Ixodes scapularis in: Anaplasma antifreeze

             Winter can be a difficult time for many organisms, particularly invertebrates. While most arthropods remain quiescent for the duration of cold conditions, the Blacklegged tick Ixodes scapularis can be found actively questing in the Northeastern United States whenever air temperatures are above freezing. Previous researchers have reported that the Antarctic tick Ixodes uriae takes thermal tolerance to extremes, capable of surviving temperatures as low as -30°C (Lee and Baust, 1987). Neelakanta, et al. (2010) investigated the Ixodes scapularis genome for clues as to how Ixodes can withstand such conditions. The research team was able to identify and isolate a novel Ixodes antifreeze protein, and soon found an interesting twist in the mechanisms at work.

The cold of winter presents many hazards: ice crystals can form, causing structural damage and inhibiting circulatory and respiratory function. Cell membranes can rupture or be disturbed by ion fluxes, osmotic regulation can be disturbed, and liposome integrity can be jeopardized in cold conditions. Antifreeze proteins, which help stabilize membranes and inhibit ice crystal formation, can be generally classified as either antifreeze proteins (AFPs) or antifreeze glycoproteins (AFGPs). AFGPs are diverse - comprising eight different subtypes - but can be identified by the presence of repeating tripeptide elements (alanine-alanine-threonine). AFPs have been identified in more than 55 terrestrial arthropods, but AFGPs in arthropods had not previously been reported (Neelakanta et al., 2010).

Searching through the Ixodes scapularis genome, the researchers were able to identify a potential antifreeze protein TC43064. Surprisingly, the protein was more similar to an afgp gene found in the Arctic cod Boreogadus saida than to known afp genes in other insects. Computer analysis of peptide-cleavage sites and subsequent deduced sequences showed an approximately 70% similarity with AFGPs found in other fish. This uniqueness earned the protein the name Ixodes scapularis AFGP (IAFGP).

The researchers then analyzed the expression, regulation, and effect of iafgp and IAFGP during tick development and under environmental conditions. [Note: iafgp refers to the gene sequence encoding the protein IAFGP]. Quantitative real-time polymerase chain reaction (QRT-PCR) measured expression in unfed nymphal and fed adult ticks, showing significantly higher expression in adults. There was no measurable difference between expression in males and females. Environmental temperature expectedly influenced gene expression, with ticks incubated at 0°C and 4°C exhibiting a four-fold increase over those incubated at 10°and 23°C. Using RNA interference (RNAi) knockouts, the research team was able to show significant (P < 0.0001) survival and mobility of ticks expressing iafgp after 25 minutes at -20°C.

The differential expression in nymphal (unfed) and adult (fed) ticks posed the question: why do adult ticks exhibit such higher rates of iafgp expression than nymphs when both nymphs and adults must overwinter? Using real-time polymerase chain reaction, elevated (3-fold) gene expression in I. scapularis infected with Anaplasma phagocytophilum ticks incubated at 23°, 10°, 4°, and 0°C was shown. Molecular analysis of cell membranes revealed 3-fold less membrane disruption in cold-treated cells of infected ticks. This research strongly suggests that Ixodes infection with Anaplasma directly affects iafgp gene expression, resulting in a mutualism conferring protection against environmental hazards.

The researchers report that the mechanism by which IAFG infers cold protection is not understood. The maintenance of membrane integrity in Anaplasma-infected cells indicate limitation of ice formation within cells and/or interactions with membrane lipids to prevent ion fluxes, liposome leakage and osmotic shrinkage. Whatever the mechanism, the role of IAFGP in protecting Ixodes scapularis during winter conditions seems clear. The mutualistic selective advantage of Anaplasma-intensified iafgp expression represents an interesting evolutionary event for arthropods and microbes as symbionts. Furthermore, the researchers suggest, this work may reveal new strategies for preventing human infection with tick-vectored diseases. 

Neelakanta, G., Sultana, H., Fish, D., Anderson, J., Fikrig, E. 2010. Anaplasma phagocytophilum induces Ixodes scapularis ticks to express an antifreeze glycoprotein gene that enhances their survival in the cold. Journal of Clinical Investigation. Vol. 120(9): 3179-3190.

Lee, R., Baust, J. 1987. Cold-hardiness in the Antarctic tick, Ixodes uriae. Physiol. Zoology. 60(4):499-506.