approach for mosquito control. Further understanding of the molecular mechanisms involved would help identify additional novel targets for vector control using ATSB.

Ixodes scapularis is the major vector of Lyme disease in the Eastern United States. Each active life stage (larva, nymph, and adult) takes a blood meal either for developing and molting to the next stage (larvae and nymphs) or for oviposition (adult females). This protein-rich blood meal is the only food taken by Ixodes ticks and therefore efficient blood digestion is critical for survival. Studies in partially engorged ticks have shown that the initial stages of digestion are carried out by cathepsin proteases within acidic digestive cells. In this study, we investigated the potential role of serine proteases in blood digestion in replete ticks. RNA interference was used for functional analysis and a trypsin-benzoyl-D, L-arginine 4-nitoanilide assay was used to measure active trypsin levels. Hemoglobinolytic activity was determined in vitro, with or without a serine protease inhibitor. Our data suggest that trypsin levels increase significantly after repletion. Knockdown of serine proteases negatively impacted blood feeding, survival, fecundity, levels of active trypsin in the midgut, and resulted in lower hemoglobin degradation. Incubation of midgut extract with a trypsin inhibitor resulted in 65% lower hemoglobin degradation. We provide evidence of the serine proteases as digestive enzymes in fully engorged, replete females. Understanding the digestive profile of trypsin during blood meal digestion in I. scapularis improves our understanding of the basic biology of ticks and may lead to new methods for tick control.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Ixodes scapularis is the major vector of Lyme disease in the eastern United States. Each active life stage (larva, nymph, and adult) takes a blood meal either for developing and molting to the next stage (larvae and nymphs) or for oviposition (adult females). This protein-rich blood meal is the only food taken by Ixodes ticks and therefore blood digestion is very important for tick survival. Most studies on blood digestion in ticks have shown that the initial stages of digestion are carried out by cathepsin proteases within acidic digestive cells. However, most of these studies have focused on partially engorged ticks. In other hematophagous arthropods, the serine proteases play an important role in blood protein degradation. In this study, we determined transcript expression of four I. scapularis serine proteases with previously characterized roles in blood digestion. RNA interference was used for functional analysis and a trypsin-benzoyl-D, L-arginine 4-nitoanilide assay was used to measure active trypsin levels. An in vitro hemoglobinolytic assay was performed with or without serine protease inhibitor. Our data suggest that trypsin levels increase significantly after blood feeding and peaked in larvae, nymphs, and adults at 3, 1, and 1 day post host detachment, respectively. The knockdown of three previously identified serine proteases by RNAi negatively impacted blood intake, survival, fecundity, levels of active trypsin in the gut and resulted in lower hemoglobin degradation in vitro. A trypsin inhibitor, PMSF, blocked the action of trypsin in the gut extract resulting in 65% lower hemoglobin degradation. We provide evidence of the serine proteases as digestive enzymes in fully engorged, replete females. Our data also demonstrated that in addition to blood digestion, these serine proteases might have a role in blood feeding success in I. scapularis.

Ixodes scapularis is the major vector of Lyme disease in the eastern United States. This species undergoes a life cycle consisting of eggs and three active stages: larva, nymph, and adult. Each active life stage takes a blood meal either for developing and molting to the next stage (larvae and nymphs) or for oviposition (adult females). This protein rich blood meal is the only food taken by Ixodes ticks. Most studies on blood digestion in ticks have shown that the initial stages of blood digestion are carried out by cathepsin proteases within endosomes of acidic digestive cells. However, in other hematophagous arthropods, the serine protease trypsin plays an important role in early protein degradation. In this study, we determined transcript expression of I. scapularis cathepsins and serine proteases, some with previously characterized roles in blood digestion. Gut pH was also determined and a trypsin-benzoyl-D, L-arginine 4-nitoanilide assay was used to measure active trypsin levels during blood digestion. Our data suggest that trypsin levels increase significantly after blood feeding and peaked in larvae, nymphs, and adults at 3, 1, and 1 days post host detachment, respectively. In addition, alkaline gut pH (8.0) conditions after I. scapularis blood feeding were similar to those required for trypsin activity in other arthropods suggesting these enzymes have an important and previously overlooked role in I. scapularis blood digestion.