This study presents the first detailed analysis of the seasonal activity of Ph. neglectus in Romania, apparently the most abundant species in the country . This is the highest number of trapped individuals of Ph. neglectus to date in Romania [3, 4, 6, 7]. The species belongs to the Ph. major complex which currently comprises five other species, widely distributed in the Old World. Phlebotomus neglectus is the only species of the complex present in the south and southeastern regions of Europe . In Romania, the species was historically reported in southwestern, southern and southeastern regions of Romania, along the Danube Valley and Bărăgan Plain [7, 16]. In other recent field studies, its presence was limited to southwestern part of Romania, in Mehedinţi Plateau, where it was present mostly peridomestically, outdoor .
The present study was conducted in the nature reserve of Canaraua Fetii, previously investigated for sand fly presence, between 1968 and 1970 . At that time, four sand fly species were present: Ph. balcanicus, and Se. minuta (the two most abundant species), also Ph. neglectus, and Ph. sergenti. In the present study, the diversity and abundance of the sand fly species appears to have changed in this location, with Ph. neglectus the most abundant species (99.63%), and a very low abundance of Ph. balcanicus (0.12%), and Se. minuta (0.25%). Phlebotomus sergenti was not present at the trapping period of the current study.
The peak activity of Ph. neglectus (mid-July) was earlier with approx. 15 days when compared to another evaluated species in Romania, Ph. perfiliewi (monomodal abundance trend with the peak activity at the beginning of August) (Fig. 7) . The earlier and longer activity frame of Ph. neglectus (southeastern Romania), compared with the one of Ph. perfiliewi (northeastern Romania) might be due to geographical position, microhabitat, and climatic differences (statistically supported in both studies) . In another recent study of Ph. mascittii, the predominant sand fly species in Central Europe, a monomodal abundance trend was also present in Austria, with variations of the peak activity to July and August . Located at a similar latitude as Romania, the sand fly activity in Austria started earlier, by late June, and ended earlier, by the end of August . A monomodal abundance trend was also observed for other sand fly species, such as Ph. ariasi in France, Ph. kandelaki, and Ph. balcanicus, in Georgia .
Considering the Balkans region, a recent review revealed that Ph. neglectus was also the most abundant species in 2014 and 2016 in Serbia, Kosovo, Bosnia and Herzegovina, Montenegro, Croatia, Slovenia, and the second most abundant species in Bulgaria, and North Macedonia . The abundance trend for Ph. neglectus can also be bimodal (two peaks of activity), a main characteristic when the species is located in the warmer, Mediterranean climate (e.g., Greece or Cyprus) . For other sand fly species, up to three density peaks and substantially longer activity periods were observed in countries with lower latitudes, such as Portugal or Turkey .
Sergentomyia minuta was the second most abundant sand fly species present between 1968 and 1970 in Canaraua Fetii . In the present study conducted after 50 years in the same location, only two specimens of Se. minuta (one male and one female) were trapped. Between 2013 and 2018, the species was not trapped in other sampling sites in Romania, despite the trapping effort [3, 4]. A possible explanation for the apparent lacking in the past years could be the trap type and the sampling date and effort. In the historical data , only sticky traps were used to collect sand flies, while in recent studies conducted in Romania between 2013 and 2018, only CDC miniature light traps were used [3, 4]. The two Se. minuta specimens (Table 1) were trapped with the sticky traps, while none were trapped with the CDC miniature light traps. Regarding the sampling date, it appears that Se. minuta was active and present at the beginning of July, while most of the trapping effort conducted in Romania so far was focused on the end of July-beginning of August . The chosen trapping period was based on several available data: another seasonality study conducted in Romania, of Ph. perfiliewi, was assessed and the results shown the peak activity at the beginning of August ; the same species, Ph. perfiliewi, was active only after the average minimum temperature for the previous 7 days was above 15°C ; in another study, under laboratory conditions, another species, Ph. papatasi (recorded also in Romania) showed no larval and pupal development at 15°C and a mean temperature of at least 18°C was necessary for successful rearing . In a more recent study, the earliest activity of Ph. mascittii was noticed after 5 consecutive days of mean temperature values above 15°C and minimum temperature values above 10°C .
In another study from eight Balkan countries (some of them neighbouring Romania), Se. minuta appeared to be one of the less abundant sand fly species in Bulgaria, North Macedonia, Serbia, Kosovo, Bosnia and Herzegovina, and Montenegro. In Slovenia, the species was not present in the evaluated trapping sites. On the other hand, in Croatia the species appeared to be the second more abundant species .
The third sand fly species present in the current study, Ph. balcanicus was one of the two most abundant species in Canaraua Fetii between 1968 and 1970 . Phlebotomus balcanicus was also identified in North Macedonia, Serbia, Kosovo, Montenegro , Turkey, Armenia and Georgia .
The changes in the sand fly composition in Canaraua Fetii might have been influenced by a series of factors, such as: (i) demographic and human behavioural changes; (ii) changes in the host species availability; (iii) ecological changes; (iv) climatic changes; (v) use of insecticides. The selected study area (Canaraua Fetii Natural Reserve) has had a sinuous path in becoming a protected area. In the past, a stone quarry with employees was running. Human activity was present and with that, the availability of domestic hosts. The domestic animal host availability has changed and transitioned to a wildlife host after the stone quarry close . This might have had influenced the changes in the sand fly fauna. Nowadays, even if the site is a protected area, a monastery was recently opened in 2012, and the tourist pilgrimage started. As a protected natural area, the abundance of animal species attracts an important number of outdoor enthusiasts. As for the insecticide use, DDT was used in Romania between 1958 and 1964 . There are no data that indicates aerial spraying over this location, but since the previous sand fly sampling (1968–1970) in the area which was done after the DDT campaign, we eventually consider that this is less likely to be the cause of sand fly species composition change.