Population dynamics of sympatric Phortica spp. and first record of stable presence of Phortica oldenbergi in a Thelazia callipaeda-endemic area of Italy

Population dynamics of sympatric Phortica spp. and first record of stable presence of Phortica oldenbergi in a Thelazia callipaeda-endemic area of Italy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Population dynamics of sympatric Phortica spp. and first record of stable presence of Phortica oldenbergi in a Thelazia callipaeda-endemic area of Italy Ilaria Bernardini, Cristiana Poggi, Daniele Porretta, Jan Máca, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5004631/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 06 Nov, 2024 Read the published version in Parasites & Vectors → Version 1 posted 7 You are reading this latest preprint version Abstract Background: Five species of the Phortica genus (Diptera: Drosophilidae) are known in Europe and the Middle East. Among these, Phortica variegata and Phortica okadai are better known for their role as vectors of the zoonotic eyeworm Thelazia callipaeda . Other species, such as Phortica semivirgo and Phortica oldenbergi , have been studied less. Given the paucity of data about these Phortica spp. vectors, we explored the population dynamics and ecology of Phortica spp. in an area highly endemic for T. callipeada (Manziana, Rome, Central Italy). Methods: Phortica spp. flies were collected over a three-year period (2018-2020) during their active season (April-October) with a sweep net while hovering around: i) a fermenting fruit bait, and ii) a human operator acting as bait. Collected flies were morphologically identified and tested for T. callipaeda infection and Wolbachia presence by PCR. Population dynamics of species collected was associated to environmental drivers through Generalized Additive Models. Results: Of the 5,564 flies collected, 90.8% were P. variegata , 9.1% were P. oldenbergi , 0.05% were P. semivirgo , and one specimen was P. okadai . Only P. variegata scored molecularly infected with T. callipaeda throughout the three-year sampling period (1.8%). Phortica oldenbergi , observed consistently during the entire sampling period, exhibited a marked preference for fruit traps, contrasting with the lachryphagous activity of P. variegata . Analysis of environmental drivers of P. oldenbergi and P. variegata population dynamics indicated temperature, wind speed, and pressure as significant factors. In addition, Wolbachia pipientis endosymbiont was detected in P. oldenbergi and P. okadai . Conclusions: For the first time, this study analysed several ecological aspects of Phortica species coexisting in a T. callipaeda endemic area, highlighting different behaviours in the same environment and the vectorial role of this zoonotic parasite. Notably, this is also the first report of the presence of P. oldenbergi in Italy and P. okadai in Europe, underscoring the importance of extensive sampling for detecting potential vectors and alien species with direct implications for vector-borne disease epidemiology. Phortica Sympatry Eyeworm Thelazia Zoonosis Lachryphagy Wolbachia Vector-borne disease Figures Figure 1 Figure 2 Figure 3 Figure 4 Background The genus Phortica Schiner (1862) (Diptera, Drosophilidae, Steganinae) counts almost 160 species distributed all over the world ( 1 ). Nearly two-thirds of these are described in East Asia, especially in southern China. The importance of this taxonomic group in medical and veterinary entomology is raising due to their role as vectors of the eyeworm Thelazia callipaeda (Railliet and Henry, 1910) (Spirurida, Thelaziidae) eyeworm, which causes infection in many mammal species ( 2 – 4 ). Indeed, while feeding around animal secretions, lachryphagous males of Phortica okadai (Máca,1977) and Phortica variegata (Fàllen,1823) transmit this eyeworm to definitive hosts such as domestic mammals, including dogs, cats, and rabbits, wildlife (e.g. foxes, hares, wolfs, mustelids, bears), and occasionally humans ( 5 – 14 ). Recently, Phortica kappa (Máca, 1977) and Phortica magna (Okada, 1960) were also described as vectors ( 15 ). Adult worms develop in the conjunctival sac of the mammals’ hosts causing mild to severe outcomes such as conjunctivitis, keratitis, corneal ulcer, and blindless if not cured ( 8 , 12 , 16 – 18 ). Thelaziosis has been historically considered endemic in Asia and for twenty years it has been spreading across Europe ( 16 , 18 ) and, recently, in United States of America ( 19 ). However, except for P. variegata , in Europe and the US ( 20 ), little is known about the distribution, ecology, and biology of the other T. callipaeda vectors. In addition to P. variegata , four more Phortica species have been reported in Europe and Near East: Phortica semivirgo (Máca,1977), which is also widely distributed ( 21 , 22 ), Phortica erinacea (Máca,1977) and Phortica goetzi (Máca,1987), which have been sporadically reported in Bulgaria/Greece and Turkey, respectively, and P. oldenbergi (Duda, 1921), of which only three specimens were collected in Germany at the beginning of the 20th century ( 23 , 24 ) and recently found in Spain ( 25 ), despite this record requires further investigations as it is based only on the morphology of females and no molecular data are available. For P. variegata , some information on its life history traits is available, and it has been reconstructed from laboratory breeding ( 26 , 27 ) and field sampling. This species is associated with oak forests ( Quercus spp.) ( 9 , 28 , 29 ) where flies are found active from April-May to October-November ( 9 , 29 ). After this period, an overwintering strategy at the adult stage has been presumed in P. variegata and P. semivirgo ( 3 , 30 ), with some adults collected in caves during winter ( 9 , 31 , 32 ). However, even the ecology of P. variegata is still under investigation, as it is still poorly known in critical aspects, such as larval ecology. Moreover, no information is available about the presence and ecological contribution of endosymbionts on Phortica species, despite there is evidence of the absence of Wolbachia pipientis (Hertig, 1936) bacteria in T. callipaeda ( 33 ). Wolbachia is an obligate intracellular maternally transmitted gram-negative genus establishing a spectrum of symbiotic relationships, ranging from parasitism to obligatory mutualism. Up to 50% of terrestrial arthropod species and several onchocercid nematodes harbour this endosymbiont in nature, including several Drosophilidae ( 34 ). In this study, we investigated the presence and the population dynamics of Phortica spp. in the ecological setting of Central Italy, Manziana, an area known for the presence of these flies and of T. callipaeda ( 29 ). Here we report, for the first time, the co-occurrence of four Phortica species, detailed data about their ecology and phenology, and the first record of a stable population of P. oldenbergi in Italy. Data about potential expansion in the distribution of these Phortica spp. are also discussed in light of the risk they may represent as competent vectors of T. callipaeda . Methods Phortica spp. sample collection and morphological identification Longitudinal samplings were performed in the oak forest of Manziana (Rome, Italy, 42°07'10.8"N − 12°06'57.6"E) during the period April-October from 2018 to 2020. Phortica spp. flies were collected with a sweep net while hovering around: i) a fermenting fruit bait for flies showing a frugivorous trophic behaviour, and ii) a human operator acting as bait for lachryphagous flies ( 8 ). Collected specimens were killed soon after being collected and stored in 70% ethanol in the field. After collection, specimens were morphologically identified by species and sex ( 23 , 35 ). In case of doubtful identification (N = 95), a molecular confirmation was carried out with conventional PCR and sequencing. Phortica spp. molecular identification Genomic DNA was isolated from single specimens using DNAzol DNA extraction protocol (MCR, Inc., Cincinnati Ohio) and then amplified with primers UEA7 (5’- TACAGTTGGAATAGACGTTGATAC-3’) and UEA10 (5’-TCCAATGCACTAATCTGCCATATTA-3’) to target the mitochondrial Cytochrome oxidase subunit I gene ( cox1 ) ( 36 ). PCR was performed in a total volume of 25µl, containing 1.5µl of DNA as a template by mixing: sterile dd H 2 O (17.00 µl), 2µl MgCl 2 [2.5mM], 2.5µl 10X reaction buffer (10X NH 4 , Meridian Bioscience, Inc., Cincinnati, Ohio), 0.8µl of dNTPs [10µM], 0.5µl of each primer [10µM], 0.20µl of Taq DNA polymerase [1u/µl]. PCR was performed in a thermal cycler (Bio-Rad, C1000 Touch) using the following cycling protocol: 95°C for 10’ (polymerase activation), followed by 31 cycles of 95°C for 1’ (denaturation), 64° for 45” (annealing), 72°C for 1’ (extension), 1’ at 72°C (final extension). The PCR 700 bp long amplified product was visualised in 1.5% agarose gel. PCR products were purified with SureClean Plus (Bioline, Meridian Bioscience) and sent to the sequencing provider (Sanger sequencing method; BMR Genomics s.r.l.). All the nucleotide sequences were screened with Chromas ( 37 ) and Mega 11 software ( 38 ). Sequence species identity was confirmed in the GenBank ® database using nucleotide Basic Local Alignment Search Tool ( BLASTn) ( 39 ). Thelazia callipaeda molecular detection A subsample of flies was tested for the presence of T. callipaeda . The cox1 region (689 bp) was amplified using primers COI_F (5’-TGATTGGTGGTTTTGGTAA-3’) and COI_R (5’- ATAAGTACGAGTATCAATATC-3’) ( 40 ). PCR was performed in a total volume of 25µl, containing 2µl of DNA as a template by mixing: sterile dd H 2 O (14.65µl), 1.5µl MgCl 2 [2.5mM], 2.5µl 10X reaction buffer (10X NH 4 , Meridian Bioscience, Inc., Cincinnati, Ohio), 2.5µl of dNTPs [10µM], 0.8µl of each primer [10µM], 0.25µl of Taq DNA polymerase [1u/µl]. PCR was performed in a thermal cycler (Bio-Rad, C1000 Touch) using the following cycling protocol: 95°C for 10’ (polymerase activation), followed by 35 cycles of 95°C for 1’ (denaturation), 55° for 30” (annealing), 72°C for 1’ (extension), 7’ at 72°C (final extension). The PCR 689 bp long amplified product was visualised in 1.5% agarose gel. Wolbachia endosymbiont molecular detection The presence of the endosymbiont Wolbachia in the collected Phortica species was verified by PCR amplifying a portion of the wsp arthropod-specific gene (600 bp) ( 41 ). The amplification reaction was carried out according to the following protocol (25µl final volume): 2.5µl 10X reaction buffer (10X \(\:{NH}_{4}\) , Meridian Bioscience, Inc., Cincinnati, Ohio), 2µl MgCl2 [2.5mM], 2.5µl \(\:\:\) dNTPs [10µM], 1µl [10µM] of primers 81F (5'-TGGTCCAATAAGTGATGAAGAAAC-3') and 691R (5'-AAAAATTAAACGCTACTCCA-3'), 0.2µl of Taq DNA polymerase [1u/µl], 1 µl of DNA template. Amplification profile: 30 cycles at 94°C for 30”, 50°C for 30”, 72°C for 30”. The amplified products were sequenced (Sanger sequencing method; BMR Genomics s.r.l.) and species identity was confirmed in the GenBank ® database using BLASTn ( 39 ). Statistical analysis Different Generalized Additive Models (hereafter named GAM-1; GAM-2; GAM-3; GAM-4) were fitted to determine the effect of the environmental parameters on the abundance of the Phortica spp. collected. The response variable was expressed as log-CPUE (the natural logarithm of Catch-Per-Unit-of-Effort, expressed as the number of collected flies/hour) to control for the variation in the sampling effort. The daily average of environmental data (temperature, humidity, barometric pressure, wind speed, dew point) were retrieved from the nearest weather station (IMANZ6, 42.106° N, 12.078° E; located 2km far from the sampling site; https://www.wunderground.com/ ). The three GAMs models differ in the response variable. In GAM-1 we considered only the log-CPUE for P. oldenbergi , while in the other two models (GAM-2 and GAM-3) the response variable was the log-CPUE of P. variegata females (from fruit traps) and males (lacryphagous), respectively. Given the limited number of P. semivirgo and a single specimen of P. okadai collected during the whole sampling period, these species were excluded from the analysis. A selection of the predictors was carried out to avoid multicollinearity. In all the GAMs the parametric coefficients were sex, bait, and atmospheric pressure, while a smooth function was applied to windspeed, dew point, humidity, and temperature. A smooth function was also estimate for the date of collection (treated as day of the year) with an interaction between the factor variable bait (fruits/net). GAMs model structure was: $$\:{Y}_{i}=\:{\alpha\:+\:f}_{k}\left({X}_{ki}\right)+{\beta\:}_{j}\left({Z}_{ji}\right)+{\epsilon\:}_{i}$$ 1 where \(\:{Y}_{i}\) is the log-CPUE, \(\:\alpha\:\) is the intercept, \(\:f\) is th k -th smooth term for the k -th non-linear predictor ( \(\:f\) ), \(\:\beta\:\) is th j -th fixed effect for the j -th linear predictor ( \(\:Z\) ), and \(\:\epsilon\:\) is the error term for the i -th observation. To test if there was a significant difference in the mean number of P. variegata flies collected on fruit bait, another GAM model (GAM-4) was fitted on the log-CPUE of flies collected with fruit bait. All statistical analyses were performed with R software (version 4.3.1) and the packages: mgcv and tidygam ( 42 , 43 ). Results In total 5,564 Phortica spp . flies were collected from 2018 to 2020 (N = 52 sampling days) in the area and morphologically identified. The most abundant species was P. variegata (90.8%), followed by P. oldenbergi (9.1%; Supplementary Figure S2). A total of three specimens of P. semivirgo (0.05%) were collected in 2018 (N = 1, by human bait) and 2020 (N = 2, by fruit bait) while a single specimen of P. okadai was collected in 2018 (0.02%, by human bait). The numbers P. variegata and P. oldenbergi collected per month, year and sampling bait are reported in Tables 1 and 2 . Table 1 Numbers of Phortica variegata collected during the sampling period. Year Month N Fruit-bait (%males) N Human-bait (%males) Total 2018 April 22 (13.6%) - May 262 (17.9%) 20 (100%) June 89 (28.1%) - July 148 (43.9%) 8 (100%) August 42 (40.5%) 2329 (100%) September 94 (43.6%) 394 (100%) October 1 (0%) 10 (100%) 3419 2019 April 5 (0%) 0 May - - June 22 (27.3%) 11 (100%) July 18 (5.6%) 749 (100%) August - 173 (100%) September 3 (66.7%) 98 (100%) 1079 2020 May 2 (0%) 16 (100%) June 12 (0%) 11 (100%) July 26 (0%) 53 (100%) August 41 (80.5%) 213 (99.5%) September 5 (0%) 151 (99.3%) October - 26 (100%) 556 Total 5054 Table 2 Numbers of Phortica oldenbergi collected during the sampling period. Year Month N Fruit-bait (%males) N Human-bait (%males) Total 2018 April 20 (0%) - May 189 (4.8%) - June 24 (4.1%) - July 31 (22.6%) - August 9 (22.2%) - September 123 (4%) 1 (100%) October - 1 (100%) 398 2019 April 7 (0%) - May 2 (0%) - June 4 (0%) 1 (0%) July 16 (0%) 1 (0%) August - - September 2 (0%) 1 (0%) October - - 32 2020 May 10 (0%) - June 18 (0%) - July 32 (0%) 2 (100%) August 7 (0%) - September 7 (0%) - October - - 76 Total 506 BLASTn sequence analysis of the cox1 gene (N = 95) confirmed the morphological identification as P. variegata (N = 84; 99.7% of nucleotide identity (ni); accession number PP990198), P. semivirgo (N = 1; 99.1% ni; accession number PP990202) and P. okadai (N = 1, 99.8% ni; accession number PP860587). Sequence analysis of P. oldenbergi (N = 9; accession number PP838740; Supplementary Text S3) showed a similar nucleotide identity with P. variegata (95.5% ni), P. okadai (95.1% ni) and P. semivirgo (94.7% ni) as sequences for this species were not available in GenBank. A total of 101 individuals (N = 74 P. variegata , N = 20 P. oldenbergi , N = 3 P. semivirgo , N = 1 P. okadai ) were analysed for the presence of Wolbachia . All P. oldenbergi and the P. okadai specimens examined scored positive for a fragment of the expected size whereas P. variegata and P. semivirgo were negative (Supplementary file, Figure S1 ). All sequences were identical and were confirmed as belonging to Wolbachia strain A (100% identity with Wolbachia endosymbiont (group A) of Melieria omissa ; accession numbers PP930348 and PP930349). Thelazia callipaeda DNA was detected in the P. variegata subsample (N = 699) showing different infection rates: 1.26% in 2018 (N = 352), 1% in 2019 (N = 100) and 2.02% in 2020 (N = 247), whereas any P. oldenbergi , P. semivirgo , and the single P. okadai were positive. The sex ratio of both P. variegata and P. oldenbergi collected each year was unbalanced (Tables 1 and 2 ), The male: female ratio for P. variegata was 6.7:1 in 2018, 24.7:1 in 2019, and 9.1:1 in 2020, while for P. oldenbergi , this ratio was 0.07:1 in 2018, 0:1 in 2019, and 0.03:1 in 2020. The sex ratio of flies collected on human bait substantially differed from those gathered on fruit – see the next paragraph. The results of the GAMs fitted on the log-CPUE of Phortica spp. showed a significant effect of the day of the year, temperature and windspeed on the mean number of flies collected (Figs. 1 and 2 ). In all models the parametric coefficient showed a significant negative effect of barometric pressure (P-value < 0.01) and no significant effects of dew point and humidity. The seasonal dynamics of P. oldenbergi showed a first peak of abundance in May-June, followed by a decrease and a second (lower) peak of September-October (GAM-1; adj. R-sq 0.46; Fig. 1 a). Similarly, P. variegata females were mainly abundant in the first part of the season (May- June) but showed a rapid decrease in June and a plateau until the end of sampling (GAM-2; adj. R-sq. 0.62; Fig. 2 a). Conversely, numbers of P. variegata males (on human bait) rapidly increased in mid-June with a peak of abundance in August-September, then slightly decreased reaching a plateau until the end of sampling (GAM-3; R-sq. adj 0.48; Fig. 3 ). It needs to be clarified whether sex-ratio or just a food preference is involved. Only P. variegata females collected on the fruit bait showed a significant trend (GAM-4; R-sq. adj 0.30; Fig. 4 ). Single specimes of P. oldenbergi collected on human bait showed subequal number of males and females, with no seasonal trend detectable (also here, the result is different from the sex ratio based mainly on the data from fruit bait). Discussion This study showed for the first time the occurrence and seasonal dynamics of Phortica species living in sympatry within a forested area of central Italy, where T. callipaeda circulates. Phortica variegata was the most abundant species, representing 90% of the collected Phortica flies, and it was the only species found positive for T. callipeda , with infection rates ranging between 1% and 2.2%. Among the less abundant species, P. oldenbergi , a newly recorded species in this geographic area, was collected throughout the sampling period, indicating its establishment in Central Italy for the first time. Phortica semivirgo , on the other hand, is considered a species native of Italian entomofauna ( 44 ), which was sporadically observed in this area. Although both P. semivirgo and P. oldenbergi tested negative for the presence of T. callipaeda during the whole study period, considering the low number of specimens collected in this study it still cannot be excluded that they could act as vectors in different ecological contexts. Finally, P. okadai , a major vector of T. callipaeda in the Asian region ( 2 ) was surprisingly recorded in the study area. The finding of a single specimen of P. okadai suggests a possible accidental introduction by fruit trading. Further sampling in surrounding areas should be performed to increase information about the possible wider presence of P. okadai at least in Central Italy, which would be of paramount relevance for T. callipaeda epidemiology, considering the major role of this vector in its original areal. The oak forest of Manziana gave us the unique opportunity to study the population dynamics and feeding behaviour of the taxa of the Phortica genus simultaneously. Population dynamics of all Phortica spp . showed a peak of abundance of females in May, possibly indicating a seasonal phase when the flies are more focused on the feeding activity to restore enough energy for reproduction after overwintering. However, a second low peak in abundance was observed in P. oldenbergi . Although not significant, this small increase in abundance might be indicative as a preparation time to obtain energy reserves in the late season before the arrival of the winter, as described in other Drosophilidae ( 45 ). The sex ratio was highly unbalanced in the studied species. Most P. oldenbergi flies collected were females caught with fruit baits, thus suggesting some connection to fermenting fruit, as observed in other Phortica species. This does not necessarily mean frugivory – adults probably, like many Drosophila spp., consume yeasts and other microorganisms and/or seek an environment for their possibly predatory larvae ( 9 ). Previous studies of various Steganinae flies have consistently reported a higher capture rate in traps baited with fermenting fruits near tree canopy ( 3 , 46 ). This applies well to P. oldenbergi , although a few specimens of both sexes of P. oldenbergi were caught with human bait, indicating that lachryphagy can occur in this species, akin to other Phortica spp. ( 47 ). Again, the low numbers of males collected in the study hampered the possibility to better clarify the feeding behaviour of P. oldenbergi males. Similarly, during this sampling only a few specimens of P. semivirgo males were collected using fermenting fruit and human bait, yielding inconclusive results. Concerning the most common species P. variegata , most specimens were males collected with human bait, consistent with prior literature ( 8 , 9 , 28 , 29 , 47 , 48 ) with their numbers rapidly increasing and peaking in August. According to previous findings ( 9 , 29 , 47 ), and consistently with this study, temperature constitutes a pivotal driver affecting the lachryphagous feeding behaviour of P. variegata males. However, a significant effect of the temperature on the mean abundance of Phortica flies on fruit bait was also observed, indicating its involvement in this feeding activity. A plateau of the abundance of both fruit baited Phortica spp. was observed between 21°C and 29°C, suggesting that at higher temperatures of the day (hottest days and hours of the year) flies are less active. This result was corroborated by the significant negative effect of the parametric coefficient of the pressure detected from all models. Indeed, high atmospheric pressure results in the absence of cloud coverage and thus increase of temperatures with a consequent decrease in flies’ abundance. Our findings agree with the previous study on the flying activity of drosophilid flies, highlighting the general avoidance of several Drosophila spp. for higher temperatures and a species-specific thermopreferences affecting the range of total daily activity of these flies ( 49 ). In addition, the models confirmed the negative effect of the wind speed on the mean abundance of collected flies, which can be reasonably explained by the higher energy required for flies for dispersal. The low numbers of P. semivirgo collected are consistent with the available literature. This species was unknown to science until 1977, and only sporadically reported in Europe, except for Switzerland, where a trapping bottle system was used to collect the flies ( 48 ). Nowadays, P. semivirgo seems more common, at least in central Europe, although the cases when it is collected in absence of P. variegata are rare ( 21 , 50 ). It is then possible that the abundance of this species was underestimated in this study due to a less efficient trapping approach. Another possible explanation for the low density of P. semivirgo in the collection could be the different vertical micro-distribution in the canopy of Phortica spp. living in sympatric conditions, as indicated in the literature ( 3 , 3 , 50 – 52 ), leading to the hypothesis that P. semivirgo may exploit upper levels of oak trees as a preferential habitat, while being less represented at ground level, where our sampling occurred. The rediscovery of a P. oldenbergi population in Europe is very surprising. In fact, before this record, the species was documented in Europe, with only tree specimens of P. oldenbergi collected and described in Germany in the early 20th century. Following this, six more female specimens were reported from Spain roughly one hundred years later ( 25 ). This led to the hypothesis that this species was accidentally introduced in Europe at the time ( 35 ) but could not settle in. Indeed, the species belongs (or is close) to the Allophortica subgenus, of which another four described species are known from the Afrotropical region ( 1 ). The rare and patchy presence of this species in Europe might be explained by two hypotheses, both yet to be demonstrated: i) it is a relict areal of its original distribution or ii) its occurrence is the consequence of new introduction events. However, the recent study indicating this species as a competent vector of T. callipaeda in laboratory conditions ( 53 ) highlights the need for further investigations to determine the vector capacity of this species in nature. The PCR amplification of a fragment of the wsp gene in the collected specimens suggests the presence of Wolbachia , belonging to the strain typical of arthropods, in both P. oldenbergi and P. okadai . At the same time, the symbiont seems to be absent in P. variegata and P. semivirgo . To the best of our knowledge, none of the Phortica species have ever been screened for the presence of this symbiont. Therefore, detecting of Wolbachia in P. oldenbergi and P. okadai is the first indicator of the symbiotic relationship this endosymbiont established with flies within the Phortica genus. Wolbachia is a widespread endosymbiotic bacterium known for its intricate relationships with various insect hosts, including drosophilids. In this family Wolbachia exhibits mutualistic and parasitic interactions, depending on the strain and host species. One of the most notable effects of Wolbachia in Diptera is cytoplasmic incompatibility, which can enhance the bacterium’s spread by favouring the reproduction of infected females over uninfected ones ( 54 ). This complex interaction highlights Wolbachia 's significant role in its hosts' evolution and ecology, influencing population dynamics and genetic diversity ( 55 ). Based on our results, assuming that this symbiont is present in only some Phortica species, it is not trivial to hypothesise the most probable mechanism for the introduction of Wolbachia in P. oldenbergi and P. okadai . The routes exploited by Wolbachia to be horizontally transferred between species are heterogeneous, including the sharing of food source in the environment ( 56 ). This might represent the most probable mechanism of introduction in the two Phortica species, which are known to also show affinity to fruit bait. Conclusions This study elucidates the ecological aspects of sympatric Phortica species within a forest in Central Italy, offering unprecedented insights into P. variegata and P. oldenbergi biology and ecology. The extensive collection and identification of these species underscore their seasonal variation of abundance and potential roles within the ecosystem, unravelling the complex interplay between environmental drivers, feeding activity and vector behaviour, especially regarding the transmission of the eyeworm T. callipaeda . In particular, the rediscovery and establishment of P. oldenbergi in Europe pose intriguing questions about its geographical origin, ecological role, and potential as a vector of T. callipaeda , deserving further investigation. Similarly, the sporadic presence of P. semivirgo and the enigmatic observation of a single specimen of P. okadai in the Manziana area highlight the complexities of species establishment in new environments, involving possible use of fruit trading. Further studies are imperative to explore the vectorial capacity of these species in natural settings, assess the impact of environmental changes on their populations, and develop strategies for monitoring and controlling their spread in relation to T. callipaeda transmission to animals and humans. Abbreviations GAM Generalized Additive Model CPUE Catch-Per-Unit-of-Effort Declarations Acknowledgments Authors would like to thank Federica Laterza, Federica Furzi and Eugenio Gabrieli for the support in sampling collection and laboratory analyses, and Prof. John Jaenike for the insightful discussions about Phortica ecology. Author contributions MP, FB, JF, RPL, JM and DO conceived the study. MP, IB, CP and DO organized the sampling plan. MP, IB, CP and SM collected data in the field. MP, IB, CP, EP, SG, VP, DP, MSL performed laboratory and molecular analyses of the specimens. IB, CP, MP analysed the results. IB, CP and MP drafted the manuscript, and all authors critically contributed to its final version. All authors read and approved the final manuscript. Funding Not applicable. Availability of data and materials Data supporting the conclusions of this article are included within the article and its additional files. The datasets used and analysed during the present study are available from the corresponding author upon reasonable request. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. References Global Biodiversity Information Facility (GBIF) [Internet]. [cited 2024 Jun 9]. Available from: https://www.gbif.org/ Jin Y, Liu Z, Wei J, Wen Y, He N, Tang L, et al. A first report of Thelazia callipaeda infection in Phortica okadai and wildlife in national nature reserves in China. Parasit Vectors. 2021 Dec;14(1):13. Máca J, Otranto D. Drosophilidae feeding on animals and the inherent mystery of their parasitism. Parasit Vectors. Nov 2014 (7): 1-8. https://doi.org/10.1186/s13071-014-0516-4 Otranto D, Lia RP, Cantacessi C, Testini G, Troccoli A, Shen JL, et al. Nematode biology and larval development of Thelazia callipaeda (Spirurida, Thelaziidae) in the drosophilid intermediate host in Europe and China. Parasitology. 2005 Jul 22;131(06):847. Bojan G, Vanja BS, Aleksandra P, Milica K, Neda B, Duško Ć. First report of eyeworm infection by Thelazia callipaeda in gray wolf (Canis lupus) from Serbia. Parasitol Res. 2019 Dec;118(12):3549–53. Deak G, Ionică AM, Mihalca AD. First report of Thelazia callipaeda Railliet & Henry, 1910 in a zoo lion (Panthera leo) (Linnaeus, 1758) from Romania. Vet Res Commun. 2023 Dec;47(4):2173–7. Nájera F, De Lucas-Veguillas J, Vela Á, López-Fernández M, Martínez-Martínez P, Mata-Huete M, et al. First report of Thelazia callipaeda in a free-ranging Iberian wolf (Canis lupus signatus) from Spain. Parasitol Res. 2020 Jul;119(7):2347–50. Otranto D, Brianti E, Cantacessi C, Lia RP, Máca J. The zoophilic fruitfly Phortica variegata : morphology, ecology and biological niche. Med Vet Entomol. 2006 Dec;20(4):358–64. Otranto D, Cantacessi C, Testini G, Lia RP. Phortica variegata as an intermediate host of Thelazia callipaeda under natural conditions: Evidence for pathogen transmission by a male arthropod vector. Int J Parasitol. 2006 Sep;36(10–11):1167–73. Otranto D, Cantacessi C, Mallia E, Lia RP. First report of Thelazia callipaeda (Spirurida, Thelaziidae) in wolves in Italy. J Wildl Dis. 2007;43(3):508–11. Otranto D, Dantas-Torres F, Mallia E, DiGeronimo PM, Brianti E, Testini G, et al. Thelazia callipaeda (Spirurida, Thelaziidae) in wild animals: Report of new host species and ecological implications. Vet Parasitol. 2009 Dec;166(3–4):262–7. Otranto D, Dutto M. Human Thelaziasis, Europe. Emerg Infect Dis. 2008 Apr;14(4):647–9. Papadopoulos E, Komnenou A, Karamanlidis AA, Bezerra‐Santos MA, Otranto D. Zoonotic Thelazia callipaeda eyeworm in brown bears ( Ursus arctos ): A new host record in Europe. Transbound Emerg Dis. 2022 Mar;69(2):235–9. Paradžik MT, Samardžić K, Živičnjak T, Martinković F, Janjetović Ž, Miletić-Medved M. Thelazia callipaeda—first human case of thelaziosis in Croatia. Wien Klin Wochenschr. 2016 Mar;128(5–6):221–3. Kitajima A, Tokiwa T, Doi K, Kotani K, Otsubo H, Kamei C, et al. New host record of Thelazia callipaeda (Nematoda: Spirurida) with a notably wide host range and shared zoonotic lineage in Japan. Parasitol Int. 2024;102913. Do Vale B, Lopes AP, Da Conceição Fontes M, Silvestre M, Cardoso L, Coelho AC. Thelaziosis due to Thelazia callipaeda in Europe in the 21st century—A review. Vet Parasitol. 2019 Nov;275:108957. Martínez-Sánchez MI, Bolívar-de-Miguel G, Cuadros-González J, González JMR. Ocular thelaziosis: a case report of an emerging zoonosis. Am J Ophthalmol Case Rep. 2021;22:101045. Otranto D, Mendoza-Roldan JA, Dantas-Torres F. Thelazia callipaeda. 2021 [cited 2024 Jul 16]; Available from: https://www.arca.fiocruz.br/handle/icict/50732 Manoj RR, White H, Young R, Brown CE, Wilcox R, Otranto D, et al. Emergence of Thelaziosis Caused by Thelazia callipaeda in Dogs and Cats, United States. Emerg Infect Dis. 2024;30(3):591. Otranto D, Iatta R, Lia RP, Cavalera MA, Màca J, Pombi M, et al. Competence of Phortica variegata from the United States as an intermediate host of the Thelazia callipaeda eyeworm. Am J Trop Med Hyg. 2018;98(4):1175. Lutovinovas E, Ivinskis P, Rimšait J. Phortica semivirgo (Máca, 1977) – new to the fauna of Lithuania (Diptera: Drosophilidae). Bulletin of the Lithuanian Entomological Society. 4 (2020): 109-113 Unterköfler MS, Dengg P, Niederbacher M, Lindorfer S, Eberle A, Huck A, et al. Occurrence of Thelazia callipaeda and its vector Phortica variegata in Austria and South Tyrol, Italy, and a global comparison by phylogenetic network analysis. Parasit Vectors. 2023 Aug 24;16(1):294. Bächli G, Vilela CR, Escher SA, Saura A. The Drosophilidae (Diptera) of Fennoscandia and Denmark. [Internet]. Brill Academic Publishers; 2004 [cited 2024 Jan 12]. Available from: https://www.cabdirect.org/cabdirect/abstract/20053205535 Máca J. Revision of Palaearctic species of Amiota subge nus Photica (Diptera, Drosophilidae). Acta Entomol Bohemoslov. 1977;74:115–30. Carles-Tolrá, M and Páez, A V., 2010. Nuevos datos dipterológicos del Parque Natural de los Alcornocales (Cádiz, España) (Insecta, Diptera). Boletín de la Sociedad Entomologica Aragonesa (S.E.A.), no 47 (2010):369-371 Bernardini I, Poggi C, Manzi S, Bezerra-Santos MA, Beugnet F, Fourie J, et al. Laboratory breeding of two Phortica species (Diptera: Drosophilidae), vectors of the zoonotic eyeworm Thelazia callipaeda. Parasit Vectors. 2022 Dec;15(1):200. Otranto D, Cantacessi C, Lia RP, Kadow ICG, Purayil SK, Dantas-Torres F, et al. First laboratory culture of Phortica variegata (Diptera, Steganinae), a vector of Thelazia callipaeda . J Vector Ecol. 2012 Dec;37(2):458–61. González MA, Bravo-Barriga D, Alarcón-Elbal PM, Álvarez-Calero JM, Quero C, Ferraguti M, et al. Development of Novel Management Tools for Phortica variegata (Diptera: Drosophilidae), Vector of the Oriental Eyeworm, Thelazia callipaeda (Spirurida: Thelaziidae), in Europe. Geden C, editor. J Med Entomol. 2022 Jan 12;59(1):328–36. Pombi M, Marino V, Jaenike J, Graham-Brown J, Bernardini I, Lia RP, et al. Temperature is a common climatic descriptor of lachryphagous activity period in Phortica variegata (Diptera: Drosophilidae) from multiple geographical locations. Parasit Vectors. 2020 Dec;13(1):89. Arme TM, Lia RP, Annoscia G, Casalino E, Pombi M, Otranto D. Survival of Phortica variegata experimentally and naturally infected with Thelazia callipaeda . Med Vet Entomol. 2020 Jun;34(2):201–6. Bächli G, Weber D. Taufliegen oder Kleine Fruchtfliegen (Insecta, Diptera, Drosophilidae) aus Höhlen des Grossherzogtums Luxemburg. Ferrantia. 2013;69:349–53. Beschovsky VL. Representatives of Diptera–Brachycera in the caves of Bulgaria. Académie Bulg Sci Bull L’Institut Zool Mus. 1972;35:23–9. Casiraghi M, Bain O, Guerrero R, Martin C, Pocacqua V, Gardner SL, et al. Mapping the presence of Wolbachia pipientis on the phylogeny of filarial nematodes: evidence for symbiont loss during evolution. Int J Parasitol. 2004;34(2):191–203. Manoj RRS, Latrofa MS, Epis S, Otranto D. Wolbachia: endosymbiont of onchocercid nematodes and their vectors. Parasit Vectors. 2021 May 7;14(1):245. Máca J. Amiota Loew (Diptera: Drosophilidae, Steganinae). Biologica (Santiago). 2003;47:247–74. Lunt DH, Zhang D ‐X., Szymura JM, Hewltt OM. The insect cytochrome oxidase I gene: evolutionary patterns and conserved primers for phylogenetic studies. Insect Mol Biol. 1996 Aug;5(3):153–65. Technelysium Pty Ltd. Chromas version 2.6.6 [Internet]. 2024 [cited 2024 Jun 9]. Available from: https://technelysium.com.au/wp/chromas/ Tamura K, Stecher G, Kumar S. MEGA11: molecular evolutionary genetics analysis version 11. Mol Biol Evol. 2021;38(7):3022–7. BLAST: Basic Local Alignment Search Tool [Internet]. [cited 2024 Jun 9]. Available from: https://blast.ncbi.nlm.nih.gov/Blast.cgi Casiraghi M, Anderson TJC, Bandi C, Bazzocchi C, Genchi C. A phylogenetic analysis of filarial nematodes: comparison with the phylogeny of Wolbachia endosymbionts. Parasitology. 2001;122(1):93–103. Zhou W, Rousset F, O’Neill S. Phylogeny and PCR–based classification of Wolbachia strains using wsp gene sequences. Proc R Soc Lond B Biol Sci. 1998 Mar 22;265(1395):509–15. Coretta S. tidygam: Tidy prediction and plotting of Generalised Additive Models. R Package Version 02 0. 2023; Wood SN. Inference and computation with generalized additive models and their extensions. TEST. 2020 Jun;29(2):307–39. Checklist [Internet]. LifeWatch Italy. [cited 2024 Jul 8]. Available from: https://www.lifewatchitaly.eu/en/initiatives/checklist-fauna-italia-en/checklist/ McCabe EA, Unfried LN, Teets NM. Survival and nutritional requirements for overwintering Drosophila suzukii (Diptera: Drosophilidae) in Kentucky. Lehmann P, editor. Environ Entomol. 2023 Dec 15;52(6):1071–81. Jones LE, Grimaldi DA. Revision of the Nearctic species of the genus Amiota Loew (Diptera: Drosophilidae). Bull Am Mus Nat Hist. 2022;458(1):1–177. Marino V, Gálvez R, Colella V, Sarquis J, Checa R, Montoya A, et al. Detection of Thelazia callipaeda in Phortica variegata and spread of canine thelaziosis to new areas in Spain. Parasit Vectors. 2018 Dec;11(1):195. Roggero C, Schaffner F, Bächli G, Mathis A, Schnyder M. Survey of Phortica drosophilid flies within and outside of a recently identified transmission area of the eye worm Thelazia callipaeda in Switzerland. Vet Parasitol. 2010;171(1–2):58–67. Ito F, Awasaki T. Comparative analysis of temperature preference behavior and effects of temperature on daily behavior in 11 Drosophila species. Sci Rep. 2022;12(1):12692. Bächli G, Schatzmann E, Haring E. On some population parameters of drosophilids in Switzerland (Diptera, Drosophilidae). 2008 [cited 2024 Jan 12]; Available from: https://www.zora.uzh.ch/id/eprint/14220 Masanori J. Toda. Vertical Microdistribution of Drosophilidae (Diptera) within various Forests in Hokkaido.: I. Natural Broad-Leaved Forest. Jpn J Ecol. 1977;27(3):207–14. Morgado ACT, do Vale B, Ribeiro P, Coutinho T, Santos-Silva S, de Sousa Moreira A, et al. First report of human Thelazia callipaeda infection in Portugal. Acta Trop. 2022;231:106436. Bezerra-Santos MA, Bernardini I, Lia RP, Mendoza-Roldan JA, Beugnet F, Pombi M, et al. Phortica oldenbergi (Diptera: Drosophilidae): A new potential vector of the zoonotic Thelazia callipaeda eyeworm. Acta Trop. 2022;233:106565. Shropshire JD, Leigh B, Bordenstein SR. Symbiont-mediated cytoplasmic incompatibility: what have we learned in 50 years? Elife. 2020;9:e61989. Ote M, Yamamoto D. Impact of Wolbachia infection on Drosophila female germline stem cells. Curr Opin Insect Sci. 2020;37:8–15. Sanaei E, Charlat S, Engelstädter J. Wolbachia host shifts: routes, mechanisms, constraints and evolutionary consequences. Biol Rev. 2021 Apr;96(2):433–53. Additional Declarations No competing interests reported. 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14:42:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5004631/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5004631/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13071-024-06526-9","type":"published","date":"2024-11-06T15:57:04+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":65893872,"identity":"16eb7678-38af-4696-b012-77b72898b8c3","added_by":"auto","created_at":"2024-10-04 06:02:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":894746,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003ePhortica oldenbergi\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e females’ population\u003c/strong\u003e \u003cstrong\u003edynamics (GAM-1).\u003c/strong\u003e Predicted log-CPUE of females collected with fruit bait in relation to the sampled months (a), temperature (b) and wind speed (c).\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-5004631/v1/56c945668991fa48aceace88.png"},{"id":65893870,"identity":"3666ae79-2660-43fa-b0d4-9e79bee2ae3d","added_by":"auto","created_at":"2024-10-04 06:02:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":843398,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003ePhortica variegata\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e females’ population\u003c/strong\u003e \u003cstrong\u003edynamics (GAM-2). \u003c/strong\u003ePredicted log-CPUE of females collected with fruit bait in relation to the sampled months (a), temperature (b) and wind speed (c).\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-5004631/v1/52b456f59694c5918adb8894.png"},{"id":65894568,"identity":"8907bd84-0997-4d6e-aa09-3aa31a13b1d6","added_by":"auto","created_at":"2024-10-04 06:10:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":634838,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003ePhortica variegata\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e males and females’ population dynamics (GAM-2 and GAM-3)\u003c/strong\u003e. Predicted log-CPUE for \u003cem\u003eP. variegata \u003c/em\u003emale (green) collected with human bait and females (red) collected with fruit bait in the sampled months.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-5004631/v1/0390e0e6f529abf61e5500b6.png"},{"id":65893869,"identity":"de8b45a8-ca00-4ae4-9a4b-22e52ee5ff77","added_by":"auto","created_at":"2024-10-04 06:02:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":535089,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFruit bait-collected \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePhortica variegata\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e population dynamics (GAM-4)\u003c/strong\u003e. Predicted log-CPUE for fruits-collected \u003cem\u003eP. variegata \u003c/em\u003emale (green) and females* (red) in the sampled months. *p-value 0.02\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-5004631/v1/0034505326a38e87496e5ac8.png"},{"id":68749815,"identity":"c620b08a-55f7-4396-8684-ebfffe4f4cfe","added_by":"auto","created_at":"2024-11-11 16:05:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4493128,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5004631/v1/f91bf3e0-9e90-467b-9d70-3120bdccd3c5.pdf"},{"id":65894569,"identity":"a4f5b432-db05-47cb-837b-805e6e29869f","added_by":"auto","created_at":"2024-10-04 06:10:53","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1416546,"visible":true,"origin":"","legend":"","description":"","filename":"SupportinginformationBernardiniPoggiPhortica.docx","url":"https://assets-eu.researchsquare.com/files/rs-5004631/v1/1faca4cc96572a8f997fe206.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Population dynamics of sympatric Phortica spp. and first record of stable presence of Phortica oldenbergi in a Thelazia callipaeda-endemic area of Italy","fulltext":[{"header":"Background","content":"\u003cp\u003eThe genus \u003cem\u003ePhortica\u003c/em\u003e Schiner (1862) (Diptera, Drosophilidae, Steganinae) counts almost 160 species distributed all over the world (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Nearly two-thirds of these are described in East Asia, especially in southern China. The importance of this taxonomic group in medical and veterinary entomology is raising due to their role as vectors of the eyeworm \u003cem\u003eThelazia callipaeda\u003c/em\u003e (Railliet and Henry, 1910) (Spirurida, Thelaziidae) eyeworm, which causes infection in many mammal species (\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Indeed, while feeding around animal secretions, lachryphagous males of \u003cem\u003ePhortica okadai\u003c/em\u003e (M\u0026aacute;ca,1977) and \u003cem\u003ePhortica variegata\u003c/em\u003e (F\u0026agrave;llen,1823) transmit this eyeworm to definitive hosts such as domestic mammals, including dogs, cats, and rabbits, wildlife (e.g. foxes, hares, wolfs, mustelids, bears), and occasionally humans (\u003cspan additionalcitationids=\"CR6 CR7 CR8 CR9 CR10 CR11 CR12 CR13\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Recently, \u003cem\u003ePhortica kappa\u003c/em\u003e (M\u0026aacute;ca, 1977) and \u003cem\u003ePhortica magna\u003c/em\u003e (Okada, 1960) were also described as vectors (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Adult worms develop in the conjunctival sac of the mammals\u0026rsquo; hosts causing mild to severe outcomes such as conjunctivitis, keratitis, corneal ulcer, and blindless if not cured (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Thelaziosis has been historically considered endemic in Asia and for twenty years it has been spreading across Europe (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e) and, recently, in United States of America (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). However, except for \u003cem\u003eP. variegata\u003c/em\u003e, in Europe and the US (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e), little is known about the distribution, ecology, and biology of the other \u003cem\u003eT. callipaeda\u003c/em\u003e vectors. In addition to \u003cem\u003eP. variegata\u003c/em\u003e, four more \u003cem\u003ePhortica\u003c/em\u003e species have been reported in Europe and Near East: \u003cem\u003ePhortica semivirgo\u003c/em\u003e (M\u0026aacute;ca,1977), which is also widely distributed (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e), \u003cem\u003ePhortica erinacea\u003c/em\u003e (M\u0026aacute;ca,1977) and \u003cem\u003ePhortica goetzi\u003c/em\u003e (M\u0026aacute;ca,1987), which have been sporadically reported in Bulgaria/Greece and Turkey, respectively, and \u003cem\u003eP. oldenbergi\u003c/em\u003e (Duda, 1921), of which only three specimens were collected in Germany at the beginning of the 20th century (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e) and recently found in Spain (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e), despite this record requires further investigations as it is based only on the morphology of females and no molecular data are available.\u003c/p\u003e \u003cp\u003eFor \u003cem\u003eP. variegata\u003c/em\u003e, some information on its life history traits is available, and it has been reconstructed from laboratory breeding (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e) and field sampling. This species is associated with oak forests (\u003cem\u003eQuercus\u003c/em\u003e spp.) (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e) where flies are found active from April-May to October-November (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). After this period, an overwintering strategy at the adult stage has been presumed in \u003cem\u003eP. variegata\u003c/em\u003e and \u003cem\u003eP. semivirgo\u003c/em\u003e (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e), with some adults collected in caves during winter (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). However, even the ecology of \u003cem\u003eP. variegata\u003c/em\u003e is still under investigation, as it is still poorly known in critical aspects, such as larval ecology.\u003c/p\u003e \u003cp\u003eMoreover, no information is available about the presence and ecological contribution of endosymbionts on \u003cem\u003ePhortica\u003c/em\u003e species, despite there is evidence of the absence of \u003cem\u003eWolbachia pipientis\u003c/em\u003e (Hertig, 1936) bacteria in \u003cem\u003eT. callipaeda\u003c/em\u003e (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). \u003cem\u003eWolbachia\u003c/em\u003e is an obligate intracellular maternally transmitted gram-negative genus establishing a spectrum of symbiotic relationships, ranging from parasitism to obligatory mutualism. Up to 50% of terrestrial arthropod species and several onchocercid nematodes harbour this endosymbiont in nature, including several Drosophilidae (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, we investigated the presence and the population dynamics of \u003cem\u003ePhortica\u003c/em\u003e spp. in the ecological setting of Central Italy, Manziana, an area known for the presence of these flies and of \u003cem\u003eT. callipaeda\u003c/em\u003e (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Here we report, for the first time, the co-occurrence of four \u003cem\u003ePhortica\u003c/em\u003e species, detailed data about their ecology and phenology, and the first record of a stable population of \u003cem\u003eP. oldenbergi\u003c/em\u003e in Italy. Data about potential expansion in the distribution of these \u003cem\u003ePhortica\u003c/em\u003e spp. are also discussed in light of the risk they may represent as competent vectors of \u003cem\u003eT. callipaeda\u003c/em\u003e.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e \u003cb\u003ePhortica\u003c/b\u003e \u003cb\u003espp. sample collection and morphological identification\u003c/b\u003e\u003c/p\u003e \u003cp\u003eLongitudinal samplings were performed in the oak forest of Manziana (Rome, Italy, 42\u0026deg;07'10.8\"N \u0026minus;\u0026thinsp;12\u0026deg;06'57.6\"E) during the period April-October from 2018 to 2020.\u003c/p\u003e \u003cp\u003e \u003cem\u003ePhortica\u003c/em\u003e spp. flies were collected with a sweep net while hovering around: i) a fermenting fruit bait for flies showing a frugivorous trophic behaviour, and ii) a human operator acting as bait for lachryphagous flies (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Collected specimens were killed soon after being collected and stored in 70% ethanol in the field. After collection, specimens were morphologically identified by species and sex (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). In case of doubtful identification (N\u0026thinsp;=\u0026thinsp;95), a molecular confirmation was carried out with conventional PCR and sequencing.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePhortica spp.\u003c/b\u003e \u003cb\u003emolecular identification\u003c/b\u003e\u003c/p\u003e \u003cp\u003eGenomic DNA was isolated from single specimens using DNAzol DNA extraction protocol (MCR, Inc., Cincinnati Ohio) and then amplified with primers UEA7 (5\u0026rsquo;- TACAGTTGGAATAGACGTTGATAC-3\u0026rsquo;) and UEA10 (5\u0026rsquo;-TCCAATGCACTAATCTGCCATATTA-3\u0026rsquo;) to target the mitochondrial Cytochrome oxidase subunit I gene (\u003cem\u003ecox1\u003c/em\u003e) (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). PCR was performed in a total volume of 25\u0026micro;l, containing 1.5\u0026micro;l of DNA as a template by mixing: sterile \u003cem\u003edd\u003c/em\u003eH\u003csub\u003e2\u003c/sub\u003eO (17.00 \u0026micro;l), 2\u0026micro;l MgCl\u003csub\u003e2\u003c/sub\u003e [2.5mM], 2.5\u0026micro;l 10X reaction buffer (10X NH\u003csub\u003e4\u003c/sub\u003e, Meridian Bioscience, Inc., Cincinnati, Ohio), 0.8\u0026micro;l of dNTPs [10\u0026micro;M], 0.5\u0026micro;l of each primer [10\u0026micro;M], 0.20\u0026micro;l of Taq DNA polymerase [1u/\u0026micro;l]. PCR was performed in a thermal cycler (Bio-Rad, C1000 Touch) using the following cycling protocol: 95\u0026deg;C for 10\u0026rsquo; (polymerase activation), followed by 31 cycles of 95\u0026deg;C for 1\u0026rsquo; (denaturation), 64\u0026deg; for 45\u0026rdquo; (annealing), 72\u0026deg;C for 1\u0026rsquo; (extension), 1\u0026rsquo; at 72\u0026deg;C (final extension). The PCR 700 bp long amplified product was visualised in 1.5% agarose gel.\u003c/p\u003e \u003cp\u003ePCR products were purified with SureClean Plus (Bioline, Meridian Bioscience) and sent to the sequencing provider (Sanger sequencing method; BMR Genomics s.r.l.). All the nucleotide sequences were screened with Chromas (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e) and Mega 11 software (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Sequence species identity was confirmed in the GenBank\u003csup\u003e\u0026reg;\u003c/sup\u003e database using nucleotide Basic Local Alignment Search Tool (\u003cem\u003eBLASTn)\u003c/em\u003e (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eThelazia callipaeda\u003c/b\u003e \u003cb\u003emolecular detection\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA subsample of flies was tested for the presence of \u003cem\u003eT. callipaeda\u003c/em\u003e. The \u003cem\u003ecox1\u003c/em\u003e region (689 bp) was amplified using primers COI_F (5\u0026rsquo;-TGATTGGTGGTTTTGGTAA-3\u0026rsquo;) and COI_R (5\u0026rsquo;- ATAAGTACGAGTATCAATATC-3\u0026rsquo;) (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). PCR was performed in a total volume of 25\u0026micro;l, containing 2\u0026micro;l of DNA as a template by mixing: sterile \u003cem\u003edd\u003c/em\u003eH\u003csub\u003e2\u003c/sub\u003eO (14.65\u0026micro;l), 1.5\u0026micro;l MgCl\u003csub\u003e2\u003c/sub\u003e [2.5mM], 2.5\u0026micro;l 10X reaction buffer (10X NH\u003csub\u003e4\u003c/sub\u003e, Meridian Bioscience, Inc., Cincinnati, Ohio), 2.5\u0026micro;l of dNTPs [10\u0026micro;M], 0.8\u0026micro;l of each primer [10\u0026micro;M], 0.25\u0026micro;l of Taq DNA polymerase [1u/\u0026micro;l]. PCR was performed in a thermal cycler (Bio-Rad, C1000 Touch) using the following cycling protocol: 95\u0026deg;C for 10\u0026rsquo; (polymerase activation), followed by 35 cycles of 95\u0026deg;C for 1\u0026rsquo; (denaturation), 55\u0026deg; for 30\u0026rdquo; (annealing), 72\u0026deg;C for 1\u0026rsquo; (extension), 7\u0026rsquo; at 72\u0026deg;C (final extension). The PCR 689 bp long amplified product was visualised in 1.5% agarose gel.\u003c/p\u003e \u003cp\u003e \u003cb\u003eWolbachia endosymbiont\u003c/b\u003e \u003cb\u003emolecular detection\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe presence of the endosymbiont \u003cem\u003eWolbachia\u003c/em\u003e in the collected \u003cem\u003ePhortica\u003c/em\u003e species was verified by PCR amplifying a portion of the \u003cem\u003ewsp\u003c/em\u003e arthropod-specific gene (600 bp) (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). The amplification reaction was carried out according to the following protocol (25\u0026micro;l final volume): 2.5\u0026micro;l 10X reaction buffer (10X \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{NH}_{4}\\)\u003c/span\u003e\u003c/span\u003e, Meridian Bioscience, Inc., Cincinnati, Ohio), 2\u0026micro;l MgCl2 [2.5mM], 2.5\u0026micro;l\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\)\u003c/span\u003e\u003c/span\u003edNTPs [10\u0026micro;M], 1\u0026micro;l [10\u0026micro;M] of primers 81F (5'-TGGTCCAATAAGTGATGAAGAAAC-3') and 691R (5'-AAAAATTAAACGCTACTCCA-3'), 0.2\u0026micro;l of Taq DNA polymerase [1u/\u0026micro;l], 1 \u0026micro;l of DNA template. Amplification profile: 30 cycles at 94\u0026deg;C for 30\u0026rdquo;, 50\u0026deg;C for 30\u0026rdquo;, 72\u0026deg;C for 30\u0026rdquo;. The amplified products were sequenced (Sanger sequencing method; BMR Genomics s.r.l.) and species identity was confirmed in the GenBank\u003csup\u003e\u0026reg;\u003c/sup\u003e database using BLASTn (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eDifferent Generalized Additive Models (hereafter named GAM-1; GAM-2; GAM-3; GAM-4) were fitted to determine the effect of the environmental parameters on the abundance of the \u003cem\u003ePhortica\u003c/em\u003e spp. collected. The response variable was expressed as log-CPUE (the natural logarithm of Catch-Per-Unit-of-Effort, expressed as the number of collected flies/hour) to control for the variation in the sampling effort. The daily average of environmental data (temperature, humidity, barometric pressure, wind speed, dew point) were retrieved from the nearest weather station (IMANZ6, 42.106\u0026deg; N, 12.078\u0026deg; E; located 2km far from the sampling site; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.wunderground.com/\u003c/span\u003e\u003cspan address=\"https://www.wunderground.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The three GAMs models differ in the response variable. In GAM-1 we considered only the log-CPUE for \u003cem\u003eP. oldenbergi\u003c/em\u003e, while in the other two models (GAM-2 and GAM-3) the response variable was the log-CPUE of \u003cem\u003eP. variegata\u003c/em\u003e females (from fruit traps) and males (lacryphagous), respectively. Given the limited number of \u003cem\u003eP. semivirgo\u003c/em\u003e and a single specimen of \u003cem\u003eP. okadai\u003c/em\u003e collected during the whole sampling period, these species were excluded from the analysis. A selection of the predictors was carried out to avoid multicollinearity. In all the GAMs the parametric coefficients were sex, bait, and atmospheric pressure, while a smooth function was applied to windspeed, dew point, humidity, and temperature. A smooth function was also estimate for the date of collection (treated as day of the year) with an interaction between the factor variable bait (fruits/net). GAMs model structure was:\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:{Y}_{i}=\\:{\\alpha\\:+\\:f}_{k}\\left({X}_{ki}\\right)+{\\beta\\:}_{j}\\left({Z}_{ji}\\right)+{\\epsilon\\:}_{i}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Y}_{i}\\)\u003c/span\u003e\u003c/span\u003e is the log-CPUE, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\alpha\\:\\)\u003c/span\u003e\u003c/span\u003e is the intercept, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:f\\)\u003c/span\u003e\u003c/span\u003e is th \u003cem\u003ek\u003c/em\u003e-th smooth term for the \u003cem\u003ek\u003c/em\u003e-th non-linear predictor (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:f\\)\u003c/span\u003e\u003c/span\u003e), \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\beta\\:\\)\u003c/span\u003e\u003c/span\u003e is th \u003cem\u003ej\u003c/em\u003e-th fixed effect for the \u003cem\u003ej\u003c/em\u003e-th linear predictor (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:Z\\)\u003c/span\u003e\u003c/span\u003e), and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\epsilon\\:\\)\u003c/span\u003e\u003c/span\u003e is the error term for the \u003cem\u003ei\u003c/em\u003e-th observation. To test if there was a significant difference in the mean number of \u003cem\u003eP. variegata\u003c/em\u003e flies collected on fruit bait, another GAM model (GAM-4) was fitted on the log-CPUE of flies collected with fruit bait. All statistical analyses were performed with R software (version 4.3.1) and the packages: \u003cem\u003emgcv and tidygam\u003c/em\u003e (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eIn total 5,564 \u003cem\u003ePhortica spp\u003c/em\u003e. flies were collected from 2018 to 2020 (N\u0026thinsp;=\u0026thinsp;52 sampling days) in the area and morphologically identified. The most abundant species was \u003cem\u003eP. variegata\u003c/em\u003e (90.8%), followed by \u003cem\u003eP. oldenbergi\u003c/em\u003e (9.1%; Supplementary Figure S2). A total of three specimens of \u003cem\u003eP. semivirgo\u003c/em\u003e (0.05%) were collected in 2018 (N\u0026thinsp;=\u0026thinsp;1, by human bait) and 2020 (N\u0026thinsp;=\u0026thinsp;2, by fruit bait) while a single specimen of \u003cem\u003eP. okadai\u003c/em\u003e was collected in 2018 (0.02%, by human bait). The numbers \u003cem\u003eP. variegata\u003c/em\u003e and \u003cem\u003eP. oldenbergi\u003c/em\u003e collected per month, year and sampling bait are reported in Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eNumbers of \u003cem\u003ePhortica variegata\u003c/em\u003e collected during the sampling period.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYear\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMonth\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN Fruit-bait (%males)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN Human-bait (%males)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2018\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eApril\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22 (13.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMay\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e262 (17.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20 (100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eJune\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e89 (28.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eJuly\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e148 (43.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8 (100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAugust\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e42 (40.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2329 (100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSeptember\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e94 (43.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e394 (100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOctober\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10 (100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3419\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2019\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eApril\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMay\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eJune\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22 (27.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11 (100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eJuly\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18 (5.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e749 (100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAugust\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e173 (100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSeptember\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3 (66.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e98 (100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1079\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2020\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMay\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16 (100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eJune\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11 (100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eJuly\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e53 (100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAugust\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e41 (80.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e213 (99.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSeptember\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e151 (99.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOctober\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e26 (100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e556\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5054\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eNumbers of\u003c/b\u003e \u003cb\u003ePhortica oldenbergi\u003c/b\u003e \u003cb\u003ecollected during the sampling period.\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYear\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMonth\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN Fruit-bait (%males)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e \u003cp\u003eN Human-bait (%males)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2018\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eApril\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMay\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e189 (4.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eJune\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24 (4.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eJuly\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e31 (22.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAugust\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9 (22.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSeptember\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e123 (4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e \u003cp\u003e1 (100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOctober\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e \u003cp\u003e1 (100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e398\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2019\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eApril\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMay\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eJune\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e \u003cp\u003e1 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eJuly\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e \u003cp\u003e1 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAugust\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSeptember\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e \u003cp\u003e1 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOctober\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2020\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMay\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eJune\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eJuly\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e32 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e \u003cp\u003e2 (100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAugust\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSeptember\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOctober\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e76\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e506\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eBLASTn sequence analysis of the \u003cem\u003ecox1\u003c/em\u003e gene (N\u0026thinsp;=\u0026thinsp;95) confirmed the morphological identification as \u003cem\u003eP. variegata\u003c/em\u003e (N\u0026thinsp;=\u0026thinsp;84; 99.7% of nucleotide identity (ni); accession number PP990198), \u003cem\u003eP. semivirgo\u003c/em\u003e (N\u0026thinsp;=\u0026thinsp;1; 99.1% ni; accession number PP990202) and \u003cem\u003eP. okadai\u003c/em\u003e (N\u0026thinsp;=\u0026thinsp;1, 99.8% ni; accession number PP860587). Sequence analysis of \u003cem\u003eP. oldenbergi\u003c/em\u003e (N\u0026thinsp;=\u0026thinsp;9; accession number PP838740; Supplementary Text S3) showed a similar nucleotide identity with \u003cem\u003eP. variegata\u003c/em\u003e (95.5% ni), \u003cem\u003eP. okadai\u003c/em\u003e (95.1% ni) and \u003cem\u003eP. semivirgo\u003c/em\u003e (94.7% ni) as sequences for this species were not available in GenBank. A total of 101 individuals (N\u0026thinsp;=\u0026thinsp;74 \u003cem\u003eP. variegata\u003c/em\u003e, N\u0026thinsp;=\u0026thinsp;20 \u003cem\u003eP. oldenbergi\u003c/em\u003e, N\u0026thinsp;=\u0026thinsp;3 \u003cem\u003eP. semivirgo\u003c/em\u003e, N\u0026thinsp;=\u0026thinsp;1 \u003cem\u003eP. okadai\u003c/em\u003e) were analysed for the presence of \u003cem\u003eWolbachia\u003c/em\u003e. All \u003cem\u003eP. oldenbergi\u003c/em\u003e and the \u003cem\u003eP. okadai\u003c/em\u003e specimens examined scored positive for a fragment of the expected size whereas \u003cem\u003eP. variegata\u003c/em\u003e and \u003cem\u003eP. semivirgo\u003c/em\u003e were negative (Supplementary file, Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). All sequences were identical and were confirmed as belonging to \u003cem\u003eWolbachia\u003c/em\u003e strain A (100% identity with \u003cem\u003eWolbachia\u003c/em\u003e endosymbiont (group A) of \u003cem\u003eMelieria omissa\u003c/em\u003e; accession numbers PP930348 and PP930349).\u003c/p\u003e \u003cp\u003e \u003cem\u003eThelazia callipaeda\u003c/em\u003e DNA was detected in the \u003cem\u003eP. variegata\u003c/em\u003e subsample (N\u0026thinsp;=\u0026thinsp;699) showing different infection rates: 1.26% in 2018 (N\u0026thinsp;=\u0026thinsp;352), 1% in 2019 (N\u0026thinsp;=\u0026thinsp;100) and 2.02% in 2020 (N\u0026thinsp;=\u0026thinsp;247), whereas any \u003cem\u003eP. oldenbergi\u003c/em\u003e, \u003cem\u003eP. semivirgo\u003c/em\u003e, and the single \u003cem\u003eP. okadai\u003c/em\u003e were positive.\u003c/p\u003e \u003cp\u003eThe sex ratio of both \u003cem\u003eP. variegata\u003c/em\u003e and \u003cem\u003eP. oldenbergi\u003c/em\u003e collected each year was unbalanced (Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), The male: female ratio for \u003cem\u003eP. variegata\u003c/em\u003e was 6.7:1 in 2018, 24.7:1 in 2019, and 9.1:1 in 2020, while for \u003cem\u003eP. oldenbergi\u003c/em\u003e, this ratio was 0.07:1 in 2018, 0:1 in 2019, and 0.03:1 in 2020. The sex ratio of flies collected on human bait substantially differed from those gathered on fruit \u0026ndash; see the next paragraph.\u003c/p\u003e \u003cp\u003eThe results of the GAMs fitted on the log-CPUE of \u003cem\u003ePhortica\u003c/em\u003e spp. showed a significant effect of the day of the year, temperature and windspeed on the mean number of flies collected (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In all models the parametric coefficient showed a significant negative effect of barometric pressure (P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and no significant effects of dew point and humidity. The seasonal dynamics of \u003cem\u003eP. oldenbergi\u003c/em\u003e showed a first peak of abundance in May-June, followed by a decrease and a second (lower) peak of September-October (GAM-1; adj. R-sq 0.46; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Similarly, \u003cem\u003eP. variegata\u003c/em\u003e females were mainly abundant in the first part of the season (May- June) but showed a rapid decrease in June and a plateau until the end of sampling (GAM-2; adj. R-sq. 0.62; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Conversely, numbers of \u003cem\u003eP. variegata\u003c/em\u003e males (on human bait) rapidly increased in mid-June with a peak of abundance in August-September, then slightly decreased reaching a plateau until the end of sampling (GAM-3; R-sq. adj 0.48; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). It needs to be clarified whether sex-ratio or just a food preference is involved. Only \u003cem\u003eP. variegata\u003c/em\u003e females collected on the fruit bait showed a significant trend (GAM-4; R-sq. adj 0.30; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Single specimes of \u003cem\u003eP. oldenbergi\u003c/em\u003e collected on human bait showed subequal number of males and females, with no seasonal trend detectable (also here, the result is different from the sex ratio based mainly on the data from fruit bait).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study showed for the first time the occurrence and seasonal dynamics of \u003cem\u003ePhortica\u003c/em\u003e species living in sympatry within a forested area of central Italy, where \u003cem\u003eT. callipaeda\u003c/em\u003e circulates. \u003cem\u003ePhortica variegata\u003c/em\u003e was the most abundant species, representing 90% of the collected \u003cem\u003ePhortica\u003c/em\u003e flies, and it was the only species found positive for \u003cem\u003eT. callipeda\u003c/em\u003e, with infection rates ranging between 1% and 2.2%. Among the less abundant species, \u003cem\u003eP. oldenbergi\u003c/em\u003e, a newly recorded species in this geographic area, was collected throughout the sampling period, indicating its establishment in Central Italy for the first time. \u003cem\u003ePhortica semivirgo\u003c/em\u003e, on the other hand, is considered a species native of Italian entomofauna (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e), which was sporadically observed in this area. Although both \u003cem\u003eP. semivirgo\u003c/em\u003e and \u003cem\u003eP. oldenbergi\u003c/em\u003e tested negative for the presence of \u003cem\u003eT. callipaeda\u003c/em\u003e during the whole study period, considering the low number of specimens collected in this study it still cannot be excluded that they could act as vectors in different ecological contexts. Finally, \u003cem\u003eP. okadai\u003c/em\u003e, a major vector of \u003cem\u003eT. callipaeda\u003c/em\u003e in the Asian region (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) was surprisingly recorded in the study area. The finding of a single specimen of \u003cem\u003eP. okadai\u003c/em\u003e suggests a possible accidental introduction by fruit trading. Further sampling in surrounding areas should be performed to increase information about the possible wider presence of \u003cem\u003eP. okadai\u003c/em\u003e at least in Central Italy, which would be of paramount relevance for \u003cem\u003eT. callipaeda\u003c/em\u003e epidemiology, considering the major role of this vector in its original areal.\u003c/p\u003e \u003cp\u003eThe oak forest of Manziana gave us the unique opportunity to study the population dynamics and feeding behaviour of the taxa of the \u003cem\u003ePhortica\u003c/em\u003e genus simultaneously. Population dynamics of all \u003cem\u003ePhortica spp\u003c/em\u003e. showed a peak of abundance of females in May, possibly indicating a seasonal phase when the flies are more focused on the feeding activity to restore enough energy for reproduction after overwintering. However, a second low peak in abundance was observed in \u003cem\u003eP. oldenbergi\u003c/em\u003e. Although not significant, this small increase in abundance might be indicative as a preparation time to obtain energy reserves in the late season before the arrival of the winter, as described in other Drosophilidae (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe sex ratio was highly unbalanced in the studied species. Most \u003cem\u003eP. oldenbergi\u003c/em\u003e flies collected were females caught with fruit baits, thus suggesting some connection to fermenting fruit, as observed in other \u003cem\u003ePhortica\u003c/em\u003e species. This does not necessarily mean frugivory \u0026ndash; adults probably, like many \u003cem\u003eDrosophila\u003c/em\u003e spp., consume yeasts and other microorganisms and/or seek an environment for their possibly predatory larvae (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Previous studies of various Steganinae flies have consistently reported a higher capture rate in traps baited with fermenting fruits near tree canopy (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). This applies well to \u003cem\u003eP. oldenbergi\u003c/em\u003e, although a few specimens of both sexes of \u003cem\u003eP. oldenbergi\u003c/em\u003e were caught with human bait, indicating that lachryphagy can occur in this species, akin to other \u003cem\u003ePhortica\u003c/em\u003e spp. (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). Again, the low numbers of males collected in the study hampered the possibility to better clarify the feeding behaviour of \u003cem\u003eP. oldenbergi\u003c/em\u003e males. Similarly, during this sampling only a few specimens of \u003cem\u003eP. semivirgo\u003c/em\u003e males were collected using fermenting fruit and human bait, yielding inconclusive results. Concerning the most common species \u003cem\u003eP. variegata\u003c/em\u003e, most specimens were males collected with human bait, consistent with prior literature (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e) with their numbers rapidly increasing and peaking in August.\u003c/p\u003e \u003cp\u003eAccording to previous findings (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e), and consistently with this study, temperature constitutes a pivotal driver affecting the lachryphagous feeding behaviour of \u003cem\u003eP. variegata\u003c/em\u003e males. However, a significant effect of the temperature on the mean abundance of \u003cem\u003ePhortica\u003c/em\u003e flies on fruit bait was also observed, indicating its involvement in this feeding activity. A plateau of the abundance of both fruit baited \u003cem\u003ePhortica\u003c/em\u003e spp. was observed between 21\u0026deg;C and 29\u0026deg;C, suggesting that at higher temperatures of the day (hottest days and hours of the year) flies are less active. This result was corroborated by the significant negative effect of the parametric coefficient of the pressure detected from all models. Indeed, high atmospheric pressure results in the absence of cloud coverage and thus increase of temperatures with a consequent decrease in flies\u0026rsquo; abundance. Our findings agree with the previous study on the flying activity of drosophilid flies, highlighting the general avoidance of several \u003cem\u003eDrosophila\u003c/em\u003e spp. for higher temperatures and a species-specific thermopreferences affecting the range of total daily activity of these flies (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). In addition, the models confirmed the negative effect of the wind speed on the mean abundance of collected flies, which can be reasonably explained by the higher energy required for flies for dispersal.\u003c/p\u003e \u003cp\u003eThe low numbers of \u003cem\u003eP. semivirgo\u003c/em\u003e collected are consistent with the available literature. This species was unknown to science until 1977, and only sporadically reported in Europe, except for Switzerland, where a trapping bottle system was used to collect the flies (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). Nowadays, \u003cem\u003eP. semivirgo\u003c/em\u003e seems more common, at least in central Europe, although the cases when it is collected in absence of \u003cem\u003eP. variegata\u003c/em\u003e are rare (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). It is then possible that the abundance of this species was underestimated in this study due to a less efficient trapping approach. Another possible explanation for the low density of \u003cem\u003eP. semivirgo\u003c/em\u003e in the collection could be the different vertical micro-distribution in the canopy of \u003cem\u003ePhortica\u003c/em\u003e spp. living in sympatric conditions, as indicated in the literature (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan additionalcitationids=\"CR51\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e), leading to the hypothesis that \u003cem\u003eP. semivirgo\u003c/em\u003e may exploit upper levels of oak trees as a preferential habitat, while being less represented at ground level, where our sampling occurred.\u003c/p\u003e \u003cp\u003eThe rediscovery of a \u003cem\u003eP. oldenbergi\u003c/em\u003e population in Europe is very surprising. In fact, before this record, the species was documented in Europe, with only tree specimens of \u003cem\u003eP. oldenbergi\u003c/em\u003e collected and described in Germany in the early 20th century. Following this, six more female specimens were reported from Spain roughly one hundred years later (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). This led to the hypothesis that this species was accidentally introduced in Europe at the time (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e) but could not settle in. Indeed, the species belongs (or is close) to the \u003cem\u003eAllophortica\u003c/em\u003e subgenus, of which another four described species are known from the Afrotropical region (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). The rare and patchy presence of this species in Europe might be explained by two hypotheses, both yet to be demonstrated: i) it is a relict areal of its original distribution or ii) its occurrence is the consequence of new introduction events. However, the recent study indicating this species as a competent vector of \u003cem\u003eT. callipaeda\u003c/em\u003e in laboratory conditions (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e) highlights the need for further investigations to determine the vector capacity of this species in nature.\u003c/p\u003e \u003cp\u003eThe PCR amplification of a fragment of the \u003cem\u003ewsp\u003c/em\u003e gene in the collected specimens suggests the presence of \u003cem\u003eWolbachia\u003c/em\u003e, belonging to the strain typical of arthropods, in both \u003cem\u003eP. oldenbergi\u003c/em\u003e and \u003cem\u003eP. okadai\u003c/em\u003e. At the same time, the symbiont seems to be absent in \u003cem\u003eP. variegata\u003c/em\u003e and \u003cem\u003eP. semivirgo\u003c/em\u003e. To the best of our knowledge, none of the \u003cem\u003ePhortica\u003c/em\u003e species have ever been screened for the presence of this symbiont. Therefore, detecting of \u003cem\u003eWolbachia\u003c/em\u003e in \u003cem\u003eP. oldenbergi\u003c/em\u003e and \u003cem\u003eP. okadai\u003c/em\u003e is the first indicator of the symbiotic relationship this endosymbiont established with flies within the \u003cem\u003ePhortica\u003c/em\u003e genus.\u003c/p\u003e \u003cp\u003e \u003cem\u003eWolbachia\u003c/em\u003e is a widespread endosymbiotic bacterium known for its intricate relationships with various insect hosts, including drosophilids. In this family \u003cem\u003eWolbachia\u003c/em\u003e exhibits mutualistic and parasitic interactions, depending on the strain and host species. One of the most notable effects of \u003cem\u003eWolbachia\u003c/em\u003e in Diptera is cytoplasmic incompatibility, which can enhance the bacterium\u0026rsquo;s spread by favouring the reproduction of infected females over uninfected ones (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e). This complex interaction highlights \u003cem\u003eWolbachia\u003c/em\u003e's significant role in its hosts' evolution and ecology, influencing population dynamics and genetic diversity (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e). Based on our results, assuming that this symbiont is present in only some \u003cem\u003ePhortica\u003c/em\u003e species, it is not trivial to hypothesise the most probable mechanism for the introduction of \u003cem\u003eWolbachia\u003c/em\u003e in \u003cem\u003eP. oldenbergi\u003c/em\u003e and \u003cem\u003eP. okadai\u003c/em\u003e. The routes exploited by \u003cem\u003eWolbachia\u003c/em\u003e to be horizontally transferred between species are heterogeneous, including the sharing of food source in the environment (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e). This might represent the most probable mechanism of introduction in the two \u003cem\u003ePhortica\u003c/em\u003e species, which are known to also show affinity to fruit bait.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study elucidates the ecological aspects of sympatric \u003cem\u003ePhortica\u003c/em\u003e species within a forest in Central Italy, offering unprecedented insights into \u003cem\u003eP. variegata\u003c/em\u003e and \u003cem\u003eP. oldenbergi\u003c/em\u003e biology and ecology. The extensive collection and identification of these species underscore their seasonal variation of abundance and potential roles within the ecosystem, unravelling the complex interplay between environmental drivers, feeding activity and vector behaviour, especially regarding the transmission of the eyeworm \u003cem\u003eT. callipaeda\u003c/em\u003e. In particular, the rediscovery and establishment of \u003cem\u003eP. oldenbergi\u003c/em\u003e in Europe pose intriguing questions about its geographical origin, ecological role, and potential as a vector of \u003cem\u003eT. callipaeda\u003c/em\u003e, deserving further investigation. Similarly, the sporadic presence of \u003cem\u003eP. semivirgo\u003c/em\u003e and the enigmatic observation of a single specimen of \u003cem\u003eP. okadai\u003c/em\u003e in the Manziana area highlight the complexities of species establishment in new environments, involving possible use of fruit trading. Further studies are imperative to explore the vectorial capacity of these species in natural settings, assess the impact of environmental changes on their populations, and develop strategies for monitoring and controlling their spread in relation to \u003cem\u003eT. callipaeda\u003c/em\u003e transmission to animals and humans.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eGAM\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGeneralized Additive Model\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eCPUE\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCatch-Per-Unit-of-Effort\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgments\u003c/p\u003e\n\u003cp\u003eAuthors would like to thank Federica Laterza, Federica Furzi and Eugenio Gabrieli for the support in sampling collection and laboratory analyses, and Prof. John Jaenike for the insightful discussions about \u003cem\u003ePhortica\u003c/em\u003e ecology.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMP, FB, JF, RPL, JM and DO conceived the study. MP, IB, CP and DO organized the sampling plan. MP, IB, CP and SM collected data in the field. MP, IB, CP, EP, SG, VP, DP, MSL performed laboratory and molecular analyses of the specimens. IB, CP, MP analysed the results. IB, CP and MP drafted the manuscript, and all authors critically contributed to its final version. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData supporting the conclusions of this article are included within the article and its additional files. The datasets used and analysed during the present\u003c/p\u003e\n\u003cp\u003estudy are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGlobal Biodiversity Information Facility (GBIF) [Internet]. [cited 2024 Jun 9]. Available from: https://www.gbif.org/\u003c/li\u003e\n\u003cli\u003eJin Y, Liu Z, Wei J, Wen Y, He N, Tang L, et al. A first report of Thelazia callipaeda infection in Phortica okadai and wildlife in national nature reserves in China. Parasit Vectors. 2021 Dec;14(1):13. \u003c/li\u003e\n\u003cli\u003eM\u0026aacute;ca J, Otranto D. Drosophilidae feeding on animals and the inherent mystery of their parasitism. Parasit Vectors. Nov 2014 (7): 1-8. https://doi.org/10.1186/s13071-014-0516-4\u003c/li\u003e\n\u003cli\u003eOtranto D, Lia RP, Cantacessi C, Testini G, Troccoli A, Shen JL, et al. Nematode biology and larval development of Thelazia callipaeda (Spirurida, Thelaziidae) in the drosophilid intermediate host in Europe and China. Parasitology. 2005 Jul 22;131(06):847. \u003c/li\u003e\n\u003cli\u003eBojan G, Vanja BS, Aleksandra P, Milica K, Neda B, Du\u0026scaron;ko Ć. First report of eyeworm infection by Thelazia callipaeda in gray wolf (Canis lupus) from Serbia. Parasitol Res. 2019 Dec;118(12):3549\u0026ndash;53. \u003c/li\u003e\n\u003cli\u003eDeak G, Ionică AM, Mihalca AD. First report of Thelazia callipaeda Railliet \u0026amp; Henry, 1910 in a zoo lion (Panthera leo) (Linnaeus, 1758) from Romania. Vet Res Commun. 2023 Dec;47(4):2173\u0026ndash;7. \u003c/li\u003e\n\u003cli\u003eN\u0026aacute;jera F, De Lucas-Veguillas J, Vela \u0026Aacute;, L\u0026oacute;pez-Fern\u0026aacute;ndez M, Mart\u0026iacute;nez-Mart\u0026iacute;nez P, Mata-Huete M, et al. First report of Thelazia callipaeda in a free-ranging Iberian wolf (Canis lupus signatus) from Spain. Parasitol Res. 2020 Jul;119(7):2347\u0026ndash;50. \u003c/li\u003e\n\u003cli\u003eOtranto D, Brianti E, Cantacessi C, Lia RP, M\u0026aacute;ca J. The zoophilic fruitfly \u003cem\u003ePhortica variegata\u003c/em\u003e : morphology, ecology and biological niche. Med Vet Entomol. 2006 Dec;20(4):358\u0026ndash;64. \u003c/li\u003e\n\u003cli\u003eOtranto D, Cantacessi C, Testini G, Lia RP. Phortica variegata as an intermediate host of Thelazia callipaeda under natural conditions: Evidence for pathogen transmission by a male arthropod vector. Int J Parasitol. 2006 Sep;36(10\u0026ndash;11):1167\u0026ndash;73. \u003c/li\u003e\n\u003cli\u003eOtranto D, Cantacessi C, Mallia E, Lia RP. First report of Thelazia callipaeda (Spirurida, Thelaziidae) in wolves in Italy. J Wildl Dis. 2007;43(3):508\u0026ndash;11. \u003c/li\u003e\n\u003cli\u003eOtranto D, Dantas-Torres F, Mallia E, DiGeronimo PM, Brianti E, Testini G, et al. Thelazia callipaeda (Spirurida, Thelaziidae) in wild animals: Report of new host species and ecological implications. Vet Parasitol. 2009 Dec;166(3\u0026ndash;4):262\u0026ndash;7. \u003c/li\u003e\n\u003cli\u003eOtranto D, Dutto M. Human Thelaziasis, Europe. Emerg Infect Dis. 2008 Apr;14(4):647\u0026ndash;9. \u003c/li\u003e\n\u003cli\u003ePapadopoulos E, Komnenou A, Karamanlidis AA, Bezerra‐Santos MA, Otranto D. Zoonotic \u003cem\u003eThelazia callipaeda\u003c/em\u003e eyeworm in brown bears ( \u003cem\u003eUrsus arctos\u003c/em\u003e ): A new host record in Europe. Transbound Emerg Dis. 2022 Mar;69(2):235\u0026ndash;9. \u003c/li\u003e\n\u003cli\u003eParadžik MT, Samardžić K, Živičnjak T, Martinković F, Janjetović Ž, Miletić-Medved M. Thelazia callipaeda\u0026mdash;first human case of thelaziosis in Croatia. Wien Klin Wochenschr. 2016 Mar;128(5\u0026ndash;6):221\u0026ndash;3. \u003c/li\u003e\n\u003cli\u003eKitajima A, Tokiwa T, Doi K, Kotani K, Otsubo H, Kamei C, et al. New host record of Thelazia callipaeda (Nematoda: Spirurida) with a notably wide host range and shared zoonotic lineage in Japan. Parasitol Int. 2024;102913. \u003c/li\u003e\n\u003cli\u003eDo Vale B, Lopes AP, Da Concei\u0026ccedil;\u0026atilde;o Fontes M, Silvestre M, Cardoso L, Coelho AC. Thelaziosis due to Thelazia callipaeda in Europe in the 21st century\u0026mdash;A review. Vet Parasitol. 2019 Nov;275:108957. \u003c/li\u003e\n\u003cli\u003eMart\u0026iacute;nez-S\u0026aacute;nchez MI, Bol\u0026iacute;var-de-Miguel G, Cuadros-Gonz\u0026aacute;lez J, Gonz\u0026aacute;lez JMR. Ocular thelaziosis: a case report of an emerging zoonosis. Am J Ophthalmol Case Rep. 2021;22:101045. \u003c/li\u003e\n\u003cli\u003eOtranto D, Mendoza-Roldan JA, Dantas-Torres F. Thelazia callipaeda. 2021 [cited 2024 Jul 16]; Available from: https://www.arca.fiocruz.br/handle/icict/50732\u003c/li\u003e\n\u003cli\u003eManoj RR, White H, Young R, Brown CE, Wilcox R, Otranto D, et al. Emergence of Thelaziosis Caused by Thelazia callipaeda in Dogs and Cats, United States. Emerg Infect Dis. 2024;30(3):591. \u003c/li\u003e\n\u003cli\u003eOtranto D, Iatta R, Lia RP, Cavalera MA, M\u0026agrave;ca J, Pombi M, et al. Competence of Phortica variegata from the United States as an intermediate host of the Thelazia callipaeda eyeworm. Am J Trop Med Hyg. 2018;98(4):1175. \u003c/li\u003e\n\u003cli\u003eLutovinovas E, Ivinskis P, Rim\u0026scaron;ait J. Phortica semivirgo (M\u0026aacute;ca, 1977) \u0026ndash; new to the fauna of Lithuania (Diptera: Drosophilidae). Bulletin of the Lithuanian Entomological Society. 4 (2020): 109-113\u003c/li\u003e\n\u003cli\u003eUnterk\u0026ouml;fler MS, Dengg P, Niederbacher M, Lindorfer S, Eberle A, Huck A, et al. Occurrence of Thelazia callipaeda and its vector Phortica variegata in Austria and South Tyrol, Italy, and a global comparison by phylogenetic network analysis. Parasit Vectors. 2023 Aug 24;16(1):294. \u003c/li\u003e\n\u003cli\u003eB\u0026auml;chli G, Vilela CR, Escher SA, Saura A. The Drosophilidae (Diptera) of Fennoscandia and Denmark. [Internet]. Brill Academic Publishers; 2004 [cited 2024 Jan 12]. Available from: https://www.cabdirect.org/cabdirect/abstract/20053205535\u003c/li\u003e\n\u003cli\u003eM\u0026aacute;ca J. Revision of Palaearctic species of Amiota subge nus Photica (Diptera, Drosophilidae). Acta Entomol Bohemoslov. 1977;74:115\u0026ndash;30. \u003c/li\u003e\n\u003cli\u003eCarles-Tolr\u0026aacute;, M and P\u0026aacute;ez, A V., 2010. Nuevos datos dipterol\u0026oacute;gicos del Parque Natural de los Alcornocales (C\u0026aacute;diz, Espa\u0026ntilde;a) (Insecta, Diptera). Bolet\u0026iacute;n de la Sociedad Entomologica Aragonesa (S.E.A.), no 47 (2010):369-371\u003c/li\u003e\n\u003cli\u003eBernardini I, Poggi C, Manzi S, Bezerra-Santos MA, Beugnet F, Fourie J, et al. Laboratory breeding of two Phortica species (Diptera: Drosophilidae), vectors of the zoonotic eyeworm Thelazia callipaeda. Parasit Vectors. 2022 Dec;15(1):200. \u003c/li\u003e\n\u003cli\u003eOtranto D, Cantacessi C, Lia RP, Kadow ICG, Purayil SK, Dantas-Torres F, et al. First laboratory culture of \u003cem\u003ePhortica variegata\u003c/em\u003e (Diptera, Steganinae), a vector of \u003cem\u003eThelazia callipaeda\u003c/em\u003e. J Vector Ecol. 2012 Dec;37(2):458\u0026ndash;61. \u003c/li\u003e\n\u003cli\u003eGonz\u0026aacute;lez MA, Bravo-Barriga D, Alarc\u0026oacute;n-Elbal PM, \u0026Aacute;lvarez-Calero JM, Quero C, Ferraguti M, et al. Development of Novel Management Tools for \u003cem\u003ePhortica variegata\u003c/em\u003e (Diptera: Drosophilidae), Vector of the Oriental Eyeworm, \u003cem\u003eThelazia callipaeda\u003c/em\u003e (Spirurida: Thelaziidae), in Europe. Geden C, editor. J Med Entomol. 2022 Jan 12;59(1):328\u0026ndash;36. \u003c/li\u003e\n\u003cli\u003ePombi M, Marino V, Jaenike J, Graham-Brown J, Bernardini I, Lia RP, et al. Temperature is a common climatic descriptor of lachryphagous activity period in Phortica variegata (Diptera: Drosophilidae) from multiple geographical locations. Parasit Vectors. 2020 Dec;13(1):89. \u003c/li\u003e\n\u003cli\u003eArme TM, Lia RP, Annoscia G, Casalino E, Pombi M, Otranto D. Survival of \u003cem\u003ePhortica variegata\u003c/em\u003e experimentally and naturally infected with \u003cem\u003eThelazia callipaeda\u003c/em\u003e. Med Vet Entomol. 2020 Jun;34(2):201\u0026ndash;6. \u003c/li\u003e\n\u003cli\u003eB\u0026auml;chli G, Weber D. Taufliegen oder Kleine Fruchtfliegen (Insecta, Diptera, Drosophilidae) aus H\u0026ouml;hlen des Grossherzogtums Luxemburg. Ferrantia. 2013;69:349\u0026ndash;53. \u003c/li\u003e\n\u003cli\u003eBeschovsky VL. Representatives of Diptera\u0026ndash;Brachycera in the caves of Bulgaria. Acad\u0026eacute;mie Bulg Sci Bull L\u0026rsquo;Institut Zool Mus. 1972;35:23\u0026ndash;9. \u003c/li\u003e\n\u003cli\u003eCasiraghi M, Bain O, Guerrero R, Martin C, Pocacqua V, Gardner SL, et al. Mapping the presence of Wolbachia pipientis on the phylogeny of filarial nematodes: evidence for symbiont loss during evolution. Int J Parasitol. 2004;34(2):191\u0026ndash;203. \u003c/li\u003e\n\u003cli\u003eManoj RRS, Latrofa MS, Epis S, Otranto D. Wolbachia: endosymbiont of onchocercid nematodes and their vectors. Parasit Vectors. 2021 May 7;14(1):245. \u003c/li\u003e\n\u003cli\u003eM\u0026aacute;ca J. Amiota Loew (Diptera: Drosophilidae, Steganinae). Biologica (Santiago). 2003;47:247\u0026ndash;74. \u003c/li\u003e\n\u003cli\u003eLunt DH, Zhang D ‐X., Szymura JM, Hewltt OM. The insect cytochrome oxidase I gene: evolutionary patterns and conserved primers for phylogenetic studies. Insect Mol Biol. 1996 Aug;5(3):153\u0026ndash;65. \u003c/li\u003e\n\u003cli\u003eTechnelysium Pty Ltd. Chromas version 2.6.6 [Internet]. 2024 [cited 2024 Jun 9]. Available from: https://technelysium.com.au/wp/chromas/\u003c/li\u003e\n\u003cli\u003eTamura K, Stecher G, Kumar S. MEGA11: molecular evolutionary genetics analysis version 11. Mol Biol Evol. 2021;38(7):3022\u0026ndash;7. \u003c/li\u003e\n\u003cli\u003eBLAST: Basic Local Alignment Search Tool [Internet]. [cited 2024 Jun 9]. Available from: https://blast.ncbi.nlm.nih.gov/Blast.cgi\u003c/li\u003e\n\u003cli\u003eCasiraghi M, Anderson TJC, Bandi C, Bazzocchi C, Genchi C. A phylogenetic analysis of filarial nematodes: comparison with the phylogeny of Wolbachia endosymbionts. Parasitology. 2001;122(1):93\u0026ndash;103. \u003c/li\u003e\n\u003cli\u003eZhou W, Rousset F, O\u0026rsquo;Neill S. Phylogeny and PCR\u0026ndash;based classification of Wolbachia strains using wsp gene sequences. Proc R Soc Lond B Biol Sci. 1998 Mar 22;265(1395):509\u0026ndash;15. \u003c/li\u003e\n\u003cli\u003eCoretta S. tidygam: Tidy prediction and plotting of Generalised Additive Models. R Package Version 02 0. 2023; \u003c/li\u003e\n\u003cli\u003eWood SN. Inference and computation with generalized additive models and their extensions. TEST. 2020 Jun;29(2):307\u0026ndash;39. \u003c/li\u003e\n\u003cli\u003eChecklist [Internet]. LifeWatch Italy. [cited 2024 Jul 8]. Available from: https://www.lifewatchitaly.eu/en/initiatives/checklist-fauna-italia-en/checklist/\u003c/li\u003e\n\u003cli\u003eMcCabe EA, Unfried LN, Teets NM. Survival and nutritional requirements for overwintering \u003cem\u003eDrosophila suzukii\u003c/em\u003e (Diptera: Drosophilidae) in Kentucky. Lehmann P, editor. Environ Entomol. 2023 Dec 15;52(6):1071\u0026ndash;81. \u003c/li\u003e\n\u003cli\u003eJones LE, Grimaldi DA. Revision of the Nearctic species of the genus Amiota Loew (Diptera: Drosophilidae). Bull Am Mus Nat Hist. 2022;458(1):1\u0026ndash;177. \u003c/li\u003e\n\u003cli\u003eMarino V, G\u0026aacute;lvez R, Colella V, Sarquis J, Checa R, Montoya A, et al. Detection of Thelazia callipaeda in Phortica variegata and spread of canine thelaziosis to new areas in Spain. Parasit Vectors. 2018 Dec;11(1):195. \u003c/li\u003e\n\u003cli\u003eRoggero C, Schaffner F, B\u0026auml;chli G, Mathis A, Schnyder M. Survey of Phortica drosophilid flies within and outside of a recently identified transmission area of the eye worm Thelazia callipaeda in Switzerland. Vet Parasitol. 2010;171(1\u0026ndash;2):58\u0026ndash;67. \u003c/li\u003e\n\u003cli\u003eIto F, Awasaki T. Comparative analysis of temperature preference behavior and effects of temperature on daily behavior in 11 Drosophila species. Sci Rep. 2022;12(1):12692. \u003c/li\u003e\n\u003cli\u003eB\u0026auml;chli G, Schatzmann E, Haring E. On some population parameters of drosophilids in Switzerland (Diptera, Drosophilidae). 2008 [cited 2024 Jan 12]; Available from: https://www.zora.uzh.ch/id/eprint/14220\u003c/li\u003e\n\u003cli\u003eMasanori J. Toda. Vertical Microdistribution of Drosophilidae (Diptera) within various Forests in Hokkaido.: I. Natural Broad-Leaved Forest. Jpn J Ecol. 1977;27(3):207\u0026ndash;14. \u003c/li\u003e\n\u003cli\u003eMorgado ACT, do Vale B, Ribeiro P, Coutinho T, Santos-Silva S, de Sousa Moreira A, et al. First report of human Thelazia callipaeda infection in Portugal. Acta Trop. 2022;231:106436. \u003c/li\u003e\n\u003cli\u003eBezerra-Santos MA, Bernardini I, Lia RP, Mendoza-Roldan JA, Beugnet F, Pombi M, et al. Phortica oldenbergi (Diptera: Drosophilidae): A new potential vector of the zoonotic Thelazia callipaeda eyeworm. Acta Trop. 2022;233:106565. \u003c/li\u003e\n\u003cli\u003eShropshire JD, Leigh B, Bordenstein SR. Symbiont-mediated cytoplasmic incompatibility: what have we learned in 50 years? Elife. 2020;9:e61989. \u003c/li\u003e\n\u003cli\u003eOte M, Yamamoto D. Impact of Wolbachia infection on Drosophila female germline stem cells. Curr Opin Insect Sci. 2020;37:8\u0026ndash;15. \u003c/li\u003e\n\u003cli\u003eSanaei E, Charlat S, Engelst\u0026auml;dter J. \u003cem\u003eWolbachia\u003c/em\u003e host shifts: routes, mechanisms, constraints and evolutionary consequences. Biol Rev. 2021 Apr;96(2):433\u0026ndash;53. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Phortica, Sympatry, Eyeworm, Thelazia, Zoonosis, Lachryphagy, Wolbachia, Vector-borne disease","lastPublishedDoi":"10.21203/rs.3.rs-5004631/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5004631/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground: Five species of the \u003cem\u003ePhortica\u003c/em\u003e genus (Diptera: Drosophilidae) are known in Europe and the Middle East. Among these, \u003cem\u003ePhortica variegata\u003c/em\u003eand \u003cem\u003ePhortica okadai\u003c/em\u003e are better known for their role as vectors of the zoonotic eyeworm \u003cem\u003eThelazia callipaeda\u003c/em\u003e. Other species, such as \u003cem\u003ePhortica semivirgo\u003c/em\u003e and \u003cem\u003ePhortica oldenbergi\u003c/em\u003e, have been studied less. Given the paucity of data about these \u003cem\u003ePhortica\u003c/em\u003espp. vectors, we explored the population dynamics and ecology of \u003cem\u003ePhortica\u003c/em\u003espp. in an area highly endemic for \u003cem\u003eT. callipeada\u003c/em\u003e (Manziana, Rome, Central Italy).\u003c/p\u003e\n\u003cp\u003eMethods: \u003cem\u003ePhortica\u003c/em\u003e spp. flies were collected over a three-year period (2018-2020) during their active season (April-October) with a sweep net while hovering around: i) a fermenting fruit bait, and ii) a human operator acting as bait. Collected flies were morphologically identified and tested for \u003cem\u003eT. callipaeda\u003c/em\u003e infection and \u003cem\u003eWolbachia\u003c/em\u003e presence by PCR. Population dynamics of species collected was associated to environmental drivers through Generalized Additive Models.\u003c/p\u003e\n\u003cp\u003eResults: Of the 5,564 flies collected, 90.8% were \u003cem\u003eP. variegata\u003c/em\u003e, 9.1% were \u003cem\u003eP. oldenbergi\u003c/em\u003e, 0.05% were \u003cem\u003eP. semivirgo\u003c/em\u003e, and one specimen was \u003cem\u003eP. okadai\u003c/em\u003e. Only \u003cem\u003eP. variegata\u003c/em\u003e scored molecularly infected with \u003cem\u003eT. callipaeda \u003c/em\u003ethroughout the three-year sampling period (1.8%). \u003cem\u003ePhortica oldenbergi\u003c/em\u003e, observed consistently during the entire sampling period, exhibited a marked preference for fruit traps, contrasting with the lachryphagous activity of \u003cem\u003eP. variegata\u003c/em\u003e. Analysis of environmental drivers of \u003cem\u003eP. oldenbergi\u003c/em\u003e and \u003cem\u003eP. variegata\u003c/em\u003epopulation dynamics indicated temperature, wind speed, and pressure as significant factors. In addition, \u003cem\u003eWolbachia pipientis\u003c/em\u003e endosymbiont was detected in \u003cem\u003eP. oldenbergi\u003c/em\u003e and \u003cem\u003eP. okadai\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eConclusions: For the first time, this study analysed several ecological aspects of \u003cem\u003ePhortica\u003c/em\u003e species coexisting in a \u003cem\u003eT.\u003c/em\u003e \u003cem\u003ecallipaeda\u003c/em\u003e endemic area, highlighting different behaviours in the same environment and the vectorial role of this zoonotic parasite. Notably, this is also the first report of the presence of \u003cem\u003eP. oldenbergi\u003c/em\u003e in Italy and \u003cem\u003eP. okadai\u003c/em\u003e in Europe, underscoring the importance of extensive sampling for detecting potential vectors and alien species with direct implications for vector-borne disease epidemiology.\u003c/p\u003e","manuscriptTitle":"Population dynamics of sympatric Phortica spp. and first record of stable presence of Phortica oldenbergi in a Thelazia callipaeda-endemic area of Italy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-04 06:02:48","doi":"10.21203/rs.3.rs-5004631/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-09-18T21:33:13+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-06T16:37:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"31018464670410559130042924090877086065","date":"2024-09-06T11:31:11+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-09-04T15:09:53+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-09-04T14:24:37+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-09-04T07:19:10+00:00","index":"","fulltext":""},{"type":"submitted","content":"Parasites \u0026 Vectors","date":"2024-08-30T14:40:55+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b8f71849-dc15-48cf-80a3-87265b6d4d77","owner":[],"postedDate":"October 4th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-11-11T15:59:18+00:00","versionOfRecord":{"articleIdentity":"rs-5004631","link":"https://doi.org/10.1186/s13071-024-06526-9","journal":{"identity":"parasites-and-vectors","isVorOnly":false,"title":"Parasites \u0026 Vectors"},"publishedOn":"2024-11-06 15:57:04","publishedOnDateReadable":"November 6th, 2024"},"versionCreatedAt":"2024-10-04 06:02:48","video":"","vorDoi":"10.1186/s13071-024-06526-9","vorDoiUrl":"https://doi.org/10.1186/s13071-024-06526-9","workflowStages":[]},"version":"v1","identity":"rs-5004631","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5004631","identity":"rs-5004631","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}