Diplostomum species

Ubels, John L., DeJong, Randall J., Hoolsema, Brittany, Wurzberger, Amy, Nguyen, Thuy-Tien, Blankespoor, Harvey D. & Blankespoor, Curtis L., 2018, Impairment of retinal function in yellow perch (Perca flavescens) by Diplostomum baeri metacercariae, International Journal for Parasitology: Parasites and Wildlife 7 (2), pp. 171-179 : 174-175

3.2. Identification of Diplostomum species by DNA sequencing

Sequences from the barcode region of COI were obtained from 66 individual metacercariae (GenBank MF142160, MF142162 - MF142201 and MF142204 - MF142228). The length of quality sequences obtained varied from 523 nt to 781 nt. Of the 66 metacercariae that were successfully sequenced, 17 were from spottail shiners (6 host fish), 7 from common shiners (3 host fish), and 42 from perch (16 host fish).

Sequence and phylogenetic analyses revealed that the choroid of perch was infected only with Diplostomum baeri and that the metacercariae from one perch lens were of this species. There were also two instances of Diplostomum baeri infecting shiners, but otherwise the shiners were infected with the following species known only from studies employing DNA sequencing: Diplostomum sp.1 , Diplostomum sp.3 , and Diplostomum sp.4 ( Désilets et al., 2013; Locke et al., 2010b; Locke et al., 2015) (see Supplementary Fig. 1 for phylogenetic results).

3.3. Effect of Diplostomum baeri infection on the yellow perch electroretinogram

To determine whether infection of the choroid of the perch eye with Diplostomum baeri , resulting in localized damage to the pigment epithelium and photoreceptor layer, affects visual function, the electroretinogram was recorded from infected eyes of fish from Douglas Lake. These eyes were heavily infected, with the average number of metacercariae in retinas used for ERG recordings 89 ± 43 (range 30–180, n = 9 fishes). Controls were uninfected eyes of perch from Paradise Lake.

Uninfected perch had typical ERGs with a negative a-wave and rapidly rising b-wave ( Fig. 5 ). In contrast, the ERG waveforms from infected fish were markedly altered, with attenuation of b-wave amplitude and increased latency ( Fig. 5 ). Statistical analysis showed that infection did not have a significant effect on the a-wave amplitude ( Fig. 6 ), perhaps due to variability related to severity of infection, a variable that cannot be controlled. However, as discussed below, this apparent lack of an effect on the a-wave must be interpreted with caution. The amplitude of the b-wave was significantly attenuated in infected eyes as compared to eyes with normal retinas ( Fig. 6 ). There was a strong trend toward an increase in a-wave latency in infected eyes (p = 0.05 <p ≤ 0.06) ( Curran-Everett and Benos, 2004) while the b-wave latency was significantly increased ( Fig. 6 ). (see Supplementary Table 1 for detailed statistical information).

The flicker fusion frequency of normal retinas was as high as 40 Hz and as low as 5–10 Hz in perch eyes infected by Diplostomum baeri ( Fig. 7 ). The mean flicker fusion frequency of infected retinas was 15.8 ± 3.8 Hz (n = 7) as compared to 23.0 ± 9.2 Hz (n = 10) for normal retinas This difference was statistically significant (t -test, p <0.05).

Figures

Fig. 1. ERG recording chamber. The eyecup sits on filter paper saturated with Ringer solution making contact with the reference electrode. The recording electrode is in the vitreous humor of the eyecup. The LED was placed 4 cm from the eyecup.

Fig. 5. Representative electroretinograms from eyes of an uninfected yellow perch (Perca flavescens) and a perch infected with Diplostomum baeri. Arrows indicate the a-wave and the b-wave. The ERG waves were analyzed for amplitude and latency (time to peak). Note reduction in ERG b-wave amplitude and increase in b-wave latency recorded from the infected eye. Stimulus intensity units = μJ/cm2, time base = 0.27 s/div, amplitude = 15.5 mV/div.

Fig. 6. Effect of Diplostomum baeri infection on the electroretinogram of yellow perch (Perca flavescens) as compared to recordings from normal fish. Infection had no effect on the a-wave but significantly reduced b-wave amplitude. Infection caused a strong trend toward an increase in latency (time to peak) of the a-wave and significantly increased the latency of the b-wave. (*p ≤ 0.05; # 0.05 <p ≤ 0.06; t-test, n = 10).

Fig. 7. Flicker fusion frequency of the electroretinogram is reduced by Diplostomum baeri infection. A. Responses to stimulation at 10 Hz, 20 Hz and 40 Hz in an uninfected yellow perch (Perca flavescens). Flicker fusion is reached at 40 Hz. B. Responses to stimulation at 5 Hz and 10 Hz in an infected perch. Flicker fusion is reached at 10 Hz. Time base = 0.27 s/div, amplitude = 15.5 mV/div.