in the field and the use of larvae collected directly from the river. Mosquito larvae (Aedes aeg.vpti) are commonly used to assess the potency of B.t.H14 formulations, by comparison with a standard, but it is widely acknowredged now that the efficacy on brack fly and mosquito larvae may differ significantly. In order to improve the quality and potency of B.t.H14 formulations ""nt to ocP, Abbott has sponsored the development of a new bioassay system by Dr. Raymond Noblet and his collaborators of Clemson University' The new bioassay system is based on the use of a standard laboratory orbital shaker that can generate current in a series of round flasks placed on top which contain black tly larvae. This system is in its final phase of development and since I was attending the AMCA meeting in New orleans, a side trip was organized by Mr. Ernest Dankwa of Abbott to the laboratory of Dr' Noblet. The visit tooK place on the 26/3/1991, with the following participants: - Dr. Raymond Noblet, professor of Medical Entomology at Clemson University. - Dr. william Barton, in charge of the bioassay system project at clemson' - Mr. Elmer Gray, assistant of Dr' Barton' - Dr. Brian Melin, in charge of B.t.H14 development at Abbott' - Mr. Dale Cooper, in charge of fermentatlon processes at Abbott' The visit started with a demonstration of the bioassay system in action; Dr. Barton and Mr. Gray were performing bioassays with a B.t.H14 formulation using sinuliun pictipes, a species that breeds in that area. one orbltal shaker unit measures approximately 1m x 0.75m x O.Sm (L x W x H). The system ls noiseless and there is no vibration of the unit once the internal counterweights are balanced. The top plate is fitted with clamps that can hold 40 round flasks of 250 mL fitted with wide mouths. Each flask contains 150 mL of distilled and demineralized water plus the black f ly larvae. The water speed ls control by a REqu I 5 AVR ISgI MEMORANDUM at\or'l d H"a'1 th OrEanization - Onchocerclasrs Control pr69r36;1e _ Insectictde Research Untt knob on the front panel, rrext to a digital display of the speed tn R.p.M.. The maximum speed is in the order of 350 RpM. The second part of the visrt was the laboratory where Dr. Noblet and his colleagues intend to rear biack flres. Therr sr-stem rs largely the same as that ofDr. E.w. cupp, who was a pioneer in the development of black fly rea.ng systems. Dr. Barton lroDes to be able to orovrde his bioassay system with the rearing unit usino .Srmrr/iun vittatun. The visit ended with a discussion about the shaker bioassay system. Dr.Barton provided the parttcipants with a dossier of results obtained in the lastfew months' several parameters need to oe cali b ratetJ oefore the system isdeemed fully operatronal: type and amount of water per fiask. number of larvaeper flask' choice of black fly species. acceotable size for larvae, water speed. cul'atrorl of expusLrr e to B.t.h l4, temDerature, number of concentrattons and number of replicates per concentration. The present selection of parameters forlate instar s' pic'tipr:s larvae is: 150 mL of distilled deionized and demineralized water ner f !ask, ia lar,,zae oer f lask. 5 rnri. acclimation at .1 55 Rp14 for attachment,12 h exoosure at 350 RPM artd 23-25 c ( rocm temperature ). 6 concentrations ofB.t.H r4, 6 replicates per concentration, 4 control flasks. The probit anal-vsis results obtained bv Dr. Barton show much similrtude with the results obtained wrth other types cf lab systems: the slope of the probit regression line is weak ((1.5) and the coefficient of variatron of the LC5os goes up tc 50%' I my oprnlorl the species usec for the test. s. pictipes, is partly responsible for thrs; it breeds in waterfalls and demands extremely fast water speed in order to feed normally. if the water speed is sub-optrmal, larvae wrllfeed on and off, or small differences ln water speed between assays will generatelarge differences In the filtration rate of larvae. s.pictipe.s also has stout, widely spaced fan rays and probably feeds more eff icienily on large particles ( >5oum );therefore differences in particle sizes of B.t.H14 formulations havrng equalamounts of toxin will bring differences in calculated LC50s; moreover, formulations with larger particles might fare better in the bioassay, while the reverse is truefor most formulations in river conditions. Using s. r,ittatu, as a test species would probably solve these problems. especially if it can be reared in the lab.one particular aspect of the protocol used in clemson is the very small amount of water per larva in each flask, and the continuous exposure for a longperiod of time (12 h).Although implying minimal manipulations, this protocol hasthe disadvantage of rendering the mortarity in each frask density-dependent forthe lowest concentrations of 8.t.H14, a situatron which is never found in the field,in open water-circuit bioassay systems, or in bioassay systems with a short exposure time followed by a change of water. This is why I proposed in clemsonthat the larvae could be exposed to the B.t.Ht4 formulations for a period of 20-30min, and then put in observation for a few hours in a cool-box, without anywater current' This would have the advantage of removing the density-dependent aspect of mortality because of the shorter feeding time and higher concentrationsof B't'H14, and the advantage of freeing the shaker earlier. It would eventuallybe possible to run one set of 40 flasks per hour; the limlting factor would thenonly be the manpower and availability of larvae. If this protocol works, theshaker bioassay system would represent a 1oX increase of the screening potential when compared with standard mini-gutter bioassays.Mr Dankwa and Dr. Melin have stated that Abbott is ready to have twoshaker units statroned in the ocP area in order to confrrm potency ratings Page 2 I I I Wor'ld Health Organization - Onchocerciasis Control Programme - Insecticide Research Unit obtained in North America. These units could very easily be operated at the field station in Soub16, where electricity and larvae are available. Considering that Mr. Dankwa and Dr. Melin will be visiting the OCP in July, Abbott should be encouraged to send the two shaker units as soon as possible, which would enable IRU/OCP to perform the calibration steps and be ready to test the f rrst formulations by the end of June. In conclusion, the progress accomplished by Clemson University and Abbott in the development of a new black tiioassay system is signif icant. It opens a much wider field of possibilities for insecticide screening on black fly larvae and should help accelerate the development of more potent B.t.H14 formulations. Page 3