Plasmodium cytb

Gonz, Merit, alez-Olvera, Hernandez-Colina, Arturo, Himmel, Tanja, Eckley, Lindsay, Lopez, Javier, Chantrey, Julian, Baylis, Matthew & Jackson, Andrew P., 2022, Molecular and epidemiological surveillance of Plasmodium spp. during a mortality event affecting Humboldt penguins (Spheniscus humboldti) at a zoo in the UK, International Journal for Parasitology: Parasites and Wildlife 19, pp. 26-37 : 31-32

3.6. Cytochrome b ( cytb ) sequence clustering and phylogenetic analysis

The data set contained 496 cytb sequences derived from mosquitoes trapped at Chester Zoo, two sequences obtained from wild birds as well as 23 penguin-derived sequences from Chester Zoo (n = 8), London Zoo (n = 2), Paignton Zoo (n = 9), Blackpool Zoo (n = 3) and Cotswold Wildlife Park (n = 1). Penguin sequences are listed in Table 3 and all sequences produced in this study are catalogued in Table F (see S1 List) .

From all the sequences produced in this study, 442 clustered in four different groups, each containing a single reference sequence from GenBank with an associated lineage ( Table 4). One cluster corresponds to the LINN1 lineage and contains 288 sequences, including 272 mosquito-derived sequences, six Chester Zoo penguin sequences from 2017 (n = 4), 2013 (n = 1) and 2018 (n = 1), two wild birds (Eurasian blackbirds), and eight penguins from Paignton Zoo (n = 6), Blackpool Zoo (n = 1) and London Zoo (n = 1). A second cluster corresponds to the SYAT05 lineage, containing 126 mosquito-derived sequences and one penguin-derived sequence from the Cotswold Wildlife Park collection. A third cluster corresponds to the GRW11 lineage, which contains 17 sequences including 13 mosquito-derived sequences, one penguin-derived sequence from Chester Z´ele´et al. (2014), and three from Paignton Zoo (n = 1), Blackpool Zoo (n = 1) and London Zoo (n = 1). The last cluster is that of the SGS1 lineage, comprising nine mosquito-derived sequences and one penguin sequence from Blackpool Zoo. From the remaining 79 mosquito-derived sequences, 72 formed their own cluster, each of which may represent a new lineage, and 7 clustered together without a reference sequence suggesting they are one new lineage. Given that we cannot entirely rule out sequencing error, we took a conservative approach to interpreting the number of distinct lineages among mosquito-derived sequences, counting only those that were observed in at least two individual mosquitoes ( Table 4) .

All of the lineages that clustered with our sequences belong to known parasite morphospecies. The lineage LINN1 belongs to P. matutinum ; this group contained 4/5 Plasmodium sequences recovered from penguins that died at Chester Zoo in 2017. The lineage SYAT05, corresponds to the species P. vaughani , which includes one case from a Humboldt penguin from Costwold Wildlife Park (GeneBank accession number MW813998), and is the first P. vaughani infection reported in penguins.

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clustering result. Of the species circulating in vectors at Chester Zoo in 2017, only P. matutinum was observed in infected dead penguins during the mortality event.

The lineages GRW11 and SGS1 both derive from P. relictum , and neither of them were found in any of the dead penguins during the 2017 outbreak; although P. relictum was detected in 2018 in a penguin that survived infection (GeneBank accession number MW813999).

A maximum likelihood cytb phylogeny displaying the relationships among mosquito- and penguin-derived sequences is shown in Fig. 4 . Nodes’ robustness was assessed with an SH-like log-Likelihood ratio metric ( Anisimova and Gascuel, 2006); non-parametric bootstrapping was carried out, but the tree topology was generally not supported by bootstrap values> 50 due to the relatively small number of characters available. Nevertheless, the topology resolves established morphospecies and largely agrees with previous estimates of their phylogenetic relationships (Valkiunas ¯et al., 2007; Valkiunas ¯and Iezhova, 2017), which provides some external validation of the topology. The tree shows how mosquito, wild-bird and all penguin-derived Plasmodium sequences obtained from Chester Zoo and another four UK locations adopt one of three positions within the topology, each well supported by a log-Likelihood ratio test. These clusters include Malavi sequence vouchers for P. relictum , P. matutinum and P. vaughani , confirming the

Figures

Fig. 4. Maximum likelihood phylogeny of Plasmodium spp. cytb sequences. The phylogeny was estimated from a 378bp multiple sequence alignment using a GTR+Γ+І model (α = 0.488; proportion of invariant sites = 0.248). The tree is rooted with an outgroup of Leucocytozoon sequences (boxed). Node accuracy is indicated by an SH-like log-Likelihood ratio metric; bootstrap values greater than 0.5 are displayed in the tree. Novel sequences obtained in this study are shaded with their corresponding reference sequence; P. matutinum (MK443241), P. vaughani (MK652243) and P. relictum (JN164731). The clusters contain sequences derived from penguins, mosquitoes or wild birds, which is indicated by a penguin, mosquito or a bird symbol. One wild bird sequence is present in the P. matutinum cluster (OM912814); and three (MW814062, MW814149, MW814045), two (MW814453, MW814028) and two (MW814503, MW814500) mosquito sequences are present in the P. matutinum, P vaughani and P. relictum clusters respectively. The rest of the sequences in those clusters correspond to 23 novel sequences from penguins infected in the UK, indicating their origin (CZ: Chester Zoo, LZ: London Zoo, PZ: Paignton Zoo, BZ: Blackpool Zoo, CWP: Cotswold Wildlife Park) and year of sampling, if not 2017. Other shade sequences correspond to recognized morphospecies. P. vaughani cluster represents 145 novel sequences, P. matutinum cluster represents 345 novel sequences and P. relictum clusters represents 31 novel sequences. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)