In response to brood parasitism, which significantly reduces the reproductive success of avian hosts, hosts have evolved the ability to recognize and reject parasitic eggs. Many studies have revealed the influencing factors of egg recognition and egg rejection, and recent study found that host personality, along the bold-shy continuum, can influence egg rejection behavior. In addition, the egg rejection behavior of hosts differed among populations, suggesting that individuals with different personalities may evolve various adaptive behaviors under different parasitic pressures, but there has been few relevant experiments to verify this. In the present study, two populations of the Japanese tit Parus minor distributed in China were used to examine whether there were differences in egg rejection behavior of individuals with different personalities. The results showed that egg rejection rates of bold individuals (who remained in the nest box when encountering human invaders) and shy individuals (who immediately flew away from the nest box when encountering human invaders) were similar in both populations of the Japanese tit, and there were also no significant differences in egg rejection rates between individuals of the same personality in the two populations. This study suggests that host personality, along the bold-shy continuum, does not account for egg rejection behavior in the Japanese tits. This may be due to either other factors that affect egg rejection behavior counteracting the effect of personality, or the presence of multiple personalities that act together in the Japanese tits counteracting the effect of a single personality trait.

During the breeding season (April-June) of year 2022, when Japanese tits were found occupying nest boxes, breeding nests were regularly monitored to determine the dates of the first eggs. Female Japanese tits usually started incubation the day after laying their last egg (Zhang et al. 2020), and therefore, we defined the date when the females laid their last eggs as day 0 of the incubation period. After determining that the Japanese tits had entered the incubation period, we began an escape behavior test and a foreign egg recognition experiment among the female Japanese tits.

In order to measure escape behavior, researchers slowly approached the nest box from about 10 m and gazed at the hole to record whether the females flew away, though the orientation of the approaching was adjusted according to location of focal nest boxes. If the female did not fly out from nest boxes when the experimenter went under the tree hung up nest box, we climbed the tree. We defined females that flew away from the nest box as shy individuals and those that did not as bold individuals during this process. If the females were not in the nest at the time of the first experiment, this experiment was repeated on the second day until escape behavior of the incubating females was recorded. 

         After waiting for the departure of the females, we placed one foreign egg in the nest cup after clutch completion (i.e., on the first day when the host began to incubate) (Figure 1a), while recording the experiment date and the clutch size in the experimental nest. In this study, most egg experiments were conducted on the day when the clutch of tits were completed or the next day, and only a small number of nests could not be determined for their incubation time. However, none of the experimental nests hatched after 6 days of the egg recognition experiment (see below), suggesting that all of these nests should have been in the early to mid-incubation period (i.e., 10-15 days for Japanese tits) at the time of egg experiments. The foreign eggs used in the experiment were the commercial non-fertilized eggs of white-rumped munia (Lonchura striata), colored with a blue non-toxic and odorless marker, which were slightly smaller in size than the experimental tit species (see Liu et al. 2019 for the exact egg sizes of Japanese tits and white-rumped munias). We set the length of the egg rejection experiment to 6 days, and considered females of the experimental nest not to reject foreign eggs if the incoming eggs remained in the experimental nest and the nest was not abandoned within 6 days. Females of the experimental nest were considered to reject foreign eggs if within 6 days, the incoming eggs disappeared or remained in the nest box but the females did not incubate them (i.e., buried under the nest material or not in the nest cup, as shown in Figure 1b) (i.e., Yang et al. 2015; Liang et al. 2016; Liu et al. 2019). Some studies have suggested that nest abandonment is also one of the ways host birds reject foreign eggs (Hanley et al. 2016), but nest abandonment was not found in our experiments. If the experimental nest succumbed to predation within 6 days, experimental results from that nest were not counted.

We used generalized linear models (GLMs) (logit-link function) to analyze the impact of escape behavior of the female Japanese tits on whether they rejected foreign eggs. The egg rejection behavior of the females (rejected=1; accepted=0) was the response variable. For analyses on the same population (i.e., in DZ or ZJ), escape behavior (escaped: shy; did not escape: bold), clutch size, and date of egg experiment were the explanatory variables. For analyses on the two populations combined, escape behavior, location, clutch size, and date of egg experiment were the explanatory variables. Likelihood ratio tests were used to obtain p-values. Data analysis was completed using R 4.1.0 software, with a two-tailed test and a significance level of α=0.05.