Survival and moulting of Amblyomma variegatum nymphs under cold conditions of the Malagasy highlands

Although Amblyomma variegatum is now regularly recorded up to 1600 m in altitude in the Malagasy highlands, where it was previously reported not to persist without a constant supply of ticks introduced from lower infested regions, some parts of the highlands remain tick-free. Studies were carried out to verify whether the cold climate prevailing in these areas in June–September could prevent the survival and moulting of nymphs, the tick life stage present in the environment at this period. Cohorts of engorged A. variegatum nymphs were released from June to August in six different sites (three in 2010, altitudes 1200–1415 m; three in 2011, altitudes 1585–1960 m) which were reported to be either tick-infested (two in 2010, one in 2011) or tick-free. The ticks were placed in cages driven into the soil and open at the bottom so that they could hide in the soil or root network. Of the 1975 nymphs released in 2010 and the 1494 released in 2011, 86% and 85% were recovered, respectively. Twenty to 23% of the recovered ticks were dead, and some of them were obviously predated; predation also likely contributed to the disappearance of the non-recovered ticks. When the rainy season started in October, 59% of the newly moulted adults were still alive in the cages. The moulting period lasted up to 20 weeks, depending on the site and release period. As verified in 2011, unfed nymphs could also survive the cold season. Various A. variegatum life stages are thus able to survive the adverse cold and/or dry seasons: unfed nymphs, engorged nymphs in developmental diapause, moulted adults in behavioural diapause as observed previously. Strong variation in mortality and recovery rates was observed between cages, highlighting the importance of the micro-environment and micro-climate for tick survival. The minimum temperature recorded in the field sites varied from 1.1 °C to 6.8 °C, but the tick-free sites were not the coldest ones; they were, however, those for which the temperature remained below 10 °C for the longest time over the study period. Recovery and mortality rates in the tick-free sites were similar to those of the tick-infested sites: the temperatures recorded during the study periods did not prevent ticks from surviving and moulting although it did delay the metamorphosis. Low temperature alone can therefore not explain the persistence of tick-free areas in the highlands. To further monitor survival, cohorts of engorged nymphs were also maintained in an incubator at 3.6 °C, 6.2 °C or 12.8 °C. More than 50% mortality was observed after 6 days at 3.6 °C, and after 15 days at 6.2 °C, whereas 18 days at 12.8 °C only delayed moulting. The collected survival, moulting and climatic data presented in this study should help to develop a predictive model to assess the distribution of A. variegatum according to climate characteristics.