Raising the bar: genus-specific nested PCR improves detection and lineage identification of avian haemosporidian parasites.

Avian haemosporidian parasites are useful model organisms to study the ecology and evolution of parasite-host interactions due to their global distribution and extensive biodiversity. Detection of these parasites has evolved from microscopic examination to PCR-based methods, with the mitochondrial cytochrome b gene serving as barcoding region. However, standard PCR protocols used for screening and identification purposes have limitations in detecting mixed infections and generating phylogenetically informative data due to short amplicon lengths. To address these issues, we developed a novel genus-specific nested PCR protocol targeting avian haemosporidian parasites. The protocol underwent rigorous testing utilizing a large dataset comprising blood samples from Malagasy birds of three distinct Passeriformes families. Furthermore, validation was done by examining smaller datasets in two other laboratories employing divergent master mixes and different bird species. Comparative analyses were conducted between the outcomes of the novel PCR protocol and those obtained through the widely used standard nested PCR method. The novel protocol enables specific identification of Plasmodium, Haemoproteus (Parahaemoproteus), and Leucocytozoon parasites. The analyses demonstrated comparable sensitivity to the standard nested PCR with notable improvements in detecting mixed infections. In addition, phylogenetic resolution is improved by amplification of longer fragments, leading to a better understanding of the haemosporidian biodiversity and evolution. Overall, the novel protocol represents a valuable addition to avian haemosporidian detection methodologies, facilitating comprehensive studies on parasite ecology, epidemiology, and evolution.

Higher body condition with infection by Haemoproteus parasites in Bananaquits (Coereba flaveola).

Parasite transmission is a heterogenous process in host-parasite interactions. This heterogeneity is particularly apparent in vector-borne parasite transmission where the vector adds an additional level of complexity. Haemosporidian parasites, a widespread protist, cause a malaria-like disease in birds globally, but we still have much to learn about the consequences of infection to hosts' health. In the Caribbean, where malarial parasites are endemic, studying host-parasites interactions may give us important insights about energetic trade-offs involved in malarial parasites infections in birds. In this study, we tested the consequences of Haemoproteus infection on the Bananaquit, a resident species of Puerto Rico. We also tested for potential sources of individual heterogeneity in the consequences of infection such as host age and sex. To quantify the consequences of infection to hosts' health we compared three complementary body condition indices between infected and uninfected individuals. Our results showed that Bananaquits infected by Haemoproteus had higher body condition than uninfected individuals. This result was consistent among the three body condition indices. Still, we found no clear evidence that this effect was mediated by host age or sex. We discuss a set of non-mutually exclusive hypotheses that may explain this pattern including metabolic syndrome, immunological responses leading to host tolerance or resistance to infection, and potential changes in consumption rates. Overall, our results suggest that other mechanisms, may drive the consequences of avian malarial infection.

Chewing lice of Bearded Reedling (Panurus biarmicus) and diversity of louse-host associations of birds in reed beds in Slovakia.

A total of 1,621 wild birds representing 34 species were examined for chewing lice in reed beds in southwestern Slovakia during the pre-breeding migration 2008-2009 and 2016-2019. A total of 377 (23.3%) birds representing 15 species were parasitized by 26 species of chewing lice of 12 genera. Dominant genera were Penenirmus (with dominance 32.6%) and Menacanthus (29.4%), followed by Brueelia (12.6%), Acronirmus (10.8%), Philopterus (7.7%), and Myrsidea (4.2%). We evaluated 33 host-louse associations including both 1) host-generalist, parasitizing more than one host species and host-specific lice, occurring only on a single host species, and 2) lice species with large range geographic distribution, reported across the range of the distribution of their hosts and lice species with only occasional records from a limited area within the range of their hosts. The Bearded Reedling, Panurus biarmicus (Linnaeus, 1758), was parasitized by two species of chewing lice, Menacanthus brelihi Balát, 1981 and Penenirmus visendus (Zlotorzycka, 1964), with conspicuously different prevalences (5.6% vs. 58.2%, respectively; n = 251). New material enabled us to redescribe both species of lice: the first one is resurrected from previous synonymy as a valid species. A fragment of the mitochondrial cytochrome oxidase I gene was sequenced from these two species in order to assess their relative phylogenetic position within their genera. Our study demonstrates the importance of an adequate identification of parasites, especially on rarely examined and endangered hosts.

Title: Mallophages de la Panure à moustaches (Panurus biarmicus) et diversité des associations mallophages-hôtes des oiseaux dans les roselières en Slovaquie. Abstract: Au total, 1 621 oiseaux sauvages représentant 34 espèces ont été examinés à la recherche de mallophages dans les roselières du sud-ouest de la Slovaquie au cours de la migration de pré-reproduction 2008­2009 et 2016­2019. Parmi ceux-ci, 377 oiseaux (23,3 %), représentant 15 espèces, étaient parasités par 26 espèces de mallophages de 12 genres. Les genres dominants étaient Penenirmus (avec une dominance de 32,6 %) et Menacanthus (29,4 %), suivis de Brueelia (12,6 %), Acronirmus (10,8 %), Philopterus (7,7 %) et Myrsidea (4,2 %). Nous avons évalué 33 associations mallophage-hôte comprenant à la fois 1) des espèces de mallophages généralistes, parasitant plus d'une espèce hôte, et des mallophages spécifiques, présents uniquement sur une seule espèce hôte et 2) des espèces de mallophages ayant une large répartition géographique, signalées à travers l'étendue de la répartition de leurs hôtes, et des espèces de mallophages avec seulement des observations occasionnelles dans une zone limitée à l'intérieur de l'aire de répartition de leurs hôtes. La Panure à moustaches, Panurus biarmicus (Linnaeus, 1758), était parasitée par deux espèces de mallophages, Menacanthus brelihi Balát, 1981 et Penenirmus visendus (Zlotorzycka, 1964), avec des prévalences nettement différentes (respectivement 5,6 % et 58,2 %, n = 251). Du nouveau matériel nous a permis de redécrire les deux espèces de mallophages, la première étant ressuscitée de la synonymie précédente en tant qu'espèce valide. Un fragment du gène mitochondrial de la cytochrome oxydase I a été séquencé à partir de ces deux espèces afin d'évaluer leur position phylogénétique relative au sein de leurs genres. Notre étude démontre l'importance d'une identification adéquate des parasites, en particulier sur les hôtes rarement examinés et menacés.

Linking avian malaria parasitemia estimates from quantitative PCR and microscopy reveals new infection patterns in Hawai'i.

Plasmodium parasites infect thousands of species and provide an exceptional system for studying host-pathogen dynamics, especially for multi-host pathogens. However, understanding these interactions requires an accurate assay of infection. Assessing Plasmodium infections using microscopy on blood smears often misses infections with low parasitemias (the fractions of cells infected), and biases in malaria prevalence estimates will differ among hosts that differ in mean parasitemias. We examined Plasmodium relictum infection and parasitemia using both microscopy of blood smears and quantitative polymerase chain reaction (qPCR) on 299 samples from multiple bird species in Hawai'i and fit models to predict parasitemias from qPCR cycle threshold (Ct) values. We used these models to quantify the extent to which microscopy underestimated infection prevalence and to more accurately estimate infection patterns for each species for a large historical study done by microscopy. We found that most qPCR-positive wild-caught birds in Hawaii had low parasitemias (Ct scores ≥35), which were rarely detected by microscopy. The fraction of infections missed by microscopy differed substantially among eight species due to differences in species' parasitemia levels. Infection prevalence was likely 4-5-fold higher than previous microscopy estimates for three introduced species, including Zosterops japonicus, Hawaii's most abundant forest bird, which had low average parasitemias. In contrast, prevalence was likely only 1.5-2.3-fold higher than previous estimates for Himatione sanguinea and Chlorodrepanis virens, two native species with high average parasitemias. Our results indicate that relative patterns of infection among species differ substantially from those observed in previous microscopy studies, and that differences depend on variation in parasitemias among species. Although microscopy of blood smears is useful for estimating the frequency of different Plasmodium stages and host attributes, more sensitive quantitative methods, including qPCR, are needed to accurately estimate and compare infection prevalence among host species.

Haemosporidian parasite infections of Malagasy Philepittidae and Nectariniidae are driven by phylogeny rather than ecology.

The nectarivorous common sunbird asity (Neodrepanis coruscans) is phylogenetically closely related to the frugivorous velvet asity (Philepitta castanea), yet it shares similar habitat and foraging behaviour as the Malagasy sunbirds (Cinnyris spp.). As ecological factors have been shown to influence blood parasite prevalence, it should be tested whether parasite abundance, prevalence and diversity of N. coruscans are more similar to the sunbirds than to its relative. Therefore, blood samples (n = 156) and smears (n = 60) were tested for different blood parasites (Haemosporida, trypanosomes, filarioid nematodes) using molecular and microscopic methods. High prevalence of haemosporidian parasites was observed in all bird taxa, with rates ranging from 23% in N. coruscans to 84.6% in C. notatus. The Malagasy Cinnyris spp. exhibited a high occurrence of mixed haemosporidian infections (>76%) with various specialized lineages. Within the Philepittidae family, no Haemoproteus infection was detected and just a few cases of mixed infections. Nectariniidae species predominantly had specialized haemosporidian lineages, while Philepittidae had infections mainly caused by generalist lineages. These findings emphasize the diverse range of blood parasites in Nectariniidae, while additionally highlighting the high diversity of trypanosomes and filarioid nematodes in Philepittidae. Additionally, several newly discovered haemosporidian lineages, Trypanosoma isolates and filarioid nematode isolates were identified. Notably, Philepittidae exhibited a lower prevalence of avian haemosporidian parasites compared to Nectariniidae, possibly due to potential resistance mechanisms. Despite N. coruscans sharing similar habitat and behavioural ecology with both Cinnyris spp., it closely resembles its relative, P. castanea, in all aspects of haemosporidian parasitism.

Host specificity of passerine Lankesterella (Apicomplexa: Coccidia).

Lankesterella parasites are blood coccidians that have recently gained attention as their records in common passerine species emerge. To date, their occurrence has been molecularly confirmed in several passerine genera, mainly among members of the families Paridae and Acrocephalidae. Despite their relatively high prevalence in some host populations, their life cycles remain unclear, mosquitoes or mites being the proposed vectors. The aim of this study was to reveal Lankesterella host specificity, focusing mainly on parasites of tit and warbler species (families Paridae and Acrocephalidae). We have determined the 18S rRNA gene sequences of Lankesterella from 35 individuals belonging to eight different host species. Phylogenetic analysis revealed that passerine Lankesterella are host-specific, with specificity at the host genus or species level. Besides Lankesterella, Isospora sequences were obtained from avian blood as well, pointing out the need for barcoding.

Species of Reticulipeurus Kéler, 1958 (Phthiraptera, Ischnocera, Oxylipeurus-complex) parasitic on species of Arborophila, with description of a new subgenus and three new species.

A new subgenus and three new species of parasitic lice (Phthiraptera: Ischnocera) in the Oxylipeurus-complex are described and illustrated. Forcipurellus new subgenus is part of the genus Reticulipeurus Kéler, 1958, and only includes species parasitic on partridges of the genus Arborophila Hodgson, 1837. The new species are: Reticulipeurus (Forcipurellus) diki new species ex Arborophila rubrirostris (Salvadori, 1879); Reticulipeurus (Forcipurellus) bracatus new species ex Arborophila atrogularis (Blyth, 1849); Reticulipeurus (Forcipurellus) longistylus new species ex Arborophila rufogularis guttata Delacour & Jabouille, 1928 and two other host subspecies. In addition, we redescribe the species Reticulipeurus (Forcipurellus) formosanus (Uchida, 1917) ex Arborophila crudigularis (Swinhoe, 1864) and Reticulipeurus (Forcipurellus) nitzschi Kéler, 1958 ex Arborophila torqueola torqueola (Valenciennes, 1825) and Arborophila torqueola millardi (Baker, 1921). Also, we briefly discuss four additional species that could not be described based on the specimens examined. A key to identify all species in the subgenus Forcipurellus is provided, as well as an emendation to include Forcipurellus in a previously published key to the Oxylipeurus-complex.

Exo-erythrocytic development of two Haemoproteus species (Haemosporida, Haemoproteidae), with description of Haemoproteus dumbbellus, a new blood parasite of bunting birds (Emberizidae).

Avian haemosporidians are widespread parasites categorized into four families of the order Haemosporida (Apicomplexa). Species of the subgenus Parahaemoproteus (genus Haemoproteus) belong to the Haemoproteidae and are transmitted by Culicoides biting midges. Reports of death due to tissue damage during haemoproteosis in non-adapted birds have raised concerns about these pathogens, especially as their exo-erythrocytic development is known for only a few Haemoproteus spp. More research is needed to better understand the patterns of the parasites' development in tissues and their impact on avian hosts. Yellowhammers Emberiza citrinella (Emberizidae) and common house martins Delichon urbicum (Hirundinidae) were screened for Haemoproteus parasites by microscopic examination of blood films and PCR-based testing. Individuals with single infection were selected for histological investigations. H & E-stained sections were screened for detection and characterization of the exo-erythrocytic stages, while chromogenic in situ hybridization (CISH) and phylogenetic analysis were performed to confirm the Haemoproteus origin and their phylogenetic relationships. Haemoproteus dumbbellus n. sp. was discovered in Emberiza citrinella single-infected with the lineage hEMCIR01. Meronts of H. dumbbellus n. sp. developed in various organs of five of six tested individuals, a pattern which was reported in other Haemoproteus species clustering in the same clade, suggesting this could be a phylogenetic trait. By contrast, in Delichon urbicum infected with the Haemoproteus lineage hDELURB2, which was linked to the more distantly related parasite Haemoproteus hirundinis, only megalomeronts were found in the pectoral muscles of two of six infected individuals. All exo-erythrocytic stages were confirmed to be Haemoproteus parasites by CISH using a Haemoproteus genus-specific probe. While the development of meronts seems to be typical for species of the clade containing H. dumbbellus, further investigations and data from more species are needed to explore whether a phylogenetic pattern occurs in meront or megalomeront formation.

New reports of parasitism by Synhimantus (Dispharynx) nasuta (Rudolphi, 1819) (Nematoda: Acuariidae) in wild birds in Brazil.

The aim of this study was to register the first occurrence of Synhimantus (Dispharynx) nasuta (Nematoda: Acuariidae) in some species of wild birds in Brazil. In addition, the aim was to deepen the knowledge about the morphology of this species through analysis using scanning electron microscopy. Nematodes were collected in wild birds of the species Turdus leucomelas, T. rufiventris, Mimus saturninus, Pitangus sulphuratus, Megascops choliba, Tyto furcata, and Falco sparverius. The morphological and morphometric data observed in the nematodes prove that these parasites are S. (D.) nasuta. This study also provides morphological data from light microscopy and scanning electron microscopy (SEM), as well as the morphometry of this nematode in each host species. Therefore, the current study confirms the first record of this nematode in F. sparverius and T. furcata in South America and, at the same time, these findings expand the host range of this parasite species worldwide, through the first records in M. choliba, M. saturninus, T. leucomelas and T. rufiventris.

First evidence for development of Plasmodium relictum (Grassi and Feletti, 1891) sporozoites in the salivary glands of Culex modestus Ficalbi, 1889.

The competence of insect vectors to transmit diseases plays a key role in host-parasite interactions and in the dynamics of avian malaria and other haemosporidian infections (Apicomplexa, Haemosporida). However, the presence of parasite DNA in the body of blood-sucking insects does not always constitute evidence for their competence as vectors. In this study, we investigate the susceptibility of wild-caught mosquitoes (Culex spp.) to complete sporogony of Plasmodium relictum (cyt b lineage SGS1) isolated from great tits (Parus major L., 1758). Adult female mosquitoes were collected with a CO2 bait trap overnight. A set of 50 mosquitoes was allowed to feed for 3 h at night on a single great tit infected with P. relictum. This trial was repeated on 6 different birds. The bloodfed mosquitoes that survived (n = 68) were dissected within 1-2 days (for ookinetes, n = 10) and 10-33 days post infection (for oocysts and sporozoites, n = 58) in order to confirm the respective parasite stages in their organs. The experiment confirmed the successful development of P. relictum (cyt b lineage SGS1) to the stage of sporozoites in Culex pipiens L., 1758 (n = 27) and in Culex modestus (n = 2). Our study provides the first evidence that C. modestus is a competent vector of P. relictum isolated from great tits, suggesting that this mosquito species could also play a role in the natural transmission of avian malaria.

Integrative taxonomy for the traditional coccidians (Chromista: Miozoa: Eimeriidae) from island canaries (Aves: Passeriformes: Fringillidae): Worldwide distribution, morphological and molecular characterization, revaluations and establishment of junior synonyms.

Island canaries Serinus canaria (Linnaeus) are finches native to the North Atlantic Islands, however, they have a worldwide distribution in captivity due to their relevance as a pet bird. Coccidians are the most reported parasites of passerines worldwide, both in the wild and in captivity, being frequently associated with disease in passerines kept in rehabilitation centers and commercial breeders. This study aimed to identify coccidians from island canaries kept in captivity in Brazil. Three hundred and fifteen genomic DNA extracted from fecal samples of island canaries from different breeders from Southern and Southeastern Brazil were used to perform a nested PCR assay to amplify a partial fragment of the 28S small subunit ribosomal RNA gene (28S) of Isospora spp. Microscopic screening and morphological identification of Isospora oocysts was performed in fecal samples corresponding to PCR positive DNA samples. Fecal samples have been formalin-stored for approximately four years. Positivity rate for both microscopy and PCR was 10.5% (33/315). Posteriorly, Isospora serini (Aragão, 1933) Box, 1975 and Isospora canaria Box, 1975 were morphologically identified from fresh fecal samples of island canaries maintained by a breeder in the State of São Paulo, Southeastern Brazil, providing a genotypic characterization via sequencing of the mitochondrial cytochrome c oxidase subunit 1 (COI) and 28S genes. The 28S and COI sequences referring to the morphological identification of I. canaria was, respectively, 100% and 99% similar to sequences deposited as Isospora serinuse Yang, Brice, Elliot & Ryan, 2015 from island canaries kept in a rehabilitation center in Australia. The COI sequence referring to the morphological identification of I. serini was 100% similar to a sequence of an extraintestinal Isospora, corroborating this identification/sequencing since I. serini is the first isosporan with an extra-intestinal cycle demonstrated. The comparison of morphological and molecular data from I. canaria and I. serini from this study with published data of Isospora spp. from canaries worldwide, allowed the specific identification from preliminary generic identifications, correction of misidentifications, as well as the establishment of junior synonyms. Finally, this study provides morphological and molecular data that ensure the correct identification of the two Isospora spp. from island canaries in future studies worldwide.

A New Isospora (Apicomplexa: Eimeriidae) from the Dark-Eyed Junco, Junco hyemalis (L.) (Passeriformes: Passerellidae), in West-Central Arkansas, USA.

PURPOSE: Many passeriform birds are excellent hosts of coccidian parasites but little is known of the coccidians infecting members of the family Passerellidae, particularly from the dark-eyed junco, Junco hyemalis (Linnaeus, 1758). Here, we report from morphometric and mensural data a new species of Isospora from J. hyemalis in Arkansas, USA. METHODS: During November 2020, seven adult J. hyemalis were taken with a mist net from Polk County, Arkansas, USA, and their faecal samples examined for coccidian parasites. Samples were placed in individual vials containing aqueous potassium dichromate. They were examined for coccidia after flotation in Sheather's sugar solution, measured, and photographed. RESULTS: A single J. hyemalis (14%) was found to be passing a new species of Isospora. Oöcysts of Isospora pittmani n. sp. were spheroidal to subspheroidal with a smooth bi-layered wall, measured (L × W) 24.8 × 23.8 µm, and had a length/width (L/W) ratio of 1.04; a micropyle and oöcyst residuum was absent but typically one to up to four polar granule(s) was present. Sporocysts are ovoidal to ellipsoidal and measured 15.0 × 10.0 µm, L/W 1.5; a flattened Stieda body was present as well as a barely discernible sub-Stieda body but para-Stieda bodies were absent. The sporocyst residuum was composed of various-sized granules dispersed between and across the sporozoites. A spheroidal to ellipsoidal posterior refractile body and spheroidal anterior refractile body occasionally seen, but a nucleus was not observed. Only two previous isosporans have been reported from passerellid birds and the new species can readily be distinguished from them. CONCLUSION: This is the first coccidian reported from J. hyemalis and, more importantly, the first isosporan known from any member of the family Passerellidae in North America.

Chigger mite (Acariformes: Trombiculidae) infestation in reed passerine birds in Central Europe: a case of the bearded tit Panurus biarmicus.

Larval trombiculid (chigger) mites are common ectoparasites of terrestrial vertebrates including humans, causing itching and skin inflammation known as trombiculiasis. Investigation of their diversity, distribution and seasonal abundance is therefore important from a veterinary and public health point of view. Although researchers have paid increased attention to these parasites in recent years, there is still little ecological data available on chiggers associated with birds inhabiting different types of habitats such as wetlands, for example. In 2021, we investigated the mite fauna in a specialist reedbed passerine, the bearded tit (Panurus biarmicus), and their effects on this host in the south-west Slovakia, Central Europe. A total of 1134 larvae of 1 mite species Blankaartia acuscutellaris were found in 99 out of 267 examined bearded tits. Juveniles were more infested than adult birds, but no differences were found between sexes. The larvae of mites first appeared on the host during the second half of June and peaked in the second half of July. After that, their numbers decreased gradually until October. Despite the relatively high prevalence and intensity of mite infestation in the bearded tit, no differences in body condition between infested and uninfested birds suggest that infestation by B. acuscutellaris may not have serious negative effects on the host health. Bearded tits can therefore be a reliable indicator of the presence of the chigger mites in wetland habitats.

Redescription of Isospora vanriperorum Levine, 1982 (Apicomplexa: Eimeriidae) from the Northern Cardinal, Cardinalis cardinalis (L.) (Passeriformes: Cardinalidae), in North Texas, USA.

Little is known about the coccidian parasites of North American birds of the family Cardinalidae. A single isosporan, Isospora vanriperorum was previously described over 40 years ago from the northern cardinal, Cardinalis cardinalis from Hawaii. Since then, it was also reported in the green-winged saltator, Saltator similis from Brazil but the identification was invalidated recently. Feces were collected in July 2022 from two C. cardinalis found dead in Lamar County, Texas, USA. One sample contained oöcysts representing I. vanriperorum. Oöcysts are spheroidal to subspheroidal with a smooth uni-layered wall, measure (L × W) 23.8 × 22.4 µm and have a length/width (L/W) ratio of 1.04; a micropyle and oöcyst residuum are absent but polar granule(s) are present. Sporocysts are ovoidal and measure 15.5 × 10.5 µm, L/W 1.5; a knob-like Stieda body and a rounded sub-Stieda body are present. The sporocyst residuum is more often composed of various-sized granules dispersed between and across the sporozoites; it is less often in a compact rounded mass. This isosporan has now been reported from the northern cardinal for the second time but most importantly, from the mainland USA for the first time.

A new isosporan (Apicomplexa: Eimeriidae) from barn swallow, Hirundo rustica (Aves: Passeriformes: Hirundinidae: Hirundininae), in Oklahoma, USA.

Compared to other commonly examined passerine birds for coccidian (Apicomplexa) parasites, little is available on the coccidia of birds of the family Hirundinidae, including barn swallows (Hirundo rustica). Feces was collected in May 2022 from two nesting H. rustica in McCurtain County, Oklahoma, USA; the samples contained oöcysts representing a new species of Isospora. Oöcysts of Isospora zimmermani n. sp. are subspheroidal to ovoidal with a smooth bi-layered wall, measure (L × W) 22.8 × 20.8 µm, and have a length/width (L/W) ratio of 1.1; a micropyle and oöcyst residuum were absent but polar granule(s) are present. Sporocysts are ellipsoidal to ovoidal and measure 15.2 × 9.6 µm, L/W 1.6; a knob-like Stieda body and a rounded sub-Stieda body are present. The sporocyst residuum is composed of compact spheroid with a dense, irregular mass of finer granules lying between and dispersed among the sporozoites. The new species represents only the second isosporan reported from H. rustica but the first from the USA, and fourth species documented from the avian family Hirundinidae. This article was registered in the Official Register of Zoological Nomenclature (ZooBank) as urn:lsid:zoobank.org:pub:1F1E71C3-56E8-4EA7-A9B0-1E585FAA9DE0.

A mass rearing cost calculator for the control of Culex quinquefasciatus in Hawai'i using the incompatible insect technique.

BACKGROUND: Hawai'i's native forest avifauna is experiencing drastic declines due to climate change-induced increases in temperature encroaching on their upper-elevation montane rainforest refugia. Higher temperatures support greater avian malaria infection rates due to greater densities of its primary vector, the southern house mosquito Culex quinquefasciatus, and enhance development of the avian malaria parasite Plasmodium relictum. Here we propose the use of the incompatible insect technique (IIT) or the combined IIT/sterile insect technique (SIT) for the landscape-scale (i.e., area-wide) control of Cx. quinquefasciatus, and have developed a calculator to estimate the costs of IIT and IIT/SIT applications at various sites in Hawai'i. METHODS: The overall cost of the infrastructure, personnel, and space necessary to produce incompatible adult males for release is calculated in a unit of ~ 1 million culicid larvae/week. We assessed the rearing costs and need for effective control at various elevations in Hawai'i using a 10:1 overflooding ratio at each elevation. The calculator uses a rate describing the number of culicids needed to control wild-type mosquitoes at each site/elevation, in relation to the number of larval rearing units. This rate is a constant from which other costs are quantified. With minor modifications, the calculator described here can be applied to other areas, mosquito species, and similar techniques. To test the robustness of our calculator, the Kaua'i-specific culicid IIT/SIT infrastructure costs were also compared to costs from Singapore, Mexico, and China using the yearly cost of control per hectare, and purchasing power parity between sites for the cost of 1000 IIT/SIT males. RESULTS: As a proof of concept, we have used the calculator to estimate rearing infrastructure costs for an application of IIT in the Alaka'i Wilderness Reserve on the island of Kaua'i. Our analysis estimated an initial investment of at least ~ $1.16M with subsequent yearly costs of approximately $376K. Projections of rearing costs for control at lower elevations are ~ 100 times greater than in upper elevation forest bird refugia. These results are relatively comparable to those real-world cost estimates developed for IIT/SIT culicid male production in other countries when inflation and purchasing power parity are considered. We also present supplemental examples of infrastructure costs needed to control Cx. quinquefasciatus in the home range of 'i'iwi Drepanis coccinea, and the yellow fever vector Aedes aegypti. CONCLUSIONS: Our cost calculator can be used to effectively estimate the mass rearing cost of an IIT/SIT program. Therefore, the linear relationship of rearing infrastructure to costs used in this calculator is useful for developing a conservative cost estimate for IIT/SIT culicid mass rearing infrastructure. These mass rearing cost estimates vary based on the density of the targeted organism at the application site.

Chewing lice of the Brueelia-complex (Phthiraptera: Ischnocera) parasitic on members of the Campephagidae (Aves: Passeriformes), with description of a new subgenus and 14 new species.

Fourteen new species of chewing lice in the Brueelia-complex are described, from hosts in the Campephagidae. In addition, Malardifax a new subgenus of Guimaraesiella Eichler, 1949 is described for the species occurring on minivets (Campephagidae: Pericrocotus). The new species and their hosts are: Guimaraesiella (Guimaraesiella) nouankaoensis n. sp. from Coracina caledonica seiuncta Mayr Ripley, 1941 and C. caledonica thilenii (Neumann, 1915); Guimaraesiella (Guimaraesiella) sphagmotica n. sp. from Coracina caeruleogrisea strenua (Schlegel, 1871) and C. caeruleogrisea adamsoni Mayr Rand, 1936; Indoceoplanetes (Capnodella) kamphaengphetensis n. sp. from Lalage melaschistos avensis (Blyth, 1852); Indoceoplanetes (Capnodella) saucia n. sp. from Edolisoma montanum montanum (Meyer, 1874); Indoceoplanetes (Capnodella) subarcens n. sp. from Edolisoma melas melas (Lesson, 1828); Indoceoplanetes (Indoceoplanetes) cinitemnina n. sp. from Edolisoma melas melas (Lesson, 1828); Indoceoplanetes (Indoceoplanetes) ephippiformis n. sp. from Edolisoma montanum montanum (Meyer, 1874); Indoceoplanetes (Indoceoplanetes) fodincana n. sp. from Coracina papuensis oriomo Mayr Rand, 1936, C. papuensis angustifrons (Salvadori, 1876), and C. papuensis elegans (Ramsay, 1881); Indoceoplanetes (Indoceoplanetes) incisoma n. sp. from Coracina macei siamensis (Baker, 1918) and C. macei rexpineti (Swinhoe, 1863); Indoceoplanetes (Indoceoplanetes) microgenitalis n. sp. from Coracina caeruleogrisea strenua (Schlegel, 1871); Indoceoplanetes (Indoceoplanetes) pterophora n. sp. from Coracina macei nipalensis (Hodgson, 1836); Indoceoplanetes (Indoceoplanetes) saburrata n. sp. from Coracina lineata ombriosa (Rothschild Hartert, 1905); Indoceoplanetes (Indoceoplanetes) wandoensis n. sp. from Coracina novaehollandiae melanops (Latham, 1802); and Indoceoplanetes (Indoceoplanetes) zambica n. sp. from Coracina pectoralis (Jardine Selby, 1828). Checklists and keys to the louse species of the Brueelia-complex parasitic on campephagid hosts are provided.

Transcriptional response of individual Hawaiian Culex quinquefasciatus mosquitoes to the avian malaria parasite Plasmodium relictum.

BACKGROUND: Plasmodium parasites that cause bird malaria occur in all continents except Antarctica and are primarily transmitted by mosquitoes in the genus Culex. Culex quinquefasciatus, the mosquito vector of avian malaria in Hawai'i, became established in the islands in the 1820s. While the deadly effects of malaria on endemic bird species have been documented for many decades, vector-parasite interactions in avian malaria systems are relatively understudied. METHODS: To evaluate the gene expression response of mosquitoes exposed to a Plasmodium infection intensity known to occur naturally in Hawai'i, offspring of wild-collected Hawaiian Cx. quinquefasciatus were fed on a domestic canary infected with a fresh isolate of Plasmodium relictum GRW4 from a wild-caught Hawaiian honeycreeper. Control mosquitoes were fed on an uninfected canary. Transcriptomes of five infected and three uninfected individual mosquitoes were sequenced at each of three stages of the parasite life cycle: 24 h post feeding (hpf) during ookinete invasion; 5 days post feeding (dpf) when oocysts are developing; 10 dpf when sporozoites are released and invade the salivary glands. RESULTS: Differential gene expression analyses showed that during ookinete invasion (24 hpf), genes related to oxidoreductase activity and galactose catabolism had lower expression levels in infected mosquitoes compared to controls. Oocyst development (5 dpf) was associated with reduced expression of a gene with a predicted innate immune function. At 10 dpf, infected mosquitoes had reduced expression levels of a serine protease inhibitor, and further studies should assess its role as a Plasmodium agonist in C. quinquefasciatus. Overall, the differential gene expression response of Hawaiian Culex exposed to a Plasmodium infection intensity known to occur naturally in Hawai'i was low, but more pronounced during ookinete invasion. CONCLUSIONS: This is the first analysis of the transcriptional responses of vectors to malaria parasites in non-mammalian systems. Interestingly, few similarities were found between the response of Culex infected with a bird Plasmodium and those reported in Anopheles infected with human Plasmodium. The relatively small transcriptional changes observed in mosquito genes related to immune response and nutrient metabolism support conclusions of low fitness costs often documented in experimental challenges of Culex with avian Plasmodium.

Molecular phylogenetics of the avian feather louse Philopterus-complex (Phthiraptera: Philopteridae).

The avian feather louse Philopterus-complex (Phthiraptera: Ischnocera: Philopteridae) currently contains 12 genera that have been grouped together because of shared morphological characteristics. Although previously lumped into a single genus (Philopterus), more recent morphological treatments have separated the group into several different genera. Here we evaluate the status of these genera using DNA sequence data from 118 ingroup specimens belonging to ten genera in the Philopterus-complex: Australophilopterus Mey, 2004, Cinclosomicola Mey 2004, Clayiella Eichler, 1940, Corcorides Mey, 2004, Mayriphilopterus Mey, 2004, Paraphilopterus Mey 2004, Philopteroides Mey 2004, Philopterus Nitzsch, 1818, Tyranniphilopterus Mey, 2004, and Vinceopterus Gustafsson, Lei, Chu, Zou, and Bush, 2019. Our sampling includes 97 new louse-host association records. Our analyses suggest that the genus Debeauxoecus Conci, 1941, parasitic on pittas (Aves: Pittidae), is outside of the Philopterus-complex, and that there is strong support for the monophyly of a group containing the remaining genera from the complex. Some diverse genera, such as Philopterus (sensu stricto) and Mayriphilopterus are supported as monophyletic, whereas the genera Australophilopterus, Philopteroides, and Tyranniphilopterus are not. The present study is the largest phylogenetic reconstruction of avian lice belonging to the Philopterus-complex to date and suggests that further generic revision is needed in the group to integrate molecular and morphological information.

The First Isospora Species (Apicomplexa: Eimeriidae) Described from the Northern Yellow-Shafted Flicker (Colaptes auratus luteus) in Ontario, Canada.

PURPOSE: Members of the genus Isospora commonly infect a wide variety of wild birds. By combining morphometric and genetic data, we are able to differentiate and describe a new apicomplexan parasite, Isospora picoflavae n. sp., from the Northern Yellow-shafted Flicker (Colaptes auratus luteus) in Ontario, Canada. METHODS: Unsporulated oocysts were observed in intestinal contents obtained during necropsy; these oocysts completed exogenous sporulation within 7 days at room temperature. Complete nuclear 18S rDNA and complete mitochondrial genome sequences were obtained from this previously undescribed Isospora sp. to compare with related coccidia. RESULTS: Sporulated oocysts were subspherical in shape measuring an average of 22.7 × 21.7 µm (n = 53; mean shape index 1.05) containing a near-spherical polar granule. Sporocysts were ovoidal to ellipsoidal in shape, measuring an average size of 17.0 × 10.5 µm (n = 9; mean shape index 1.62). Sporocyst residuum was concentrated in an irregular, spherical mass in the middle of each sporocyst. The isolated oocysts differed morphologically from other Isospora species reported to infect members of the family Picidae. Based on phylogenetic analyses using either combined nuclear 18S rDNA and mitochondrial cytochrome c oxidase I (COI) sequences or complete mitochondrial genome sequences, this Isospora sp. n. isolated from the Northern Flicker grouped within a clade containing Isospora species described from various passeriform hosts. CONCLUSION: Based on combined morphological and molecular data, the oocysts found in the gastrointestinal contents of Colaptes auratus luteus represent a new species of Isospora named herein as Isospora picoflavae n. sp.