Can thermoregulation explain differences in habitat selection and distribution range in Calopteryx damselflies?

We studied the body temperatures of Calopteryx virgo and Calopteryx splendens under both field and laboratory conditions using an insect thermometer. These damselflies are ideal for studying thermoregulation, because they are closely related, but vary in colouration and size. We have two different datasets in this research: the field data and the laboratory data.

The field data was collected in 2000 (during mid-June to the end of July) in three different Calopteryx populations in Central Finland, two of which were sympatric (Pitkäjoki and Mustajoki) and one of which was allopatric (Vasarajoki). For the field dataset, damselflies were captured during daytime when the temperature is typically highest (10:00-16:00) and only in sunny weather conditions with no rain or wind. They were captured with a standard butterfly net. All the individuals were observed for 2-3 minutes prior the capturing by the same person (JS). We selected only resting individuals to negate any that might increase endothermic heat generation caused by activation (e.g. flying or fighting).

We used an insect temperature probe (a copper-constant thermocouple Type MT-29/1B with time constant 0.015 s mounted in a 29 -gauge hypothermic needle, accuracy ±0.1°C, Physitemp Instruments, New Jersey) to measure the body temperatures of the damselflies. The tip of the thermometer was set under the thorax of a damselfly immediately after each damselfly was captured. The flight activity of the insect depends on the temperature of the flight muscles, which are located on the thorax and for this reason we chose this position for the temperature probe. Immediately after measuring the body temperature of the individual, the ambient temperature was measured 1 m above ground or water from with the same temperature probe. After the body temperature was measured, wing length of the damselfly and wing spot size of the males were measured by Mitutoyo digital caliper (to the nearest ±0.01 mm). The same person (JS) did all the measurements for all the individuals to prevent measurement errors in measuring accuracy. After the measuring, each damselfly individual was marked with a silver marker (Edding ® 780) to prevent the same individuals from being captured multiple times. Then they were freed back to the same locations where they were originally captured.

The laboratory data was collected 2022-2012 (during early June to mid-August) in two sympatric population in Southwestern Finland (Mietoinen and Tarvasjoki).

In this dataset, damselflies were collected using a standard butterfly net and only on sunny days in calm weather conditions with no wind or rain. In each day, ten individuals were captured and placed separately in 0.5-liter plastic jars, equipped with tiny ventilation holes and a damp piece of tissue paper to maintain humidity. To minimize stress, only one damselfly was placed in one jar. During transport to the laboratory, all the jars were kept in a Styrofoam cooler with two ice bricks, to maintain a temperature between 5–15 °C to prevent heat shock. At the university, the damselflies were placed in a cooling room set at 10 °C. All the individuals were measured in the laboratory on the same day they were captured.

In the laboratory, we used the same insect temperature probe as in the field measurements. First, the temperature probe was placed below the thorax of the damselfly. A natural roosting posture of the damselflies was mimicked by placing a test tube below the damselfly. Damselfly was bound to the test tube with a narrow thread to prevent it from escaping.

The entire set-up was placed in a white Styrofoam box. The damselfly body temperature was cooled down to 3-4 °C temperature by placing ice bricks in the box. The white box was selected for the experiment to prevent radiation absorbing to the walls and to direct most of the radiation to the damselfly. Then the Styrofoam box was placed under a light source in a dark room. The sodium lamp (IDMAN, 681 20 SON-T-400, 1 x SON-T 400 W, 230 W, IP 23) was used as the light source in this experiment because it is radiating the same spectrum of light as the Sun, including UV and infra-red radiation.

At the beginning of the experiment, the body temperature of the damselfly was allowed to rise to 5 °C by removing the ice bricks from the box. From that point we started measuring the time to the point when the damselfly started to activate by flapping its wings. At that point we measured the activation time and body temperature of the damselfly and then the damselfly was removed from the set-up. Wing length of the damselfly and wing spot size of the males were measured by the same Mitutoyo digital caliper (to the nearest ±0.01 mm) as in the field study. The laboratory measurements were done by only one person (LL) to prevent the measurement errors in measuring accuracy between different persons. After this, wings of the damselflies were marked with tiny numbers by using the same silver marker as in the field study section (Edding ® 780) to prevent capturing and measuring the same individuals twice. After the laboratory experiment, all the damselflies were transported and freed back to their original populations on the same day.