Abstract
In ticks, the digestion of blood occurs intracellularly and proteolytic digestion of hemoglobin takes place in a dedicated type of lysosome, the digest vesicle, followed by transfer of the heme moiety of hemoglobin to a specialized organelle that accumulates large heme aggregates, called hemosomes. In the present work, we studied the uptake of fluorescent metalloporphyrins, used as heme analogs, and amitraz, one of the most regularly used acaricides to control cattle tick infestations, by Rhipicephalus (Boophilus) microplus midgut cells. Both compounds were taken up by midgut cells in vitro and accumulated inside the hemosomes. Transport of both molecules was sensitive to cyclosporine A (CsA), a well-known inhibitor of ATP binding cassette (ABC) transporters. Rhodamine 123, a fluorescent probe that is also a recognized ABC substrate, was similarly directed to the hemosome in a CsA-sensitive manner. Using an antibody against conserved domain of PgP-1-type ABC transporter, we were able to immunolocalize PgP-1 in the digest vesicle membranes. Comparison between two R. microplus strains that were resistant and susceptible to amitraz revealed that the resistant strain detoxified both amitraz and Sn-Pp IX more efficiently than the susceptible strain, a process that was also sensitive to CsA. A transcript containing an ABC transporter signature exhibited 2.5-fold increased expression in the amitraz-resistant strain when compared with the susceptible strain. RNAi-induced down-regulation of this ABC transporter led to the accumulation of metalloporphyrin in the digestive vacuole, interrupting heme traffic to the hemosome. This evidence further confirms that this transcript codes for a heme transporter. This is the first report of heme transport in a blood-feeding organism. While the primary physiological function of the hemosome is to detoxify heme and attenuate its toxicity, we suggest that the use of this acaricide detoxification pathway by ticks may represent a new molecular mechanism of resistance to pesticides.
Highlights
Ticks and tick-borne diseases began to be considered economic and public health concerns at the end of the nineteenth century, when the number of cattle increased in an attempt to feed a growing human population [1]
Digest cells of the tick midgut degrade hemoglobin and direct heme into an intracellular pathway that involves digestive vacuoles and leads to heme accumulation in hemosomes. To test whether this heme intracellular transport is dependent on an ATP binding cassette (ABC) transporter, digest cells isolated from the midgut of R. microplus were incubated with Rhodamine 123, a PgP protein (ABCB) transporter substrate that has been validated for membrane transport studies [38, 39]
The participation of an ABC transporter in heme metabolism in typical eukaryotic cells has been shown in mitochondria, where an ABC transporter was implicated in the uptake of a heme precursor, coproporphyrinogen III, but not in heme transport directly [51]
Summary
Ticks and tick-borne diseases began to be considered economic and public health concerns at the end of the nineteenth century, when the number of cattle increased in an attempt to feed a growing human population [1]. The free heme that is produced by this process is transferred from these vacuoles to the cytosol and subsequently moved to a very specialized organelle called hemosome, wherein heme aggregates accumulate [6]. This process is responsible for alleviating digest cells—and the tick as a whole—from the potentially deleterious effects of heme. In the case of the digest cell of the tick midgut, an intracellular pathway dedicated to heme transport from digestive vesicles to hemosomes has been implicated as a key aspect of heme detoxification, the molecular nature of the mechanisms involved in transport across cellular membranes or through the cytosol remains poorly understood
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