Assessment of the population structure of the South African sardine Sardinops sagax using a multi-method approach and the morphological and molecular characterization of a stock-discriminating digenean parasite biotag of the genus Cardiocephaloides

An understanding of the population structure of commercially exploited species is essential for effective fisheries management. Fish stocks are typically identified based on the observation of differences in genetic and/or phenotypic characteristics between fish from discrete units. Recent deliberations on the management of the South African purse-seine sardine fishery recognises the likely existence of two stocks, one on the west and another on the south coasts, following studies that have documented spatial variability in several phenotypic characteristics of this species around South Africa. Those studies typically examined spatial variability in a single characteristic (e.g. gill raker morphology and meristics, body shape, otolith shape, vertebral count, parasite loads), but the application of multiple stock identification methods to the same individual fish has been recommended in order to maximize the likelihood of correctly inferring and identifying fish stocks. This study seeks to assess the population structure of South African sardine Sardinops sagax using a combination of stock identification methods including meristic, morphometric and parasite markers. Most of the sardine samples used in this study were obtained off the west and south coasts of South Africa during pelagic surveys conducted in 2013 and 2014 by Fisheries Branch of the Department of Agriculture, Forestry and Fisheries, with some additional samples obtained from commercial fisheries. Fish caught from the west and south coasts are presumed to be part of the putative western and southern stocks respectively. The variables examined included body shape, gill arch length, gill raker spacing, number of gill rakers, otolith shape indices (otolith circularity and form factor), the number of vertebrae, and the abundance of a 'tetracotyle' type metacercarian parasite found in the eyes of sardine; these were first analysed individually and then collectively (excluding body shape data) in a multivariate analysis to test their effectiveness in discriminating between sardine from the putative western and southern sardine stocks. Analysis using geometric morphometrics revealed significant differences in body shape between sardine from the putative western and southern stocks. Results of GLM analyses indicated that gill arch length, the number of gill rakers, otolith form factor and circularity, and parasite abundance were effective univariate discriminators of sardine stocks. No significant difference in gill raker spacing and the number of vertebrae was found between individuals from the two stocks. A Stock Identification Index (SDI) of 0.75 derived from the univariate analyses, as well as results of the multivariate analysis of data provided strong evidence for the existence of two mixing stocks, therefore, supporting the two-stock hypothesis. The five multivariate classification models used in the study showed varying degree of allocation success. Overall classification accuracy ranged from a low 47% in the Linear Discriminant Analysis model to highs of 82% and 91% in the Classification Tree Analysis and Random Forest models, respectively. Both CTA and RF revealed the combination of variables with the strongest spatial discriminatory power to be the number of vertebrae and abundance of the 'tetracotyle' type metacercarian parasite. These results agree with those of previous studies and further support the inclusion of sardine population structure into management strategies for the purse-seine fishery for South African sardine. Given the importance of the 'tetracotyle' type metacercaria in the discrimination of South African western and southern sardine stocks, further studies were undertaken to identify and describe this parasite using light and scanning microscopy, as well as molecular tools. Such information will assist in the definitive identification of the first intermediate host of the parasite, presently hypothesized to be a sub-tidal gastropod occurring off the west coast only. Definitive identification of the first intermediate host (or hosts) will enable the full life cycle of this parasite to be determined and the parasite endemic area to be identified; a critical knowledge gap in application of the parasite biotag approach to stock discrimination using this 'tetracotyle' type metacercarian parasite. Metacercariae collected from fresh sardine sampled from a commercial landing in Gans Baai, South Africa, were manually excysted, relaxed in warm water, fixed in 70% ethanol and stained with haematoxylin for light microscopy. The metacercarial body is oval-shaped, measuring 762 - 967 x 512 - 677μm. It is divided by transverse folds into a forebody, midbody and hindbody. Diagnostic features include the unique, large excretory bladder lobes situated on the lateral sides of the body; two large pseudosuckers in the anterior part of the midbody; an acetabulum which is larger than the oral sucker; and a large lobulated holdfast organ in the posterior half of the midbody. These features are those of the metacercariae of the genus Cardiocephaloides, confirming the previous hypothesis regarding the genus of this digenean biotag. Analysis of partial 28S rDNA region sequence data showed that the metacercariae and the adult Cardiocephaloides found in the African penguin Spheniscus demersus are of the same species, likely C. physalis. This study is the first documentation of the morphological and molecular characterization of the stock-discriminating Cardiocephaloides metacercaria found in the eyes of Sardinops sagax in South Africa.