Manship, Brigitte.M (2009) Aspects of development and feeding in 'Paragnathia formica' (Hesse, 1864). (PhD thesis), Kingston University, .
Abstract
Gnathiid isopods (peracarida: Isopoda: Gnathiidae) are marine ectoparasites, in which the juvenile stages associate with fishes for several hours to days to feed on blood, plasma and tissue fluids, in order to complete their intricate life cycles. These cycles each include two juvenile moults and a final metamorphosis into non-feeding, adult stages. The blood meal ingested provides resources for adult survival, reproduction and embryological development. In the model gnathiid species Paragnathia formica, embryological development occurs in internal uterine sacs of the adult female, rather than in a brood pouch, as in most Peracarida. In this study, by means of traditional histology, nuclear staining and scanning electron microscopy, changes occurring throughout embryological development to the pre-hatching juvenile stages were examined, including gastrulation and three post-gastrulation stages. Loss of the egg shell/membrane as well as an intra-uterine moult were observed, and conversion of the yolk mass into larval tissues and organs was recorded. The internal morphology of the digestive tract was examined in third stage juveniles to track changes in cells lining the digestive glands with blood meal digestion. Dark, sub-cuticular cells were also detected prior to the metamorphosis into adult stages, which may function in the moulting process. The final moult accompanied dramatic tissue re-arrangement in the male cephalosome, during which the small head of praniza 3 juveniles adapted for feeding, transformed into an enlarged cephalon with prominent mandibles. The structural trait of the mandibles with other morphological male characteristics, form the basis of the taxonomic classification and identification of individuals within the Family Gnathiidae. The parasitic juvenile stages, if caught separately from males, are difficult to identify due to intra-specific polymorphisms. Thus attempts were made to link adult and juvenile stages of a variety of species including P.formica, using two approaches, namely telson morphometries and molecular typing. However, adult and juvenile telsons varied within a single species, and were often too conserved between species to be used as identification tools morphometric ally. On the other hand, molecular typing methods were more successful in identifying the host fishes on which juveniles had fed, rather than identifying the ectoparasite species themselves. This anomaly was possibly due to the long-lasting storage of nucleated fish red blood cells in the anterior hindgut of P.formica juveniles before being processed. Blood meal digestion was studied using in situ histochemistry and spectrophotometric proteinase assays on fed (praniza 3) juveniles, with unfed (zuphea 3) stages used as controls. It demonstrated predominantly protein breakdown by cathepsin-like cysteine proteases within the digestive glands during early, mid- and late digestion, with only limited activity in the anterior hindgut and rectal vesicle. Furthermore, serine protease inhibitors were partially purified, from first stage (zuphea 1) unfed P.formica juveniles using chromatographic techniques. These partially purified inhibitors were shown to display anticoagulant activities, possibly to facilitate feeding of the ectoparasite by preventing the host haemostatic reactions. This project succeeded in complementing information previously gathered on the developmental biology of Pi formica, with novel observations made of intra-uterinë moults during embryological development in females, of dark, sub-cuticular cells growing as digestion progressed in fed final stage juveniles, and of tissue re-arrangement occurring in the cephalosome during the final juvenile metamorphosis into males. Furthermore, a potentially novel species of Gnathia was discovered after microscopy observations of telsons, and discriminant function analysis was applied to gnathiid telson measurements for the first time to separate different species statistically. Finally, both biochemical investigations, namely on digestive proteinases and anticoagulant compounds in P.formica, were original, with new fmdings including protein digestion by cathepsin-like enzymes in the digestive glands offed fmal stage juveniles, and trypsin inhibitors displaying anticoagulant properties in unfed first stage juveniles.
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