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Microbial communities within metazoans are increasingly linked with development, health and behaviour, possibly functioning as integrated evolutionary units with the animal in which they live. This would require microbial communities to show some consistency both ontogenetically (across life stages) and geographically (among populations). We characterise the bacteriome of the parasitic trematode Philophthalmus attenuatus, which undergoes major life cycle transitions, and test whether its bacteriome remains consistent on developmental and spatial scales. Based on sequencing the prokaryotic 16S SSU rRNA gene, we compared the parasite bacteriome (i) across three life stages (rediae in snails, cercariae exiting snails, adults in birds) in one locality and (ii) among three geographic localities for rediae only. We found that each life stage harbours a bacteriome different from that of its host (except the adult stage) and the external environment. Very few bacterial taxa were shared among life stages, suggesting substantial ontogenetic turnover in bacteriome composition. Rediae from the three different localities also had different bacteriomes, with dissimilarities increasing with geographical distance. However, rediae from different localities nevertheless shared more bacterial taxa than did different life stages from the same locality. Changes in the bacteriome along the parasite’s developmental history but some degree of geographical stability within a given life stage point toward non-random, stage-specific acquisition, selection and/or propagation of bacteria.

Philophthalmus is a genus of globally distributed parasitic eye flukes with some members of the genus found in disparate locales. In particular, Philophthalmus gralli, a zoonotic trematode, appears to be a relatively new introduction to the Americas, facilitated by spillover from the invasive snails Melanoides tuberculata (red-rimmed melania) and Tarebia granifera (quilted melania), which were introduced via the aquarium trade, and perhaps furthered by avian dispersal. Given that two known intermediate hosts of Philophthalmus flukes are actively expanding their range as a result of human activities, we hypothesize that this spread is also associated with the spread of Philophthalmus flukes. To address this, we systematically reviewed the literature and examined whether the global expansion of P. gralli flukes is associated with the spread of invasive snails M. tuberculata and T. granifera. Here, we show that (1) specimens of P. gralli are only found in intermediate snail hosts M. tuberculata or T. granifera, suggesting intermediate host specificity for these 2 species, and (2) specimens of P. gralli have rarely been found outside the ranges (native and introduced) of M. tuberculata or T. granifera. Given the importance of distribution information of parasites in the role of identifying parasite invasions, we also review the known distribution of all Philophthalmus species. Considering recent outbreaks in humans and wild and domestic animal species, the continued spread of Philophthalmus presents a potential threat to veterinary and public health and conservation.

1. Colonial organisms with division of labour are assumed to achieve increased colony-level efficiency in task performance through functional specialisation of individuals into distinct castes. In social insects, ratios of individuals in different castes can adjust adaptively in response to external threats. However, whether flexibility in caste ratio also occurs in other social organisms with division of labour remains unclear. Some parasitic trematodes, in which clonal colonies within the snail intermediate host comprise a reproductive caste and a soldier caste, offer good systems to test the general nature of adaptive caste ratio adjustments. 2. Using the trematode Philophthalmus sp. as model, we test whether trematode colonies shift their composition towards more soldiers when exposed to a sustained risk of invasion by a competitor parasite species, and/or when experiencing sustained, active competition. We also quantify the colony-level fitness impact of caste ratio adjustments, measured as the colony's output of larval infective stages. 3. We conducted two long-term laboratory experiments on within-snail trematode colonies. First, snails harbouring Philophthalmus colonies were exposed to different levels of invasion risk by another trematode species, Maritrema novaezealandense. Second, the structure of Philophthalmus colonies was quantified after a year-long period of within-snail competition with the other trematode species. 4. When facing the risk of invasion by a competitor, independently of the level of risk, Philophthalmus colonies showed a significant shift towards producing more soldiers, resulting in altered caste ratio. Similarly, when experiencing actual competition by another trematode established in the same snail, Philophthalmus colonies also adjusted by producing significantly more soldiers. Greater investments in defense via more soldiers had negative impacts on the establishment and size of the competitor's colonies. Nevertheless, the presence of the competitor reduced the fitness (output of infective stages) of Philophthalmus colonies, although the production of more soldiers mitigated that effect. 5. Our findings demonstrate that trematode colonies with division of labour are capable of adaptive caste ratio adjustments in response to both the perceived threat of competition and actual competition, with trade-offs against reproductive success only apparent when soldier numbers are very high. Combined with results on social insects, our study suggests parallel adaptations of colonial organisms in phylogenetically disparate organisms.

Background Parasitic conjunctivitis caused by Philophthalmus spp. is a common ophthalmic disease in birds, with localized outbreaks occurring worldwide. There is no consensus on treating this disease; mechanical removal is considered a standard recommendation, but is associated with disease relapses within days or weeks. Methods From 2015 to 2020, we examined 4295 Larus michahellis and Larus fuscus gulls in southern Portugal for the presence of Philophthalmus spp. Due to the need to treat dozens of infected gulls in the rescue station, we tested three treatment regimens aimed at targeting Philophthalmus lucipetus in the infected gulls: (I) the ophthalmic application of levamisole; (II) the oral application of milbemycin in combination with praziquantel; and (III) the subcutaneous application of ivermectin. Results The outbreak of philophthalmosis in gulls in southern Portugal has been ongoing since the first cases were reported in 2015–2016. The prevalence of philophthalmosis has fluctuated annually, peaking a maximum of 10.3% in L. fuscus in 2017 and at 2.1% in L. michahellis in 2016. The infection intensity peaked at a median of 11.5 eye-flukes per host bird in L. fuscus in 2016 and a median of six eye-flukes per host bird in L. michahellis in 2017. Nine gulls were infected with >50 eye-flukes. None of the treatment options were effective at treating P. lucipetus infections: the numbers of eye-flukes in the infected birds did not decrease, and the clinical signs of the disease did not change. Conclusions An outbreak of philophthalmosis in southern Portugal has massively affected two species of gulls in the region. Two previously suggested philophthalmosis treatments (ocular levamisole and praziquantel given orally), as well as a third mode of treatment with a previously failed compound (ivermectin administered subcutaneously) were used. However, the treatments did not affect the numbers of P. lucipetus in the eyes of the treated gulls. Further research should address ophthalmic gel formulations or sub-conjunctival delivery mode for antihelminthic drugs that are effective against Philophthalmus spp. in vitro. Graphical abstract

Two cases of human philophthalmosis have been reported in Japan. Gravid flukes removed from the eyes of the patients were broken, but their morphological characteristics suggest that an unknown species of the genus Philophthalmus is involved as a pathogen for humans. The mitochondrial DNA barcode of the human eye fluke enabled us to discover its larval stage from the Japanese mud snail, Batillaria attramentaria. The discovered cercaria had previously been temporarily described as “Philophthalmid sp. I.” In this study, we examined the infection status of B. attramentaria with Philophthalmid sp. I found on a muddy seashore of the Seto Inland Sea, Japan, and the resulting metacercariae were experimentally administered to Japanese quails to develop them into the gravid adult stage. The complete specimens of the adult and larval stages allowed us to describe a new species. Based on morphological and molecular analyses, Philophthalmus hechingeri n. sp. is proposed for the human-infecting eye fluke in Japan. The natural definitive hosts of the new species are unknown. However, the habitat of B. attramentaria suggests that shorebirds (seagulls, sandpipers, and plovers) might be the possible candidates.

Philophthalmid eyeflukes are cosmopolitan parasites of birds and occasionally of mammals, including humans. A gravid adult of Philophthalmus sp. was found from the bulbar conjunctiva of a 64-yr-old woman in Japan, who was diagnosed with acute conjunctivitis. The parasite was morphologically most similar to Philophthalmus hegeneri, but distinctive in lacking an esophagus and in having clearly lobed testes. The DNA sequence analysis of genes for nuclear 28S ribosomal RNA and mitochondrial cytochrome c oxidase subunit 1 supported the identification at generic level. The morphological and molecular analyses strongly suggest that the eyefluke from a human in Japan should be treated as an undescribed species of Philophthalmus. The occurrence of human philophthalmosis is very rare. As far as we know, a total of 11 human cases have been reported worldwide to date.

Shell damage and parasitic infections are frequent in gastropods, influencing key snail host life‐history traits such as survival, growth, and reproduction. However, their interactions and potential effects on hosts and parasites have never been tested. Host–parasite interactions are particularly interesting in the context of the recently discovered division of labor in trematodes infecting marine snails. Some species have colonies consisting of two different castes present at varying ratios; reproductive members and nonreproductive soldiers specialized in defending the colony. We assessed snail host survival, growth, and shell regeneration in interaction with infections by two trematode species, Philophthalmus sp. and Maritrema novaezealandense, following damage to the shell in the New Zealand mud snail Zeacumantus subcarinatus. We concomitantly assessed caste‐ratio adjustment between nonreproductive soldiers and reproductive members in colonies of the trematode Philophthalmus sp. in response to interspecific competition and shell damage to its snail host. Shell damage, but not parasitic infection, significantly increased snail mortality, likely due to secondary infections by pathogens. However, trematode infection and shell damage did not negatively affect shell regeneration or growth in Z. subcarinatus; infected snails actually produced more new shell than their uninfected counterparts. Both interspecific competition and shell damage to the snail host induced caste‐ratio adjustment in Philophthalmus sp. colonies. The proportion of nonreproductive soldiers increased in response to interspecific competition and host shell damage, likely to defend the parasite colony and potentially the snail host against increasing threats. These results indicate that secondary infections by pathogens following shell damage to snails both significantly increased snail mortality and induced caste‐ratio adjustments in parasites. This is the first evidence that parasites with a division of labor may be able to produce nonreproductive soldiers according to environmental factors other than interspecific competition with other parasites. We assessed snail host survival, growth, and shell regeneration in interaction with infections by two trematode species following damage to the shell. We concomitantly assessed caste‐ratio adjustment between nonreproductive soldiers and reproductive members in colonies of the trematode Philophthalmus sp. in response to interspecific competition and shell damage to its snail host. Shell damage, but not parasitic infection, significantly increased snail mortality, likely due to secondary infections by pathogens. The proportion of nonreproductive soldiers increased in response to interspecific competition and host shell damage. This is the first evidence that parasites with a division of labor may be able to produce nonreproductive soldiers according to environmental factors other than interspecific competition with other parasites.

The Galapagos sea lion (Zalophus wollebaeki) is an otariid species endemic to the Galapagos archipelago and is currently listed as endangered. The ocular trematode Philophthalmus zalophi was recently reported to affect the survival of juvenile Galapagos sea lions on Santa Cruz Island, resulting in marked ophthalmic changes. This study evaluated the ophthalmic disease and histopathologic effects of P. zalophi on juvenile Galapagos sea lions in the largest rookery located on San Cristóbal Island. Twenty juvenile Galapagos sea lions (10 male and 10 female) were evaluated among five sites in the rookery El Malecón. Ophthalmic examination, including fluorescein staining and evaluation of the adnexa, cornea, and sclera, were performed on each eye. The presence, number, and location of ocular parasites were determined, and parasites were collected for identification. Conjunctival biopsy was performed on 11 animals: 2 that lacked parasites and gross lesions and 9 with both parasites and gross lesions. All parasites collected were confirmed as P. zalophi and identified in 80% (16/20) of the study animals and 70% (28/40) of the examined eyes. Philophthalmus zalophi was most frequently found attached to the nictitating membrane but also located on the palpebral conjunctiva or cornea. The most common clinical signs were varying degrees of conjunctival hyperemia (28/40 eyes), most frequently of the nictitating membrane and mucoid ocular discharge (12/40 eyes). The number of parasites was significantly associated with the degree of conjunctival hyperemia (P < 0.001). Histopathology of conjunctival biopsies revealed organized lymphoid follicles and lymphoplasmacytic infiltrates. The histopathologic changes and gross lesions were likely due to the parasite's attachment to the conjunctiva. This study provides additional details of P. zalophi infection in juvenile Galapagos sea lions. Further research is warranted to detail the life cycle of this parasite, transmission to sea lions, and potential treatment protocols.

Purpose Species of the genus Philophthalmus are eye flukes with a complex taxonomy, which began to be improved with the help of molecular data only recently. However, most described species have never been placed into a phylogenetic context. In this study, eye flukes previously found on kelp gulls, Larus dominicanus , from Brazil and identified as P hilophthalmus lacrymosus  were subjected to molecular analysis. Methods For the molecular analyses, we analyzed parasites found in six infected gulls (one worm per bird) collected from different municipalities of the state of Santa Catarina, Brazil. We carried out the amplification and sequencing of the partial region of the 28S and cox 1 genes and the data obtained were compared with sequences available to philophthalmid species and subjected to phylogenetic analysis. Results The isolates of P. lacrymosus from Brazil grouped in well-supported clades with five other species of Philophthalmus with sequences available for comparison. Interspecific divergences of 0.1–1.6% in 28S and 8.2–14.9% in c ox 1 were found in relation to other isolates of Philophthalmus spp. Two cox 1 haplotypes differing in one nucleotide (0.1%) were found between the six eye flukes isolates in gulls from different localities. The Brazilian isolates grouped in a subclade with parasites identified as P. lacrymosus in Portugal; however, the molecular divergences found in cox 1 (8.2–8.5%) strongly suggest that these isolates belong to different species. The phylogenetic trees obtained and the intergeneric divergences to species of the genera Cloacitrema and Parorchis did not support the validity of the genus Natterophthalmus , for which P. lacrymosus was proposed as the type species in the past. Conclusion As P. lacrymosus was described from Brazil, we recommend that this name be applied to the South American isolates and that the Portuguese isolates be provisionally considered as Philophthalmus sp., a probable cryptic species. Moreover, data obtained supports the previous morphology-based synonymizing between Natterophthalmus and Philophthalmus . Considering our results and most of previous reports of P. lacrymosus in South America, we suggest this species presents a marine life cycle. Graphical Abstract

A division of labour occurs in colonies of the trematode Philophthalmus sp. within their first intermediate hosts. Two castes exist: one which reproduces and one which does not reproduce. It has been hypothesized that the benefit of the non-reproductive caste is in competitive interactions. Evidence for this from past experiments with Philophthalmus sp. colonies has been contradictory: the non-reproductive caste appears to benefit the colony in some way but not necessarily by combating interspecific competitors. The aims of this study were to consider intraspecific competition as a possible cause of the division of labour in Philophthalmus sp. colonies. Results show that mixed genotype infections occur in Philophthalmus sp. infected hosts and thus intraspecific competition is likely. Furthermore, the total number of individuals per colony is reduced in mixed genotype infections, indicating that intraspecific competition reduces colony fitness. However, the results do not indicate that the division of labour in Philophthalmus sp. plays a role in competitive interactions as the ratio of small, non-reproductive to large, reproductive individuals is unaffected by the presence of intraspecific competition. This is the first study to identify and quantify intraspecific competition in Philophthalmus sp., and to assess its selective role in this species’ division of labour.