Development of Novel Management Tools for Phortica variegata (Diptera: Drosophilidae), Vector of the Oriental Eyeworm, Thelazia callipaeda (Spirurida: Thelaziidae), in Europe.

Lachryphagous males of Phortica variegata (Fallén, 1823) are gaining increasing attention in Europe, as they act as vectors of the nematode Thelazia callipaeda Railliet & Henry, 1910, causal agent of thelaziosis, an emergent zoonotic disease. Currently, there are no effective control strategies against the vector, and surveillance and monitoring rely on time-consuming and nonselective sampling methods. Our aim was to improve the knowledge about the population dynamics and the chemical ecology of the species. A total of 5,726 P. variegata flies (96.4% males and 3.6% females, mostly gravid) were collected in field experiments during June-September of 2020 in an oak forest in northern Spain. Our results indicate that 1) by means of sweep netting a significantly higher number of captures were found both around the collector´s body and in the air than at ground level; 2) a positive relationship was detected between the abundance of Phortica flies and temperature, with two significant peaks of abundance at 24 and 33°C; 3) the blend of red wine and cider vinegar was the most attractive bait; 4) yellow traps captured fewer flies compared to black and transparent traps; and 5) a significant reduction toward vinegar and wine was detected in presence of the phenolic monoterpenoid carvacrol. In addition, all the males (n = 690) analyzed by both molecular detection and dissection resulted negative for the presence of T. callipaeda larvae. Overall, these findings provide a better understanding of the vector in terms of monitoring and management strategies.

[Ocular thelaziosis in a dog in Germany - an autochthonous case?] / Okuläre Thelaziose bei einem Hund in Deutschland ­ ein autochthoner Fall?

Thelazia callipaeda is a vector-borne nematode transmitted by Phortica spp. (fruit flies) and one of the causes of mild to severe conjunctivitis and keratitis in dogs, cats, rabbits and humans. It has been formerly known as the oriental eye worm based on its geographic occurrence. By now, it has been shown to be endemic in several southern and eastern European countries as well as extending its geographic distribution further throughout Europe. In the present case report, T. callipaeda infection was diagnosed in a female dog from Germany. The dog was referred by a local veterinarian due to a treatment-resistant conjunctivitis. A comprehensive ophthalmologic examination revealed 3 adult eye worms in the conjunctival sac as well as on the bulbar side of the nictitating membrane of the left eye. These were identified by morphological features and molecular techniques as T. callipaeda and represented the primary cause of the dog's unilateral blepharospasm, hyperemic conjunctiva and epiphora. Treatment consisted of manual collection of the adult worms and the administration of moxidectin/imidacloprid as spot on (Advocate®, Bayer). All clinical signs resolved within one week after treatment. In Germany, ocular thelaziosis still represents a rare disease. Usually, it is diagnosed in cats and dogs either imported from abroad or accompanying the owners on holiday travels to endemic countries. The dog presented in this case report was born in Germany. Except for a one-week stay in the Netherlands 11 months before the symptoms began, it had never traveled abroad. Considering the prepatency of T. callipaeda, an autochthonous transmission in this case is highly probable.

A first report of Thelazia callipaeda infection in Phortica okadai and wildlife in national nature reserves in China.

BACKGROUND: Thelazia callipaeda is a zoonotic parasitic nematode of the family Thelaziidae, with Phortica okadai as its intermediate host and only confirmed vector in China. China has the largest number of human cases of thelaziosis in the world. It is generally believed that infected domestic animals (dogs and cats) are the most important reservoir hosts of T. callipaeda, and thus pose a direct threat to humans. At present, there is little research or attention focused on the role of wildlife in the transmission cycle of thelaziosis in nature reserves. METHODS: We selected locations in four national nature reserves across China to monitor P. okadai and wildlife. We used a fly-trap method to monitor P. okadai density. Morphological analysis of the parasites collected from the conjunctival sac of the infected wildlife was undertaken as the first step in species identification, and polymerase chain reaction (PCR) was used for species confirmation. RESULTS: In 2019, the density of P. okadai in Foping National Nature Reserve in China increased sharply, and infected P. okadai were newly found in the reserve. Giant panda, wild boar, leopard cat, and black bear were found to be newly infected with T. callipaeda (one individual of each species). A total of four worms were collected, one from each species of wildlife. The four worms were identified as T. callipaeda by their morphological characteristics; species identification was confirmed by PCR amplification. CONCLUSIONS: To the best of our knowledge, this is the first report of T. callipaeda infection in P. okadai as well as in a variety of wildlife, including giant panda, in nature reserves in China. These results indicate that there is a transmission cycle of T. callipaeda among wildlife in these nature reserves. The increasing number of case reports of thelaziosis in wildlife suggest a likely risk of T. callipaeda infection for the inhabitants of villages situated around nature reserves.

Survival of Phortica variegata experimentally and naturally infected with Thelazia callipaeda.

Knowledge of the effects that Thelazia callipaeda (Spirurida, Thelaziidae) infection has on the survival of its vector Phortica variegata (Drosophilidae, Steganinae) is scarce. The present study aimed to: (a) assess the rate of infection between experimentally infected (EI) and not experimentally infected (NEI) flies and (b) determine how T. callipaeda infection may affect the survival of P. variegata. In addition, fat composition was evaluated in flies that died during overwintering. Molecular analysis showed that T. callipaeda prevalence in flies that died before experimental infection, plus those from the NEI group, is 0.75% (i.e. 11 out of 1462 individuals). The EI group showed a significantly higher positivity to T. callipaeda (i.e. 51 out of 682 individuals; 7.48%) compared with the NEI group (i.e. 9 out of 750 individuals; 1.2%). Thelazia callipaeda DNA was detected until 147 days after experimental infection. This demonstrates that larvae of this eyeworm may survive in the fly for a significant period of the winter. Fat composition analysis showed that flies produced more unsaturated than saturated fatty acids during diapause, probably because unsaturated fatty acids remain in a liquid state at lower temperatures, providing anti-freeze properties to survive winter.

Thelaziosis due to Thelazia callipaeda in Europe in the 21st century-A review.

Thelazia callipaeda was first described at the beginning of the 20th century in Asia, but this eyeworm is now frequently reported in Europe in the 21st century. To date, thelaziosis has been described in the following European countries (in order of appearance): Italy, France, Germany, Switzerland, Spain, Portugal, Belgium, Bosnia and Herzegovina, Croatia, Serbia, Romania, Greece, Bulgaria, Hungary, Slovakia, the United Kingdom, Turkey and Austria. The infected vertebrate host species include domestic carnivores (dogs and cats), wild carnivores (red foxes, wolves, beech martens, wildcats and golden jackals), lagomorphs (brown hares and wild European rabbits) and humans. In Europe, 11 cases of human thelaziosis have been reported, the majority of which are autochthonous. However, some of them have been imported, a fact which highlights the importance of surveillance policies to restrict cross-border spread of the parasite. The objectives of this article are to review key aspects of the epidemiology of T. callipaeda, summarise animal and human cases in Europe and emphasise the importance of education and awareness among veterinarians, physicians (particularly ophthalmologists) and animal, in order to owners to tackle this zoonosis.

Predicting the distribution of Phortica variegata and potential for Thelazia callipaeda transmission in Europe and the United Kingdom.

BACKGROUND: Male fruitflies Phortica variegata (Drosophilidae, Steganinae) are the intermediate host of the zoonotic nematode Thelazia callipaeda (Spirurida, Thelaziidae). More than 10 years ago, when T. callipaeda was confined to remote regions of southern Italy, ecological niche models were used to predict the potential distribution of P. variegata across Europe and the likely risk of the nematode spreading through infected dogs travelling to/from endemic regions. As predicted, over the last 10 years T. callipaeda has spread rapidly across Europe. Recently, we identified the potential for its introduction to the UK through infected dogs travelling to/from endemic regions of mainland Europe. METHODS: Here updated information is used to re-evaluate the model-predicted European, and specifically, UK distribution to determine the likelihood of T. callipaeda becoming established. Additionally, the UK distribution of P. variegata was further investigated through snapshot fly trapping at model-predicted locations. RESULTS: Ecological niche modelling using Genetic Algorithm for Rule-set Prediction (GARP) analysis suggests a European range similar to that described previously, with some indication of potential spread further eastward. Finer scale UK mapping suggested that P. variegata presence was limited mostly to southern England, but highlighted regions where P. variegata has not been documented previously. The arbitrary fly trapping identified activity of P. variegata at two locations where the species has been found previously late in the season. No specimens were collected at model-predicted locations, although habitat suitable for the species was identified. CONCLUSIONS: GARP-model prediction of P. variegata distribution suggests presence of suitable conditions in previously undocumented regions of the UK and Europe and highlight the possibility for further spread of T. callipaeda across Europe, including the UK. Further work to validate the P. variegata UK model with field data will help improve its accuracy in predicting suitable areas, whilst surveillance of sylvatic definitive host species in such locations is advised to monitor for evidence of autochthonous T. callipaeda transmission.

Detection of Thelazia callipaeda in Phortica variegata and spread of canine thelaziosis to new areas in Spain.

BACKGROUND: The fruit fly Phortica variegata (Drosophilidae: Steganinae) feeds on the ocular secretions of animals and humans, and has been described as an intermediate host of the eye worm Thelazia callipaeda (Spirurida: Thelaziidae) in Italy. Despite the increased detection of T. callipaeda in many European countries, information about its vector role in natural conditions is still limited. In the Iberian Peninsula, thelaziosis caused by T. callipaeda has been reported in dogs, cats, red foxes, wild rabbits and humans. METHODS: In the last seven years, we have detected increased numbers of cases of canine thelaziosis at three locations in mainland Spain: Site 1, La Vera region (Cáceres Province, central-western Spain; 51 cases); Site 2, El Escorial municipality (Madrid Community, central Spain; 23 cases); and Site 3, Miraflores de la Sierra municipality (Madrid Community, central Spain; 41 cases). Site 1 is considered endemic for T. callipaeda while the other two sites have been recently recognised as risk zones for T. callipaeda infection. RESULTS: From June 2016 to September 2017, 2162 flies were collected and morphologically identified as Phortica spp. (Site 1, n = 395; Site 2, n = 1544; and Site 3, n = 223). Upon dissection, third-stage T. callipaeda larvae were found in two out of 155 flies examined from Site 1, and both these larvae tested molecularly positive for the eye worm. Of the 395 flies collected from Site 1, 371 were molecularly processed for arthropod species identification and T. callipaeda detection. All 371 flies were identified as P. variegata and 28 (7.5%; 95% CI: 4.8-10%) tested positive for T. callipaeda DNA haplotype 1. CONCLUSIONS: Our findings indicate that T. callipaeda circulates among dogs and P. variegata in Spain, where zoonotic cases have been also reported. The co-existence of canine thelaziosis and Phortica spp. in geographical areas previously considered free of the eye worm indicates a risk of infection for both animals and humans living in this region.

Competence of Phortica variegata from the United States as an Intermediate Host of the Thelazia callipaeda Eyeworm.

Over the past 20 years, Thelazia callipaeda (the oriental eyeworm) has become endemic in Europe, infecting domestic and wild carnivores and humans. The vector of this nematode, the fruit fly Phortica variegata, has recently been discovered in the United States, and its vector competence is demonstrated for T. callipaeda in this article, therefore representing a potential new threat for infection of carnivores and humans in the United States.

First report of Thelazia callipaeda infection in wild European rabbits (Oryctolagus cuniculus) in Portugal.

BACKGROUND: Thelazia callipaeda is a zoonotic nematode that affects the eyes of domestic and wild animals, including dogs, cats and red foxes. This parasitic eye worm is transmitted by Phortica variegata, which is a zoophilic fruit fly spread in Europe. Two wild European rabbits (Oryctolagus cuniculus) found dead in north-eastern Portugal were submitted to necropsy. RESULTS: Both animals presented gross lesions compatible with haemorrhagic viral disease. Eye examination revealed the presence of six worms (three in each animal, on both eyes). Out of the six nematodes, five females and one male were morphologically and molecularly identified as T. callipaeda. CONCLUSIONS: This is the first report of T. callipaeda in wild rabbits from Portugal, which reveals a new host for this parasite in southern Europe and emphasizes the importance of including thelaziosis in the differential diagnosis of ocular alterations in both animals and humans from areas where the eye worm is endemic.

Transmission of the eyeworm Thelazia callipaeda: between fantasy and reality.

BACKGROUND: Thelazia callipaeda is transmitted by Phortica variegata, a drosophilid that feeds on lachrymal secretions of mammals. Scientific information on human thelaziosis is still relatively limited, mainly for physicians and ophthalmologists. Indeed, the literature is full of misleading information on the transmission of T. callipaeda to humans. FINDINGS: A recent paper reported a case of human intraocular infestation in a patient from Karnataka. The information presented in that article as well as in other articles in the international literature is outdated and incorrect in several instances, mostly regarding to the localization of T. callipaeda in the host, its biology and routes of transmission. CONCLUSIONS: Physicians and ophthalmologists should be aware that T. callipaeda is larviparous and transmitted exclusively by secretophagous flies. These flies buzz around the eyes of animals and humans at the daytime, landing on the eyes and releasing the infective larvae on the host conjunctiva. That is the only possible way of transmission of T. callipaeda.

Drosophilidae feeding on animals and the inherent mystery of their parasitism.

Insect evolution, from a free to a parasitic lifestyle, took eons under the pressure of a plethora of ecological and environmental drivers in different habitats, resulting in varying degrees of interactions with their hosts. Most Drosophilidae are known to be adapted to feeding on substrates rich in bacteria, yeasts and other microfungi. Some of them, mainly those in the Steganinae subfamily, display a singular behaviour, feeding on animal tissues or secretions. This behaviour may represent an evolving tendency towards parasitism. Indeed, while the predatory attitude is typical for the larval stages of a great proportion of flies within this subfamily, adult males of the genera Amiota, Apsiphortica and Phortica display a clearly zoophilic attitude, feeding on the lachrymal secretions of living mammals (also referred as to lachryphagy). Ultimately, some of these lachryphagous species act as vectors and intermediate hosts for the spirurid nematode Thelazia callipaeda, which parasitizes the eyes of domestic and wild carnivores and also humans. Here we review the scientific information available and provide an opinion on the roots of their evolution towards the parasitic behaviour. The distribution of T. callipaeda and its host affiliation is also discussed and future trends in the study of the ecology of Steganinae are outlined.

A role for nematocytes in the cellular immune response of the drosophilid Zaprionus indianus.

The melanotic encapsulation response mounted by Drosophila melanogaster against macroparasites, which is based on haemocyte binding to foreign objects, is poorly characterized relative to its humoral immune response against microbes, and appears to be variable across insect lineages. The genus Zaprionus is a diverse clade of flies embedded within the genus Drosophila. Here we characterize the immune response of Zaprionus indianus against endoparasitoid wasp eggs, which elicit the melanotic encapsulation response in D. melanogaster. We find that Z. indianus is highly resistant to diverse wasp species. Although Z. indianus mounts the canonical melanotic encapsulation response against some wasps, it can also potentially fight off wasp infection using two other mechanisms: encapsulation without melanization and a non-cellular form of wasp killing. Zaprionus indianus produces a large number of haemocytes including nematocytes, which are large fusiform haemocytes absent in D. melanogaster, but which we found in several other species in the subgenus Drosophila. Several lines of evidence suggest these nematocytes are involved in anti-wasp immunity in Z. indianus and in particular in the encapsulation of wasp eggs. Altogether, our data show that the canonical anti-wasp immune response and haemocyte make-up of the model organism D. melanogaster vary across the genus Drosophila.

Survey of Phortica drosophilid flies within and outside of a recently identified transmission area of the eye worm Thelazia callipaeda in Switzerland.

Phortica drosophilid flies are the intermediate hosts and vectors of the eye worm Thelazia callipaeda. This nematode originates from Asia and was first detected in southern Europe in 1989. The aim of the study was to assess the presence and the population dynamics of Phortica flies in a recently discovered new endemic area (Ticino, Southern Switzerland, south of the Alps) of T. callipaeda (site 1), at its border (site 2), at higher altitudes (beyond 1100 meters above sea level) within (site 3) or outside (site 4) the endemic area, and in a site north of the Alps (site 5). Flies were captured using two types of fruit-baited traps, the bait being changed once per week, and by netting around the eyes of a dog and human. A total of 1695 Phortica flies were collected. One of the fruit-baited traps, which can easily be assembled with cheap components, was found to be efficient for catching Phortica spp. At site 1, 644 such flies were collected with this trap during 34 weekly catches from April to October. The number of flies caught was highest at site 2 (n=903) and it was significantly lower (n=36) at site 5 north of the Alps. Virtually no Phortica at all were caught at higher altitudes (sites 3 and 4). Females were all in all predominant in the traps, accounting for 72.6% of Phortica flies (1150/1584), although males became dominant late in the season (male/female ratio 1.26 in October). In contrast, 80.2% of Phortica flies collected around the eyes of dog and human baits by netting (n=111) were males. No female at all was captured by netting until September. PCR for T. callipaeda was negative with all Phortica flies. Morphological examination of the 523 male flies based on features of the eye margin and the number of particular genital sensilla identified 89.1% P. semivirgo, 5.7% P. variegata but also 5.2% intermediate forms. Genetic analyses of partial mitochondrial cox1 and rDNA internal transcribed spacer 1 sequences revealed that these three morphotypes were genetically not distinguishable. This study confirms the presence of Phortica spp. north to the Alps and therefore the potential risk of T. callipaeda infection outside the currently known endemic region, depending on local abundance and longevity of the drosophilid vectors.

[Amiota magna as an intermediate host of Thelazia callipaeda under laboratory conditions].

A number of flies around the eyes of a person or around a fruit bait were collected from Huangshan Mountain, and experimentally infected by newborn larvae of Thelazia callipaeda. After 20 days, the flies were examined for T. callipaeda. Following dissection, 3 (30%, 3/10) of Amiota magna, and 55 (21.6%, 55/255) of A. okada were found infected by T. callipaeda. The susceptibility of T. callipaeda is similar in the two species fruit flies (chi2=0.0584, P> 0.05). The rabbits were infected by infective larvae of T. callipaeda from A. magna. At the 35th day after infection, the newborn larvae and worms of T. callipaeda were found in the conjunctival sac of rabbits. This study suggested that A. magna acts as intermediate host of T. callipaeda under laboratory conditions.

Phortica variegata as an intermediate host of Thelazia callipaeda under natural conditions: evidence for pathogen transmission by a male arthropod vector.

Knowledge about Phortica variegata (Drosophilidae, Steganinae), the intermediate host of the eyeworm Thelazia callipaeda (Spirurida, Thelaziidae), is confined to experimental studies. To investigate the role P. variegata plays in the transmission of T. callipaeda under natural conditions, the population dynamics of these flies in the natural environment and their feeding preferences (on vegetables and/or animal lachrymal secretions) were examined. From April to November 2005, a total number of 969 (557 males and 412 females) P. variegata flies were collected weekly in a region of southern Italy with a history of canine thelaziosis. The flies were identified and dissected or subjected to a PCR assay specific for a region within the ribosomal ITS-1 DNA of T. callipaeda. The zoophilic preferences of P. variegata were assessed by collecting flies around the eyes of a person or around a fruit bait. Seven hundred and twenty flies (398 males and 322 females) were dissected under a stereomicroscope; 249 flies (158 males and 91 females) that died prior to the dissection were subjected to molecular investigation. Only P. variegata males were infected with larval T. callipaeda both at dissection (six, 0.83%) and with the specific PCR (seven, 2.81%), representing a total percentage of 1.34% flies infected. Interestingly, only males were collected around the eyes, compared with a male/female ratio of 1:4 around the fruit. This survey indicated that P. variegata males act as intermediate hosts of T. callipaeda under natural conditions in Europe. Both the zoophilic behaviour of P. variegata males on lachrymal secretions and their role as vector of T. callipaeda have been discussed as they represent a peculiarity in medical and veterinary entomology. The synchrony between the fly population dynamics and the biology of the nematode in the definitive host provides an interesting model for exploring the co-evolution of Thelazia spp. with their hosts.

Nematode biology and larval development of Thelazia callipaeda (Spirurida, Thelaziidae) in the drosophilid intermediate host in Europe and China.

Thelazia callipaeda, commonly known as the 'oriental eyeworm', has been recently reported in Italy and other European countries. The insect/s that act as intermediate hosts and details of larval development inside the vector remain unclear. In order to (1) demonstrate the species of fly that may act as vector/s for T. callipaeda in southern Italy (Site A) and China (Site B) and (2) describe the larval development of the nematode in the body of flies, 847 Phortica (Drosophilidae) flies were collected from the above two sites, each with a history of human and/or canine thelaziosis. Flies were identified as Phortica variegata (245 - site A) and Phortica okadai (602 - site B), experimentally infected by 1st-stage larvae (L1), kept at different temperatures and dissected daily until day 180 post-infection (p.i.). Dead flies from site A were subjected to specific polymerase chain reaction (PCR) assay to detect T. callipaeda. To demonstrate the role of Phortica as vectors of T. callipaeda, 3rd-stage larvae (L3) recovered from the proboscis of flies were deposited onto the cornea of the eyes of dogs and rabbits. Following dissection, 3 (2.9%) of P. variegata in site A were found to be infected by L3 in the proboscis on days +14, +21 and +53 p.i., compared with 26 (18.4%) of Phortica flies recorded as being positive by PCR. Sequences from positive PCR products were 99% identical to sequences of the corresponding species available in GenBank (AY207464). At site B, 106 (17.6%) of 602 dissected P. okadai were found to be infected by T. callipaeda larvae (different stages) and in total 62 L3 were recovered from the proboscis of 34 (5.6%) flies. The shortest time in which L3 were found was at day +14, +17, +19, and +50 p.i. respectively, depending on the environmental temperatures. Of 30 flies overwintered for 6 months, 6 L3 were detected at day +180 p.i. in 3 flies (10%). The biology of larval development was reconstructed on the basis of the dissection of 602 P. okadai-infected flies and the morphology of larval stages in the insect body described. The present work provides evidence that P. variegata and P. okadai act as vectors for T. callipaeda in southern Europe and in China, respectively. The phenomenon of overwintering is described here for the first time for T. callipaeda and discussed. Finally, the relationship between T. callipaeda and its fly vector is considered in light of disease prophylaxis and to model its dissemination into habitats and environments favourable to Phortica flies.

First occurrence of parasitoid Spalangia endius (Walker) (Hymenoptera: Pteromalidae) in pupae of Zaprionus indianus Gupta (Diptera: Drosophilidae) in Brazil.

This paper reports the first occurrence of the parasitoid Spalangia endius collected in pupae of Zaprionus indianus using traps with fruit bait. The experiment was carried out at the University of Lavras in Lavras, Minas Gerais, Brazil, from November to December, 2001. A total of 4 Spalangia endius specimens were obtained from 105 Zaprionus indianus pupae. The overall prevalence of parasitism was 3.8%.