Flavonoid from Tithonia diversifolia affects the cuticle of Spodoptera litura

The 6-methoxyluteolin, a flavonoid isolated from the plant tithonia diversifolia, reduced tyrosinase activity, decreased melanin content, and caused structural changes in the cuticle of larvae of Spodoptera litura. The result, obtained by Chinese researchers, indicates the compound's potential as a base for botanical insecticides that act on melanization in lepidopteran pests.

Scientists evaluated larvae between the third and sixth instar stages. The study used concentrations of 1,625 to 100 micrograms per milliliter of 6-methoxyluteolin. The larvae received an artificial diet treated with the compound for three days, starting from the second instar. Afterwards, they were fed a diet without the compound.

The research targeted the melanization pathway. This route is involved in cuticle sclerotization, wound healing, and immune defense in insects. Tyrosinase catalyzes steps related to the oxidation of tyrosine-derived substrates. This process leads to the deposition of melanin in the integument.

Tyrosinase activity

Tyrosinase activity changed in all instars evaluated. In the third instar, the control group showed the highest activity, close to 125 units per milligram of protein. The lowest activity occurred at a concentration of 100 micrograms per milliliter. In the fourth instar, the baseline activity of the control group was lower than in the third instar. A concentration of 1,625 micrograms per milliliter significantly reduced activity. In the fifth and sixth instars, the authors recorded differences between treatments, with variation dependent on concentration.

The melanin content in the skin also decreased after treatment. The effect was most pronounced in the third instar. At this stage, the content dropped from approximately 2,2 micrograms per milligram of tissue in 3,125 micrograms per milliliter to approximately 0,4 micrograms per milligram in 100 micrograms per milliliter. The reduction was more than fivefold. In the fifth instar, the control group showed the highest content, close to 6,2 micrograms per milligram, and there were significant reductions at 6,25 and 25 micrograms per milliliter.

Macroscopic images

Macroscopic images reinforced the biochemical data. Control larvae showed dark pigmentation and defined dorsal stripes. Treated larvae showed progressive body lightening. The visual effect appeared from 3,125 micrograms per milliliter. At concentrations of 25 to 50 micrograms per milliliter, the tegument assumed a paler coloration compared to the control.

Histological analysis

Histological analysis revealed loss of melanin granules and disorganization of the cuticle. The authors prepared sections of fourth- and fifth-instar larvae, embedded in paraffin and stained with hematoxylin and eosin. In the fourth instar, the control showed a continuous cuticle and dense deposition of melanin granules on the outer surface. At 12,5 micrograms per milliliter, the structure became looser and with a lower density of granules. At 100 micrograms per milliliter, the cuticular layer became thinner and more disorganized.

In the fifth instar, the control maintained a well-defined pigmented layer. At 6,25 micrograms per milliliter, there was loosening of the cuticle and a reduction in melanin deposition. At 25 micrograms per milliliter, the authors observed structural fragmentation and irregular distribution of granules.

Thickness measurement confirmed the effect in the fourth instar. The cuticle of the control group measured 4,70 ± 0,13 micrometers. All treatments with 6-methoxyluteolin showed reduced thickness. The greatest reduction occurred at 12,5 micrograms per milliliter, with a thickness of 2,53 ± 0,13 micrometers. This value represents a 46% decrease compared to the control group.

More information at doi.org/10.3390/insects17060535