Trace element biomineralisation in the carapace in male and female Argulus japonicus.

Parasites of fishes have been shown to be effective bioindicators of the aquatic environment. Few investigations have been conducted on ectoparasite models and therefore little is known about the fate of trace elements and metals which they accumulate. In this study trace element sequestration was observed in the carapace of the fish louse, Argulus japonicus and found to relate to the sex of the parasite, as well as, the degree of sclerotization of the carapace. Adults of A. japonicus were collected from cyprinid hosts in the Vaal Dam, South Africa. Parasites were removed and flash frozen in liquid nitrogen before being sectioned with a cryomicrotome. Sections and whole mounts of parasites were prepared and treated with Phen-Green TM FL cell-permeant diacetate. Cryosections were assessed for trace elements and metals using a scanning electron microscope equipped with energy dispersive spectroscopy. Results indicated that in both male and female parasites, trace elements become bound to the carapace and produce more intense fluorescence than in soft tissues. Sexual dimorphic differences were further observed between male and female parasites. The intensity of the fluorescence signals was greater in the carapace of male parasites than in females, particularly when comparing the carapace of the ventral side of the thorax. In females, an amorphous layer of material surrounding the eggs was observed and produced an intense fluorescent signal. Levels of trace elements and metals detected were not significantly different between male and female parasites. Results observed serve as a demonstration for the first time of trace element sequestration in a freshwater crustacean parasite and possible mechanisms employed to reduce body burdens of trace elements and metals

Trace element and metal sequestration in vitellaria and sclerites, and reactive oxygen intermediates in a freshwater monogenean, Paradiplozoon ichthyoxanthon.

Exposure to metals and other trace elements negatively affects infection dynamics of monogeneans, including diplozoids, but, physiological mechanisms linked to exposure have yet to be documented. In this study sequestration of trace elements and reactive oxygen intermediate production in the monogenean, Paradiplozoon ichthyoxanthon, was demonstrated. During dissection of host fish, Labeobarbus aeneus, the gills were excised and assessed for P. ichthyoxanthon, which were removed and frozen for fluorescence microscopy or fixed for transmission electron microscopy. Trace elements were sequestered in the vitellaria and sclerites in P. ichthyoxanthon, and the presence of reactive oxygen intermediates was observed predominantly in the tegument of the parasite. Trace elements and metals identified and ranked according to weight percentages (wt%) in the vitellaria were Cu > C > Au > O > Cr > Fe > Si while for the sclerites C > Cu > O > Au > Fe > Cr > Si were identified. For most element detected, readings were higher in the vitellaria than the sclerites, except for C and O which were higher in sclerites. Specifically for metals, all levels detected in the vitellaria were greater than in sclerites. Based on the proportion of trace elements present in the vitellaria and sclerites it appears that most trace elements including metals were sequestered in the vitellaria. The results of reactive oxygen intermediate production in the tegument of the parasite suggests either trace element accumulation takes place across the tegument or results from the action of the host's immune response on the parasite. The results serve as the first demonstration of trace element sequestration and reactive oxygen intermediates in a freshwater monogenean parasite

Trace element biomineralisation in the carapace in male and female <i>Argulus japonicus</i> - Fig 1

<p><b>Whole mounts of male (a; c; e) and female (b; d; f) <i>Argulus japonicus</i> showing autofluorescence in unbleached (A) and photobleached (B) specimens, and fluorescence of Phen–Green (C</b>). (a–d) Micrographs showing the anterior region of adult male and female <i>A</i>. <i>japonicus</i> where antennules (Antl), antennae (Ant), maxillules (Ml), maxillae (Ma), and basal segment of the maxillae (Bpl) in untreated (A), photobleached (B) and Phen–Green fluorochrome (C). (e–f) Micrographs of the thoracic region of male (e) and female (f) <i>A</i>. <i>japonicus</i>. In male parasites fluorescence of untreated (A and B) and Phen–Green treated (C) reactions were present in the ventral carapace between the third and fourth swimming legs (Sl) and of the peg (P) of the podomeres of the third pair of swimming legs (Sl). In females the spines on the spermathecae (Sps) were observed for all treatments and <i>in utero</i> eggs were observed to produce a positive reaction for the Phen-Green (Cf). All images were taken using Zeiss bandpass filter set 01 (BP 365/12) at 490–520 nm. Scale bars represent 100 μm.</p

Trace element biomineralisation in the carapace in male and female <i>Argulus japonicus</i> - Fig 2

<p><b>Micrographs showing whole mount (A) and longitudinal sections (B, C, D and E) through male <i>Argulus japonicus</i>.</b> Section planes are indicated on the light micrograph of a whole mount of a male <i>A</i>. <i>japonicus</i> (A) with dotted lines indicating sections for B–E. Fluorescence of the Phen–Green probe indicates a positive reaction for divalent cations including trace metals in longitudinal sections (B–C) of the maxillule (Ml) and maxilla (Ma). The cuticle of these regions produce more intense signals than the cuticle of the carapace (Ca) of the cephalic shield. (D) A longitudinal section through the posterior region of the thorax and abdomen shows positive fluorescence for the carapace (Ca) of the lobes of cephalic shield and the testes (Ts) in the abdomen. More intense signal was observed for the portion of the carapace of the ventral side of the thorax (Vp). (E) Longitudinal section through the thorax shows positive signals for the peg (P) of the third pair of swimming legs (Sl) and the carapace of the ventral surface of the thorax (Vp). Images were acquired using Zeiss bandpass filter set 01 (BP 365/12) at 490–520 nm.</p