Hua et al. Parasites Vectors (2019) 12:579 https://doi.org/10.1186/s13071-019-3831-y Parasites & Vectors RESEARCH Open Access Morphology is not a reliable taxonomic tool for the genus Lernaea: molecular data and experimental infection reveal that L. cyprinacea and L. cruciata are conspecifc Cong J. Hua1,2, Dong Zhang1,3, Hong Zou1, Ming Li1, Ivan Jakovlić4, Shan G. Wu1, Gui T. Wang1,3 and Wen X. Li1* Abstract Background: Species belonging to the genus Lernaea are cosmopolitan parasites that can infect many diferent freshwater fsh hosts. Due to a high degree of morphological intraspecifc variability and high levels of interspecifc similarities, their classifcation is extremely difcult and controversial. Although the suitability of the shape of cephalic horns has been questioned decades ago by some experimental infection studies, this character still plays the central role in the identifcation of Lernaea spp. Methods: We used the nominal species Lernaea cyprinacea and Lernaea cruciata to test the hypothesis that the shape of the anchor can exhibit host-induced morphological variability, and that the two taxa may be synonymous. Results: We examined 517 wild or farmed specimens of fve host fsh species (four cyprinids and a mosquitofsh), and found that all 16 parasite specimens collected from mosquitofsh could be morphologically identifed as L. cruciata, whereas the remaining 25 parasite specimens were all identifed as L. cyprinacea. We experimentally infected goldfsh and mosquitofsh specimens with ofspring (copepodids) of a single L. cyprinacea specimen: the adult parasites from goldfsh were morphologically identifed as L. cyprinacea, and those from mosquitofsh as L. cruciata. We then used molecular data to corroborate that all these specimens are conspecifc. Conclusions: Our results suggest that L. cyprinacea and L. cruciata may be synonyms, misidentifed as diferent species as a result of host-induced morphological variation. Given the current shortage of molecular data for the genus Lernaea, in order to resolve the taxonomy of this genus (determine the exact number of species), future studies should aim to sequence as much molecular data as possible, and conduct further experimental infections. Keywords: Lernaea, Parasitic copepods, Phenotypic plasticity, Molecular taxonomy Background [1–3], causing lernaeosis, a disease that can cause serious Cosmopolitan parasitic copepods belonging to the pathogenic efects on their hosts. Te taxonomy of this genus Lernaea Linnaeus, 1758 (Cyclopoida: Lernaei- genus is still largely unresolved due to the existence of dae) can infect many diferent freshwater fsh species an exceptionally large number (109) of recorded nominal species [4], a small number of morphological traits useful for species identifcation, a high degree of intraspecifc *Correspondence: [email protected] morphological variability, and a lack of clear morphologi- 1 Key Laboratory of Aquaculture Disease Control, Ministry of Agriculture, cal distinction between some species [5–8]. As a result, and State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, almost half (48) of the nominal species are believed to be People’s Republic of China synonymous [4]. Full list of author information is available at the end of the article © The Author(s) 2019. 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The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Hua et al. Parasites Vectors (2019) 12:579 Page 2 of 13 Te shape of the cephalic anchors, also known as the a single sufciently reliable tool for the identifcation of “horns” or “antlers”, and their processes, has been tra- Lernaea species at disposal. ditionally used as the most reliable characteristics for Lernaea cruciata Lesueur, 1824, frst reported from taxonomic identifcation of Lernaea spp. [9, 10]. How- the body surface of the rock bass Cichla aenea [17], has ever, experimental infection studies put a major question been subsequently reported from more than a dozen fsh mark over the validity of the prevailing taxonomy of the species [18]. Te anchor of this species is X-shaped, with genus by showing that the anchor exhibits high intraspe- four simple and short arms approximately equal in size. cifc variation [5, 11]. For example, Harding [6] and Fryer Lernaea cyprinacea is the most widely distributed spe- [7] have shown that the growth and orientation of the cies of the genus [19], which also exhibits very low host anchor are afected by the anatomy of the host. As dis- specifcity, infecting a wide range of freshwater fshes, cussed by Fryer [7], Yashouv [12] collected larvae from as well as some amphibians [19, 20], but its anchoring adults settled on one host and then successfully infected apparatus is much more complex. It also has two pairs another fsh species (host not specifed by Fryer) and of arms [21], but the dorsal pair is larger than the ven- tadpoles with these larvae; most of the specimens from tral pair, it branches out into pairs of processes at the tip; carp and bufalo fsh were assignable to L. cyprinacea, but hence, some studies refer to it as the T- or Y-shaped dor- specimens from Gambusia were morphologically notably sal ramifed pair, or as “antlers” [5, 21–23]. For instance, diferent from L. cyprinacea. A subsequent study of Pod- experimental infection trials on Gambusia sp. infected dubnaya (1973) even further casted the shadow of doubt with L. cyprinacea showed a signifcant change in the over the usefulness of the anchor for species identifca- shape of the anchor. In this case, the anchor of L. cypri- tion: when diferent hosts were infected by larvae from nacea specimens developed into the adults which were a single batch of L. elegans (a synonym of L. cyprinacea) similar to those described for L. gobioptera [24] and/or eggs, adult parasites exhibited diferent anchor charac- L. cruciata [17, 25], and not the typical form of L. cypri- teristics, some of which corresponded to other described nacea. However, despite the fact that the evidence for its species of Lernaea [13]. Finally, a recent study [10], used extreme variability was presented several decades ago, 18S and 28S gene fragments to identify four Lernaea perhaps due to the absence of other morphological fea- specimens, which could be assigned to diferent spe- tures that could be used for this purpose, the shape of cies on the basis of their morphology (the authors did the anchor remains commonly used as the most useful not indicate which species), and found that the speci- morphological trait, for the identifcation of Lernaea spe- mens shared a similar genotype, so all were identifed as cies [10, 11, 16, 26–29]. Te issue of reliability of this trait L. cyprinacea. Tese results clearly demonstrate that the has not been revisited in decades, and it has never been anchor characteristics are often merely structural adap- studied using a combination of experimental infection tations of a single parasitic specimen to a diferent host and molecular tools. Our working hypothesis was that L. species, and therefore have very limited reliability for tax- cyprinacea and L. cruciata are synonyms, but the shape onomic purposes. of the anchor is prone to host-induced morphological Although molecular data (partial sequences of 18S and variability, which results in taxonomic misidentifcation. 28S rRNA) have been used in the identifcation of Ler- Tis would also prove that this morphological trait is naea species [10, 11, 14–16], their availability remains not a reliable tool for the identifcation of (at least these extremely limited. To illustrate this, in December 2018 two) Lernaea species. In a preliminary survey, specimens there were only 21 sequences for just two genes (18S and morphologically (anchor) corresponding to L. cyprinacea 28S), and a mitochondrial genome sequence, all belong- were found on four cyprinid fsh species, whereas speci- ing to a single species, L. cyprinacea, available in the mens morphologically corresponding to L. cruciata were GenBank database (Additional fle 1: Table S1). found only on Gambusia holbrooki (the eastern mos- Te reliability of the two single gene-based molecular quitofsh, referred to as mosquitofsh henceforth). To test markers (18S and 28S) that have been traditionally used our hypothesis, and the taxonomic validity of L. cruciata, in the identifcation and phylogenetic studies of Lernaea we infected diferent host species with these specimens, species remains questionable, or at least unconfrmed, and used molecular data to corroborate the identity of largely due to a limited number of studies and limited specimens collected from diferent hosts. amount of molecular data publicly available [10, 16]. Te DNA barcode marker, cox1, has not been used in Methods the identifcation or phylogenetic studies. Terefore, we Sample collection and identifcation of Lernaea cyprinacea can conclude that due to the shortage of molecular data and L. cruciata from wild fshes and the shortcomings of the anchor as a tool for taxon- In 2016 and 2017, we collected 517 fsh specimens omy and identifcation, scientists currently do not have belonging to 5 species (Carassius auratus, Cyprinus Hua et al.