Ghanim et al 1998 - Virology .pdf

296 GHANIM ET AL.PCR. Plasmid pTYH19 that contains a cloned copy ofthe TYLCV genome was used as a positive control.Figure 1B shows that the 410-bp TYLCV DNA fragmentthat was amplified from plasmid pTYH19, frominfected tomato, and from viruliferous whiteflies wasalso amplified from the progeny of the viruliferousfemales (eggs, crawlers, and adults), but not fromthose of nonviruliferous insects. These results suggestthat TYLCV is transmitted to the progeny of viruliferouswhiteflies through the egg.FIG. 1. Analysis of viruliferous whiteflies and their offspring by PCR.(A) Development stages of the whitefly Bemisia tabaci and their duration,under our experimental conditions. The first instar is the crawlerstage; the fourth instar is the pupal stage. (B) Amplification of TYLCVDNA from groups of 50 eggs, 20 crawlers and 20 adults, from offspringof viruliferous whiteflies, and from the same number of progeny ofnonviruliferous insects; infected tomato plants and plasmid pTYH19 arepositive controls. The reaction products were submitted to agarose gelelectrophoresis and stained with ethidium bromide. Thick arrow, amplifiedTYLCV DNA; thin arrow, primers.progeny of viruliferous insects for at least two generations.RESULTSPCR analysis of progeny of viruliferous whitefliesWhiteflies develop from an egg, through fournymphal instars, into an adult. Figure 1A shows thedevelopmental time course under our experimentalconditions. The association of TYLCV DNA with developingeggs of viruliferous insects was investigated.Viruliferous whiteflies were caged on eggplants andon cotton plants (about 20 insects per plant) and ontwo TYLCV nonhosts (Cohen and Nitzany, 1966; Al-Musa, 1982). The adults were collected after 5 daysand the eggs were allowed to develop. Samples fromeach developmental stage (Fig. 1A) were collected atrandom. Total DNA was extracted from groups of 10viruliferous females, 50 of their eggs, 20 crawlers, and20 adult progeny. In parallel, DNA was extracted fromthe same number of nonviruliferous females, theireggs, crawlers, and adult progeny and from an infectedtomato leaflet. All samples were subjected toThe offspring of a single viruliferous whitefly containTYLCV DNAThe question of whether TYLCV is present in all progenyof a single viruliferous insect was addressed. Viruliferouswhiteflies were allowed to lay eggs on eggplants(one insect per plant). After 5 days, the insects werecollected and eggs were allowed to develop. In accordancewith the developmental time scale of Fig. 1A,eggs, crawlers, pupae, and adults were collected in sucha fashion that all the individuals at a given developmentalstage were progeny of a single insect. DNA extractedfrom each individual was subjected to PCR amplification.Figure 2 shows the 410-bp viral DNA fragment amplifiedfrom 16 of the 23 eggs laid by a single viruliferouswhitefly. Twenty-five of the 30 crawlers issued from anotherviruliferous insect contained viral DNA as did 7 ofthe 13 pupae and 15 of the 16 adults issued from a thirdand a fourth insect. Hence, TYLCV was transmitted tosome, but not to all, progeny of viruliferous whiteflies.This analysis was extended to the progeny of nineviruliferous whiteflies. Viruliferous insects were allowedto lay eggs on eggplants and on cotton plants (one insectper plant). Eggs, first and second instars, and adultswere collected in such a fashion that all the individuals ata given developmental stage were progeny of three differentinsects. The progeny from a single insect werekept separated from those of the other insects. DNA fromeach individual was subjected to PCR amplification, asdescribed in the legend to Fig. 2. The results summarizedin Table 1 show that TYLCV DNA was amplifiedfrom some, but not from all progeny. This proportionvaried from insect to insect. TYLCV DNA was present inall of the developmental stages of the whitefly, from eggto adult. Adult progeny of three additional viruliferousinsects were used to inoculate tomato test plants (seebelow).The second-generation progeny of a singleviruliferous whitefly contain TYLCV DNAThe passage of TYLCV through the egg from the first tothe second generation of viruliferous whiteflies progenywas investigated using the methods described above.Eggs laid by whiteflies that had acquired TYLCV frominfected tomato were allowed to develop on cotton. The