Evaluation of Anion Transporters as Potential Target Sites for Insect and Nematode Control: Toxicological, Electrophysiological, and Molecular Studies

In this study, four anion transporter (AT) blockers, DIDS (4, 4′-diisothiocyanatostilbene-2, 2′-disulfonic acid), 9-AC (anthracene-9-carboxylic acid), NPPB (5-nitro-2-(3-phenylpropylamino) benzoic acid), and IAA-94 (indanyloxy acetic acid) were selected to evaluate ATs as potential target sites for insect and nematode control.

All the AT blockers showed slowly developing toxicity against second-stage larvae of Meloidogyne incognita (Kofoid and White 1919) Chitwood 1949 and adults of Caenorhabditis elegans Maupas 1900 but not against third-stage larvae of Heterorhabditis bacteriophora Poinar 1975 even at 200 ppm. Symptoms of AT blocker toxicity observed in C. elegans adults were increased pharyngeal muscle contractions and decreased locomotion. Exposure of C. elegans as fourth-stage larvae to double-stranded RNA (dsRNA) of ceclc-1 and ceclc-2 (VGCC genes coding for CeClC-1 and CeClC-2, respectively) either alone or together for 24 h decreased their expression in F1 progeny in a time-dependent manner. Reduction in expression of ceclc-2 alone or together with ceclc-1 significantly increased pharyngeal contractions and decreased locomotion in significantly higher percentage of F1 progeny. The above findings suggested AT blockers nematicidal activity primarily comes from inhibition of CeClC-2 channels, while inhibition of CeClC-1 channels may enhance this activity.

All the AT blockers showed slowly developing toxicity against adults of a susceptible strain (Oregon-R) of Drosophila melanogaster Meigen 1830, while DIDS, was equally toxic to dieldrin-resistant rdl flies. All AT blockers, except 9-AC, at 100 µM showed significant excitatory effect on desheathed central nervous system (CNS) of third-instar larvae of Drosophila, while DIDS showed a modest excitatory effect on ascending peripheral nerves. Feeding adult flies on 10% sugar solution mixed with 100 ppm of DIDS for 6 h decreased the midgut pH by 2 units approximately.

All the AT blockers inhibited the growth of larvae (in weight), increased the developmental time, and decreased survival when Ostrinia nubilalis (Hübner 1796) second-instar larvae were fed for seven days. All the AT blockers decreased the midgut alkalinity and inhibited chloride ion transport from midgut lumen into epithelia in fifth-instar larvae when fed for 3 h on treated diet. Positive correlations observed among growth, midgut alkalinity, and midgut chloride transport in AT blocker-fed larvae suggested that inhibition of chloride/bicarbonate exchangers by AT blockers may have contributed to midgut alkalinity decrease affecting the digestion and resulting in observed lethal and sublethal effects.