This study reports the first finding of Phlebotomus simici in Austria which represents the northern- and westernmost record of this species to date and further highlights the necessity of more detailed sand fly research in Austria and in Central Europe in general. Apart from prior Ph. mascittii findings in eastern parts of Austria, the sand fly fauna has remained unexplored and probably underreported in this country [5, 6].
The observation of Ph. simici in Austria is rather unexpected, as this species has never been reported in any of the bordering countries (Table 5). Prior to this study, only a single species, namely Ph. mascittii, had been recorded in Austria, as also in neighboring Slovakia. In those neighboring countries of Austria that are known to harbor more than one species, other species are found (e.g. Ph. mascittii and Ph. perniciosus in Germany or Ph. mascittii, Ph. perfiliewi, Ph. neglectus and Ph. papatasi in Hungary) but never Ph. simici. Even in the southern neighboring countries (Italy and Slovenia) that both provide relatively diverse sand fly fauna comprised by several different species, Ph. simici was never recorded. Geographically closest recent records of Ph. simici are from Serbia, which is neighboring Hungary, a direct neighbor to Austria, in the south.
Table 5
Checklist of reported sand fly species in Austria and its neighboring countries. Subgenus, author and year of description are provided at first mention of the respective species. Countries are presented in alphabetical order.
country | species | reference | GenBank COI |
Austria | Phlebotomus (Adlerius) simici NITZULESCU, 1931 | present study | yes |
| Phlebotomus (Transphlebotomus) mascittii GRASSI, 1908 | Naucke et al. 2011 [5], Poeppl et al. 2013 [6] | yes |
Czech Republic | none observed | - | - |
Germany | Phlebotomus mascittii | Oerther et al. 2020 [26] | no |
| Phlebotomus (Laroussius) perniciosus NEWSTEAD, 1911 | Naucke et al. 2004 [3] | no |
Hungary | Phlebotomus mascittii | Trájer et al. 2017 [10] | no |
| Phlebotomus papatasi | no |
| Phlebotomus neglectus | no |
| Phlebotomus perfiliewi | no |
Italy | Phlebotomus mascittii | Dantas-Torres et al. 2014 [27] | no |
| Phlebotomus perniciosus | no |
| Phlebotomus (Phlebotomus) papatasi SCOPOLI, 1786 | no |
| Phlebotomus (Laroussius) neglectus TONNOIR, 1921 | no |
| Phlebotomus (Laroussius) perfiliewi PARROT, 1930 | yes |
| Phlebotomus (Laroussius) ariasi TONNOIR, 1921 | no |
| Phlebotomus (Paraphlebotomus) sergenti PARROT, 1917 | no |
| Sergentomyia minuta | no |
Liechtenstein | none observed | - | - |
Slovakia | Phlebotomus mascittii | Dvořák et al. 2016 [8] | yes |
Slovenia | Phlebotomus mascittii | Praprotnik 2019 [28] | yes |
| Phlebotomus perniciosus | Ivović et al. 2015 [29] | no |
| Phlebotomus papatasi | no |
| Phlebotomus neglectus | no |
| Sergentomyia minuta | no |
Switzerland | Phlebotomus mascittii | Knechtli & Jenni 1989 [30] | no |
| Phlebotomus perniciosus | no |
| Sergentomyia (Sergentomyia) minuta RONDANI, 1843 | no |
Ph. simici belongs to the Adlerius NITZULESCU subgenus, which includes about 20 described as well as several undescribed species with predominantly Eurasian distribution and an assumed origin in Central Asia [31]. Ph. simici is frequently reported in Balkan [24, 25, 32, 33] and Middle Eastern countries [34, 35]. Recent reports from North Macedonia (Dvořák p.o.), Kosovo [36] and Serbia [37] point towards a northward European distribution, which is further corroborated by an older mention from Croatia [38]. Ph. simici is also mentioned in an ex-Yugoslavian study, but it is not entirely clear whether it was indeed recorded in areas which belong to Croatia today [39].
The periurban village where the Ph. simici specimen was caught in Austria is located in the Danube valley in the very eastern part of the country, belonging to the warmest parts of Austria. Microclimatic conditions in river valleys support the establishment and prevalence of local populations of sand flies north of the core area of European distributions shown by occurrence of Ph. mascittii in the Rhine valley [40]. The Danube valley has been assumed to be particularly suitable for sand fly occurrence [41]. The sampling location exhibits perfect breeding site requirements for sand flies, having several buildings with natural floors and various animal hosts, including a dog, poultry, swine, rabbits and goats, close to human dwellings. Typically, also Ph. mascittii is found at similar locations in Central Europe [2, 5, 6, 8], which raises the question if possibly these two species overlap also in other regions and more Ph. simici populations are already established and have been overlooked in the past.
The fact that only a single specimen was detected may be attributed to several factors. Firstly, even though July is usually the warmest month in Austria, abnormal weather conditions were observed in 2019, with great temperature fluctuations. The night temperature was only 15.6 °C in the trapping night and decreased in the consecutive nights, probably temporarily suspending sand fly activity. In Romania, Phlebotomus perfiliewi was observed to be active at 15 °C minimum night temperature, but no activity was observed below this temperature [42]. Secondly, this finding supposedly represents the northern distribution limit of this species and thus, low population densities and consequently small trapping numbers are to be expected. In Austria, trapping rates are generally extremely low, also for Ph. mascittii, with typical trapping numbers of less than five specimens per night [5, 6, 26]. In Slovakia only a single specimen of Ph. mascittii has been trapped, namely in 2016 [6, 8]. A comparative study by Obwaller et al. [7] reported huge differences in numbers of trapped Ph. mascittii specimens in consecutive years at two locations in Austria. These observations suggest that sand fly activity, and thus trapping success might not only depend on temperature but other factors may also play a role.
Identification of the female specimen was challenging and morphological identification was only possible to the subgenus level. Both, pharynx and spermatheca, showed typical Adlerius structures, however, spermatheca were hardly visible by light microscopy. An additional assessment of the spermatheca under UV light illuminated structures confirming the Adlerius subgenus. To our knowledge, the use of autoimmunofluorescence for sand fly identification has never been reported before. The application of this technique might add a valuable tool for the morphological examination of spermatheca. While its suitability for identification to the species level has to be further evaluated, it clearly contributes to the visualization of the otherwise often hardly visible spermatheca. The impossibility to morphologically identify the female specimen to the species level is not surprising, Adlerius females are often unidentifiable by morphology. This is particularly known for Ph. simici and Ph. brevis, two species that overlap in all morphological characters used to distinguish females of the subgenus Adlerius [31]. For example, Perrotey et al. [35] reported that females of sympatrically occurring Ph. simici and Ph. brevis in Lebanon were undistinguishable by morphological characters.
To clarify conflicting morphological identifications, molecular approaches using suitable marker genes are needed. In our study, species identification was possible by sequencing the COI gene, a classical DNA barcoding marker. Interestingly, sequence identity ranged from 95.99–99.85% with sequences of Ph. simici from Turkey and Greece, respectively. Further sequence analyses revealed a monophyletic group of three distinct lineages of Ph. simici, however, mean pairwise distances between the three lineages were unexpectedly high for within one species. In addition, interspecific distances to Ph. brevis and the unidentified Adlerius species from Turkey and Armenia were rather low (< 10%) compared to distances to the other Adlerius species (> 10%) included. This finding indicates that Ph. simici, Ph. brevis and other Adlerius spp. are genetically very close and COI might not be an ideal genetic marker for such closely related species.
COI has been a commonly used genetic marker for species identification since its introduction as “the barcoding gene” by Hebert et al. [43] and thus, sequence availability in GenBank is high and COI is frequently used for sand fly identification and interspecific comparisons [44]. Although there is no common cut-off value for species delimitation, Hebert et al. [43] observed a mean divergence value of 11.3% between species and only a small fraction showed 2% or less divergence. However, in this study we observed pairwise distances between Ph. simici, Ph. brevis and another Adlerius sp. far below 10%. In particular, the mean interspecific distance between Ph. simici and Ph. brevis was only marginally higher than the mean interspecific distances between the three observed Ph. simici lineages. This clearly indicates that Ph. simici, Ph. brevis and the yet unidentified Adlerius species have a short history of divergence and are thus challenging to differentiate by COI. In contrast, interspecific distances of Ph. simici to further Adlerius specimens included in the analysis were far above 10% and thus species easy to separate.
To corroborate our results, cyt b was used as a second genetic marker, albeit sequence availability is rather poor for Adlerius species. Cyt b is the most used genetic marker in sand fly systematics [44]. Further confirmation of species delimitation was achieved by comparing the obtained Ph. simici sequences with reference sequences of Ph. simici and Ph. brevis from Iran. Although the intraspecific distance within Ph. simici was similarly high as observed for COI, the calculated interspecific distance almost doubled the mean interspecific distance of COI between Ph. simici and Ph. brevis (9.1%) and clearly separated these two species.
Ph. simici is an assumed but unproven vector species for Leishmania infantum [31]. Even though the specimen found was tested negative for Leishmania DNA, this species has been shown to be highly anthropophilic [24], which is important for its potential relevance in Leishmania transmission to humans.
Altogether, the finding of a single Ph. simici specimen in Austria does not allow to infer on deeper population genetic structures, however, interesting results at the sequence level were obtained and should be considered in future studies. It is obvious that a single specimen cannot prove the existence of a permanent population and does not give any information on the actual population size. However, particularly eastern parts of Austria have been shown to be suitable for sand flies, which is underlined by continuous trappings of Ph. mascittii, the closest population being found in Rohrau approximately 15 km away from the location reported in this study [5, 6]. Yet, the origin and routes of dispersal are still unclear. By finding a unique but genetically very close haplotype and a shared haplotype of COI and cyt b, respectively, to a haplotype from North Macedonia, post-glacial northward recolonization from this area seems likely. This is further corroborated by recent findings in Serbia [37]. Temperatures in Central Europe during the Holocene optimum around 6,000 years ago were comparable to today and the presence of Mediterranean species in Central Europe may result from northward recolonization events from different refugial areas at that time [45]. The known distribution of Ph. simici and the high interspecific distances between the European, Turkish and Israeli lineages suggest that Ph. simici is most certainly a polycentric Balkanopontomediterranean species. The split between the European and the Turkish Ph. simici lineages might have taken place during one of several complex paleogeographic events that separated the Aegean region into eastern and western parts as demonstrated for the Transphlebotomus subgenus, where separation of the five species, including Ph. mascittii, were dated back to major biogeographic events in the Aegean region [46]. Inference on genetic divergence can be tricky and high mutation rates based on molecular clock calibrations of 5.7%/Mya [47] and 19.2%/Mya [48] have been published at population level compared to a commonly applied rate of 2.3%/Mya for mitochondrial DNA [49]. Thus, the clarification of separation events between Ph. simici lineages and between other Adlerius species should be subject of further studies including a more representative set of populations.