As previously indicated, male horseflies are captured infrequently, particularly when employing Malaise traps (Krolow et al. 2012). Consequently, it is paradoxical to observe a higher representation of 59.14% and male dominance over a span of seven months in half of the encountered species in Tiquibuzo. We understand that despite the Tiquibuzo data regarding monthly collections not being statistically significant, nonetheless the mere fact of representing almost 60% of the total collections in Tiquibuzo is puzzling considering the extensive dataset spanning nearly eight years of our studies and collections across different months in Ecuador. Undoubtedly, the results indicate, a distinct deviation from the patterns documented in the scientific literature with malaise trap. However, it has been reported that the capture rate of males tends to increase significantly with other mechanisms, such as light trapping in the forest canopy, resulting in representation levels of up to 63% (Krolow et al. 2010). Similarly, studies in salt marshes using emergence traps have consistently shown comparable proportions of male and female individuals (Cookson 1967; Rockel and Hansens 1970). These traps exhibit notable parity in the proportion of females and males collected. This suggests that certain populations have comparable sex ratios, although field observations indicate that sex ratios vary among species and seasons. We noted this phenomenon during our field collections in Tiquibuzo, where overall monthly abundances showed no significant variation, except in three instances where male prevalence exceeded that of females. This observation hints at a potential sex-related dynamic in the population structure. Similarly, our analysis using Generalized Linear Models (GLMs) revealed a direct correlation between male and female monthly abundances, as illustrated in Fig. 3. Hence, the primary inquiry should revolve around the reasons behind the elevated male capture rates observed in Tiquibuzo using Malaise traps, as well as the underlying factors contributing to the generally low capture rates of males with these traps. To tackle these inquiries, there are several hypotheses to consider, such as the males horseflies are dominant in the initial emergence, but they become a minority toward the end of the emergence period (Cookson 1967), although this pattern was not evidenced in Tiquibuzo. Also, there are instances where males have been observed to have shorter lifespans than females (Karandinos and Axtell 1967; Lane et al. 1983; Matsumura 1995). Hence, if males do indeed exhibit shorter lifespans, the likelihood of encountering and capturing them would correspondingly decrease.
The hilltopping behavior hypothesis refers to the behavior of certain insect species that select hill or mountain tops as sites for aggregation and reproduction (Skevington 2008). While this phenomenon has been well documented in some insect families, including Tabanidae (Cookson 1967; Smith et al. 1994; Braga da Rosa 2006), the available evidence for this behavior in Tabanidae is limited compared to other groups such as lepidopterans. Other reports also suggest the presence of preferred areas on hilltops, where males were captured more frequently using entomological nets, even during mating activity (Leprince et al. 1983). The study area, Tiquibuzo, is situated in a valley, therefore, the hilltopping behavior hypothesis (Braga da Rosa 2006; Yuval 2006; Skevington 2008) would not be suitable to explain the observed disruption, as the traps were placed in the valley at an altitude of 2300 m, in the foothills. In other locations, such as the Fernando de Noronha Archipelago in Brazil, a significant number of male individuals of Tabanus occidentalis were captured using Malaise traps. Apparently, the collections at this specific site were near the summit of the island and represented one of the best-preserved areas (Rafael et al. 2021), contrary to Tiquibuzo, which exhibited vegetative patches and anthropogenic activity.
The evidence suggests that certain members of the Tabanidae family display swarm mating behavior, circling flight and hovering activity, although a substantial portion of reproductive behavior in Tabanidae remains unknown. In other words, certain horsefly species have specific reproductive areas, such as hilltop clearings, where males would be present more frequently, potentially increasing their capture rate (Bailey 1948; Sullivan 1981; Mullens and Freeman 2017; Yuval 2006). However, in Tiquibuzo, no mating behavior such as swarms or concentric flights were observed (Yuval 2006).
Another hypothesis we consider is the differential foraging hypothesis between females and males, which could account for the low collection rate of females in Malaise traps. This hypothesis partially explains how the search for prey and resources can affect the capture frequency in Malaise traps. The feeding and foraging behavior of horseflies exhibits substantial differences between females and males. Female horseflies are typically facultative and can opportunistically consume blood, nectar and pollen to fulfill their energy requirements for flight, reproduction and oviposition (Leprince et al. 1983; Karolyi et al. 2014; Mullens 2019). Some of their prey, including mammals, reptiles and birds, exhibit evasion and defense behaviors to avoid horsefly bites (Limeira de Oliveira et al. 2002; Barros and Foil 2007; Caro et al. 2014; Altunsoy 2015), indicating that the foraging behavior of female horseflies can be energetically demanding, therefore, the blood intake provides them with advantages such as increased escape speed (Horváth et al. 2020). This energetic investment in foraging would enhance the likelihood of encountering prey due to an increase in flight frequency; however, it would also raise the probability of being captured in Malaise traps. In contrast to females, male horseflies display specialized feeding behavior, exclusively relying on pollen and nectar consumption. However, the energetic costs associated with foraging and reproductive activity, particularly during hovering flight, can be substantial for males (Smith et al. 1994; Smith 2013) and our understanding of male horseflies' energy expenditure and specific feeding patterns remains limited (Allan et al. 1987).
Male horseflies are frequently observed in close proximity to water bodies or moist areas along the banks of rivers or lagoons, resulting in a significant focus of the literature on their behavior in such habitats. These areas serve as aggregation sites for reproduction (Cookson 1967; Mullens and Freeman 2017). However, in Tiquibuzo, no significant bodies of water in the area, only small rivulets, eliminating a reference area for males to orient through positive polarotaxis generated by water reflections, which is crucial for male-female aggregation and courtship (Horváth et al. 2008; Herczeg et al. 2014). In the forest, certain horsefly species appears to exhibit a preference for flying higher in the tree canopy (Krolow et al. 2010). Therefore, collecting samples in the understory would reduce the likelihood of intercepting them with malaise traps.
The hypothesis that emerges as the most plausible is that of an optimal aggregation zone, suggesting that in certain areas of Tiquibuzo provides favorable conditions for development and feeding. Therefore, a higher density of male and female horseflies would increase the likelihood of capturing them with the Malaise traps, despite their inefficiency for this purpose. This suggests that these optimal aggregation zones exist despite the absence of bodies of water or hilltopping effects.
Finally, the impact of habitat reduction and forest fragmentation cannot be disregarded. Species' behaviors tend to change as their habitat is fragmented, as observed in forest patches (Debinski and Holt 2000; Harris and Johnson 2004; Baldacchino et al. 2014). A decrease in available habitat creates pressure for optimal reproductive spaces, compelling many species to modify their behavior and coexist with domestic fauna, benefiting from feeding on livestock (Barros and Foil 2007; Baldacchino et al. 2017). It is suggested that the reduction of forests and the presence of patches could alter the behavioral ecology of reproduction and foraging in horseflies, potentially resulting in a decrease in aggregation areas. Within the study area, the traps were positioned in a small forest patch; however, this patch serves as a corridor connecting to a larger forest patch, facilitating the movement of both male and female horseflies. Females would acquire resources from nearby livestock and the flora and fauna in the more densely vegetated interior of the forest, where they would collect pollen and nectar, similar to male horseflies (Barros and Foil 2007); subsequently, they would reassemble in this optimal zone. This behavior is possible because studies using marking and weighing techniques have demonstrated that male horseflies exhibit site fidelity, and return to their breeding sites after feeding activities (Smith 2013).