First record of the parasitoid wasp Ixodiphagus hookeri (Hymenoptera: Encyrtidae) infesting the tick Amblyomma nodosum (Acari: Ixodidae)

Ticks (Ixodida) are ecologically important ectoparasites that may impact human health and economic activities. Parasitoid wasps are natural enemies of ticks and a potential option for its biological control. In the present study, we recorded parasitism of the nymphs of the tick Amblyomma nodosum by the parasitoid wasp Ixodiphagus hookeri. The ticks, in turn, were parasitizing white-bearded manakins, Manacus manacus (Passeriformes: Pipridae) in the União Biological Reserve, one of the last major remnants of lowland rainforest in Rio de Janeiro state, southeastern Brazil. During the collection of ectoparasites from two manakins in this reserve, 20–30 parasitoid wasps hatched spontaneously from two of the ticks collected. The species of the tick nymphs and adult wasps were identified using morphological traits and molecular analyses. This record is the first documented case of I. hookeri parasitizing A. nodosum and the first time that parasitoid wasps have been found in ticks parasitizing birds in Brazil. This is also the first record of Ixodiphagus from southeastern Brazil in more than a century, with the only other record dating back to 1914. The record presented here provides valuable new information on the biology of this tick and its parasitoids, and new insights into this interspecific interaction. Further research on these vertebrate-tick-wasp interactions will be necessary to better understand the respective roles of the organisms involved in these processes.

species, Amblyomma nodosum Neumann, 1899, is amply distributed in the Neotropics, between Mexico and Argentina. The immature stages (larvae and nymphs) of A. nodosum parasitize birds (Guglielmone et al. 2021), while the final hosts are mammals (anteaters) of the family Myrmecophagidae, which include the genera Myrmecophaga and Tamandua (Pinheiro et al. 2015;Guglielmone et al. 2021). Amblyomma nodosum is found primarily in pristine or wellpreserved environments (Ogrzewalska et al. 2009).
Given the potential impacts of ticks on public health and economic activities, measures are often implemented for the control of the populations of these parasites. While widely used, chemical controls (i.e., acaricides) have certain limitations, due primarily to the development of resistance in the ticks (Hu et al. 1998). The cosmopolitan parasitoid wasps of the genus Ixodiphagus are known to parasitize many different tick species and, as they invariably result in the mortality of their hosts, they are widely considered to be potential agents for the biological control of tick populations (Mwangi et al. 1991;Hu et al. 1998). However, as the biology of their interspecific interactions Section Editor: Charlotte Oskam * Maria Alice S. Alves masaalves19@gmail.com is still poorly-known, in general, further research is needed to improve this potential. In the present study, we recorded the parasitism of nymphs of Amblyomma nodosum by wasps of the species Ixodiphagus hookeri (Howard, 1908). The ticks, in turn, were found parasitizing white-bearded manakins, Manacus manacus (Linnaeus, 1766) (Passeriformes: Pipridae), in the Atlantic Forest of the União Biological Reserve, in the state of Rio de Janeiro, southeastern Brazil. The União Biological Reserve, which has a total area of 7769 hectares, is one of the last major remnants of lowland rainforest in Rio de Janeiro state. During a study of ectoparasitism by ticks in the manakins (Pipridae) of this reserve, we collected two Amblyomma nymphs from two individuals of M. manacus. One tick was collected on April 7th 2018, from an individual with female plumage, and the other from a male on June 26th of the same year. Following collection, between 20 and 30 parasitoid wasps hatched spontaneously from each tick through a hole in its idiosome.
The two nymphs were morphologically identical, and their taxon was identified based on their morphology (Martins et al. 2010). The specimens were subjected to total genomic DNA extraction (Aljanabi and Martinez 1997) and the analysis of a ~ 460 bp fragment of the mitochondrial 16S rDNA gene, following the protocol described by Bitencourth et al. (2019). To determine the species, this sequence was amplified and analyzed by the maximum likelihood approach at the Laboratory of Ticks and Other Apterous Arthropods (LAC), at the Oswaldo Cruz Institute (FIOCRUZ) in Rio de Janeiro.
The adult wasps were identified from their morphology, based on Quaraishi (1958). Samples of DNA were also extracted from four specimens for the molecular confirmation of the species. For this, the genomic DNA was extracted from each sample using the Wizard® Genomic DNA Purification kit (Promega, USA) in accordance with the manufacturer's instructions. A PCR was then performed to amplify a partial (294 bp) sequence of the cytochrome c oxidase subunit 1 (COI) gene, using the primers Ihookcox1F 5′-TTA GAT GAT TAG CTT CAA TAA ATG GAAT-3′ and Ihook-cox1R 5′-CCA AAA ATT GCA AAA ACT GCT CCT AT-3′, designed from a partial I. hookeri COI sequence available in GenBank, following Ramos et al. (2015). The amplicons were sequenced in both directions using the same primers used for the PCR, with the Big Dye Terminator v.3.1 kit in a 3130 genetic analyzer (Applied Biosystems, CA, USA). A phylogenetic tree was constructed using the neighbor-joining method (Saitou and Nei 1987). Bootstrap resampling (1000 replicates) was performed for the statistical support of the reliabilities of the nodes on the trees (Felsenstein 1985) using the MEGA software, version 11 (Tamura et al. 2021). Aedes albopictus (GenBank accession number MN080750) was used as outgroup ( Supplementary Fig. 1).
The two nymphs were identified as A. nodosum based on their morphology. The analysis of the 16S rDNA sequence (GenBank accession number OP646872) obtained from one of the nymphs corroborated this classification, given that the sequence was 100% (452/452) similar to that of A. nodosum from GenBank (MW654243) and, in the phylogenetic reconstruction, it was assigned to the same clade as this ixodid species (Supplementary Fig. 2).
All the adult wasps were identified as I. hookeri based on their morphology. The PCR amplified a fragment of approximately 294 bp from all four samples (four adults) tested. The fragment sequenced (212 bp) was 99% similar to the I. hookeri sequences available in GenBank (accession number JQ315225). The sequences obtained in the present study were deposited in GenBank under accession numbers MW711784, MW711785, MW711786, and MW711787. The I. hookeri strains clustered independently in the tree branch, supported by high bootstrap value.
Our record of I. hookeri is the first documented case of wasps parasitizing A. nodosum and the first time that wasps have been found in ticks parasitizing birds in Brazil. This is also the first record of Ixodiphagus from southeastern Brazil for 104 years, given that the only previous report was that of Costa-Lima (1915), who identified I. hookeri in nymphs of Rhipicephalus sanguineus sensu lato (Latreille, 1806) collected from a domestic dog in the municipality of Rio de Janeiro in 1914. The four other published records of Ixodiphagus from Brazil were obtained from ticks parasitizing mammalian hosts (dogs and a jaguar, Panthera onca (Linnaeus, 1758)) in other regions (the north, northeast, and midwest) of this country (Table 1).
In the specific case of Ixodiphagus wasps infesting ticks found on passeriform hosts, there are records from three continents. Hoogstraal and Kaiser (1961) recorded Ixodiphagus in ticks collected from northern wheatear, Oenanthe oenanthe (Linnaeus, 1758), isabelline wheatear, Oenanthe isabellina (Temminck, 1829), and common redstart, Phoenicurus phoenicurus (Linnaeus, 1758) in Egypt, while Bowman et al. (1986) found them in field sparrow, Spizella pusilla (Wilson, 1810) in the USA. In New Zealand, Heath and Cane (2010) found the wasps in ticks collected from substrates associated with nests in a breeding colony of Stewart Island shag, Leucocarbo chalconotus (Gray, 1845).
Amblyomma nodosum is not considered to represent a potential risk to public health, primarily because there have been no reports of parasitism in humans up to now (Guglielmone et al. 2021). However, a number of different Rickettsia species have already been detected in this ixodid, including Rickettsia belli, Rickettsia amblyommatis, and Rickettsia parkeri strain NOD (see Moerbeck et al. 2018). The latter is closely related to the Atlantic rainforest strain, which is one of the bioagents of spotted fever in Brazil. This implies a potential, but still undetermined role in the epidemiology of rickettsiosis (Pinheiro et al. 2015), in particular in the enzootic cycle. When dispersed by birds, the immature forms of A. nodosum may bring this rickettsia into contact with ticks that parasitize humans (Ogrzewalska et al. 2009).
As the immature ticks of the genus Amblyomma parasitize birds (Guglielmone et al. 2021) and the parasitoid wasps are associated primarily with tick nymphs, often when engorged (Ramos et al. 2015;Santos et al. 2017), it seems likely that many more wasp species may be found in ticks infesting bird hosts. It is also important to determine which vertebrate hosts are infested by the ticks, and their relationship with the prevalence of parasitoids in these ticks, in order to better understand how the parasitoid populations are sustained and whether chemical signals emitted by the ticks attract the I. hookeri females (Buczek et al. 2021).
The type of vertebrate host may also be fundamental to the infection of the wasps by microorganisms, given that the wasp typically selects feeding nymphs and consumes the entire contents of its host (Bohacsova et al. 2016;Krawczyk et al. 2020). Adult wasps (I. hookeri) that parasitized Ixodes ricinus have tested positive for pathogens such as Rickettsia helvetica and Rickettsia monacensis (Bohacsova et al. 2016), although it is not known whether or to what extent these pathogens can be transmitted to other ticks when parasitized by infected wasps. This reinforces the importance of understanding the role that the bird hosts of the ticks play in this ecological relationship.
Manacus manacus, which was the passerine host of the A. nodosum specimens parasitized by I. hookeri in the present study, is a resident bird with disjunct populations in the Amazon and Atlantic Forest biomes (Sick 1997). This manakin tends to be associated with secondary forest, dense vegetation, and forest edges (Sick 1997). Amblyomma nodosum is known to parasitize a large number of bird species and is often found parasitizing wild birds in Brazil and, in the Atlantic Forest, it has already been detected in dozens of bird species, including M. manacus (Labruna et al. 2007).
During a comprehensive study of the ticks that parasitize birds in the Atlantic Forest of the União Biological Reserve, M.O.M.V. (unpublished data) recorded A. nodosum, Amblyomma longirostre (Koch, 1844), Amblyomma ovale Koch, 1844, and Rhipicephalus sanguineus sensu lato (s.l.). Only one of these species-R. sanguineus s.l.-was known to be parasitized by wasps (Lopes et al. 2012;Santos et al. 2017), although there are records for the genus Amblyomma, albeit with no species identification (Lopes et al. 2012).
The potential of I. hookeri for the biological control of these ticks has already been assessed in laboratory trials, based on experimental infestation (Mwangi et al. 1991). However, many gaps persist in the understanding of the biology, distribution, and ecology of these parasitoid wasps, which hampers the development of reliable biological control measures. It is possible, in fact, that there is a correlation between the prevalence of wasp parasitism and the population density of the ticks and their hosts (Krawczyk et al. 2020;Buczek et al. 2021), in addition to the influence of other factors, such as microclimatic variables (Krawczyk et al. 2020).
Our record is the first case of A. nodosum as host of parasitoid wasps. This may constitute a valuable contribution to the eventual planning of effective strategies for the control of ixodid populations, the understanding of the natural impact of the parasitoids, and even their potential role in biological control (Barros-Battesti et al. 2006;Lopes et al. 2012;Santos et al. 2017), as well as the interaction network  Lopes et al. (2012) in general. Clearly, however, further studies of vertebratetick-wasp interactions will be necessary to understand the relative roles of these organisms more fully.