This study presents the first detailed analysis of the seasonal activity of Phlebotomus mascittii. We report, to the best of our knowledge, the highest number of trapped individuals of Ph. mascittii to date and newly identified blood meal hosts of this species.
Even though Ph. mascittii is usually reported in very low numbers, it is widely distributed throughout Europe and the predominant species in Central European countries. Only sporadic findings of other sand fly species such as Phlebotomus perniciosus Newstead, 1911, in Germany [33] or Phlebotomus simici Nitzulescu, 1931, in Austria [22] have been reported. In our study, we trapped unexpectedly high numbers of Ph. mascittii, which indicates that population densities might be higher than previously reported. Clear differences in trapping numbers were observed between locations and caught specimens per trap-night ranged from 0.2 to 0.9. In Germany, an average of 0.2 caught specimens of Ph. mascittii per trap has been reported [34]. Surveys in Southern European countries usually exceed this number by multiple times, as reported from trappings in Southern Italy, where 85 sand flies of various species per trap-night were caught [35].
Higher trapping numbers, namely 184 and 158 were observed in Hummersdorf and Unterpurkla, respectively, where a chicken barn was located at the property, guaranteeing constant host availability, which might lead to increased population densities. In Southern France, trapping success of Ph. perniciosus and Phlebotomus ariasi Tonnoir, 1921, was strongly associated with host abundance and availability and an extraordinary high number of sand flies was caught at a poultry barn with many chicken [36]. Also, Cazan et al. [37] observed Phlebotomus perfiliewi Parrot, 1930, to be only present in a chicken shed, but not in other barns at the same property where cattle, horses, pigs and rabbits were kept at a farm in Romania.
Interestingly, the sex ratio was strongly biased towards females ranging from 1/2.4 to 1/16.6. The light traps used in this study are commonly used for trapping phototropic insects including sand flies, however, the effectiveness of this method varies significantly between species and sex [38]. While a shifted sex ratio towards females has been reported for Ph. mascittii [34,36,39], studies on other Phlebotomus species often report more captured males than females by light trapping [40,41]. A male biased sex ratio among Lutzomyia longipalpis Lutz & Neiva, 1912, was found to be associated with higher sand fly densities and more available hosts [42,43]. However, these factors were not influencing the sex ratio in this study when comparing trapping numbers at the four trapping locations. Clearly, trapping methods and ecological factors are associated with the sex ratio of caught specimens. However, activity peaks differed between males and females in this study, male activity peaked in late June and female activity at the end of July. Also under laboratory conditions males are observed to emerge earlier than females with shorter live spans [44]. This indicates that the time point of trapping during the active season influences observed sex ratios and should be taken into account for calculations.
To date, no studies monitoring Ph. mascittii activity over a full season have been published and this study presents the first detailed insights into the seasonal dynamics of this species. July and August are the typical months of Ph. mascittii activity in Central European [4,6,34] as well as Southern European countries [36,45]. The only exception is a climatically stable tunnel in Corsica, where winter activity of Ph. mascittii has been noticed [15]. As we observed Ph. mascittii to be active by late June in 2018 we adjusted our trapping scheme starting regular trappings at the beginning of June in 2019, thereby recording first activity already in early June. While the length of activity periods varied between locations, a mono-modal activity trend was observed at all locations with activity peaks in July at three locations and August in Ratzenau. According to Alten et al. [46] the number of peaks is associated with the number of generations, which suggests a single generation of Ph. mascittii in Austria. A mono-modal trend was also observed for Ph. ariasi in France and Phlebotomus kandelaki Shurenkova, 1929, as well as Phlebotomus balcanicus Theodor, 1948, in Georgia. Up to three density peaks and substantially longer activity periods are usually observed in countries with lower latitudes such as Portugal, Turkey, Greece or Cyprus [46].
While data on seasonal dynamics of sand flies are available from Mediterranean countries [36,46,47], data on activity at the northern boundary of sand fly occurrence are scarce. Recently, Cazan et al. [37] published a study on the seasonal dynamics of Ph. perfiliewi in Northern Romania approximately at the same latitude as locations surveyed in this study. They observed sand fly activity from July to August, being comparably shorter than observed activity periods in our study. Common mild May and June temperatures in Austria possibly contribute to early sand fly activity starting already in early June. Taking constant rising temperatures into account, even earlier sand fly activity might be observed in particularly warm years and in the future, however, this clearly needs further studies.
Also, Ph. masciittii was observed to be active at night temperatures as low as 12.8°C and first activity was noticed after mean temperatures did not drop below 15°C and 10°C minimum. These rather low temperature requirements for activity might contribute to an early start of sand fly activity in June, however, no inference on larval and pupa requirements for development can be drawn from this study, this needs experimental clarification. Kasap et al. [14] observed no larval and pupal development of Ph. papatasi at 15°C under laboratory conditions and a mean temperature of at least 18°C was necessary for successful rearing. Even though temperature is a driving factor for sand fly activity, similar thresholds for activity of adult sand flies compared to this study were observed for Ph. ariasi in France [36] and Ph. simici in Austria [22]. In Romania, Ph. perfiliewi was not active until minimum temperatures did not fall below 15°C for seven days [37].
Despite the low threshold temperature, an association between mean night temperature and sand fly abundance was observed at all locations, which indicates that Ph. mascittii actually prefers higher temperatures, but can be active at low temperatures as well. This is in concordance with other surveys, where increasing minimum temperatures were associated with higher trapping success of Ph. ariasi in France [36] and Ph. perfiliewi in Romania [37]. As Ph. mascittii occurs in temperate as well as Mediterranean regions with apparent differences in winter and summer temperatures, it might have a wider temperature tolerance than other sand fly species.
Also, relative humidity was significantly associated with sand fly abundance and peaked at approximately 80%, decreasing again with higher percentages. Compared to other species the peak at about 80% is rather high. Significant differences in temperature and humidity requirements were observed between different Phlebotomus species on Greek Aegean islands [41]. In contrast to our results for Ph. mascittii, the activity of Ph. perfiliewi declined with increasing relative humidity in a study from Romania [37].
Interestingly, an increase in barometric pressure was significantly associated with a decrease in sand fly activity, which indicates an active response of Ph. mascittii to pressure changes. Tichy et al. [48] experimentally confirmed responses of cockroaches and stick insects to humidity and pressure. The response of sand flies to changes in barometric pressure is widely assumed but data are scarce. In general, a rise in barometric pressure is associated with good weather whereas a drop is associated with poor weather including possible rain, thus, observations in our study are rather unexpected. Herczeg et al. [49] observed an increase of the activity of the blood-sucking horsefly (Tabanidae) in response to a quickly dropping air pressure prior to storms, concluding that climatic conditions shortly before storms exhibit high humidity and potential blood meal hosts move to shelters and might be more easily available for the horseflies. This might also apply for sand fly activity. Moreover, a weak negative correlation between temperature and air pressure was observed, which might lead to an indirect effect of pressure on sand fly activity through temperature. As we trapped exclusively indoors, this setting might explain a higher tolerance of Ph. mascittii to bad weather conditions. This is supported by the absence of a significant association between flight activity and wind speed. As sand flies have a weak flight ability, activity usually decreases with increasing wind speed as observed for various species in Sardinia [50]. However, indoor trapping sites provide shelter for sand flies during bad weather including rainfall and high wind speed. In Spain, trapping sites not exposed to wind showed significantly higher sand fly densities compared to exposed sites [51]. Our data suggest that temperature and humidity can be used as good predictors for sand fly abundance in prospective studies.
The permanent availability of blood host species was shown to have a promoting effect on sand fly abundance. Only five of a total of 15 collected engorged females could be successfully analyzed by either MALDI-TOF mass spectrometry or DNA sequencing, probably due to late stages of blood meal digestion in the remaining specimens. Degraded DNA makes PCR challenging already 24 hours after the blood meal [52] and even though MALDI-TOF peptide mass mapping is possible for longer periods after the blood meal [26], observed blood bolus in some specimens suggest a too advanced stage of digestion, making molecular identification of the blood meal host impossible.
Three engorged females were identified to have fed on chicken. They had been trapped in Unterpurkla and Hummersdorf, where chicken sheds are located at the respective properties. Interestingly, the other two blood meals that could be identified originated from either donkey or horse, although no equid species were present at the respective properties. However, horse barns were located in the range of a few 100 m. Sand flies usually have short flight ranges and only a few have been shown to travel more than 1000 m [53]. Altogether, our findings suggest that Ph. mascittii is a multi-host feeding species, which is further supported by observations by Grimm et al. [7], who confirmed for one Ph. mascittii specimen to have fed on human blood. In a recent study, Leishmania infantum DNA was detected in an unfed Ph. mascittii female in Lower Austria and a dog at the property was observed to be infected with L. infantum. The dog most probably had been vertically infected by its mother, which indicates that the sand fly was infected by feeding on the dog [19].
Host availability and identification is not only important for finding new breeding sites and to assess the potential for further dispersal, but also with regard to the suspected vector capacity of Ph. mascittii for L.infantum. Dogs are the main reservoir for L. infantum and they are commonly imported with asymptomatic infections from endemic countries [54]. However, reports of horses infected with L. infantum from Germany [55] and with an unidentified Leishmania species in Switzerland and Germany indicate that equids might act as reservoir hosts for Leishmania species as well. Thus, further clarification of the vector capacity of Ph. mascittii and its role on the epidemiology of leishmaniasis in Central Europe is urgently needed.