La planeación microcurricular y el docente como planificador

The two sagittal otoliths of each A. butcheri were extracted, cleaned, dried and stored in paper envelopes. The left sagittal otolith from each fish was embedded in clear epoxy resin and, using an Isomet® low-speed saw (Buehler Ltd., Lake Bluff, Illinois), cut transversely

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Note that, in order to publish the data Chapters of this thesis, there will be duplication of the general material and methods section to ensure that the methods presented in each data Chapter stands alone.

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© E. C. Ashworth

into approximately 0.3 mm sections through its primordium and perpendicular to the sulcus acusticus. The sections were cleaned, polished with wet and dry carborundum paper (grade 1 200) under tap water and mounted on microscope slides under coverslips using DePX mounting adhesive. Otolith sections were examined under reflected light against a black background using a high-resolution digital microscope camera (Leica DFC 425) mounted on a dissecting microscope (Leica MZ7.5). High-contrast digital images of the sectioned otoliths were analysed using the computer imaging package Leica Application Suite version 3.6.0 (Leica Microsystems).

The number of opaque zones in each sectioned otolith of A. butcheri was counted independently and on different occasions by E. Ashworth and P. Coulson (Fig. 2.1). On the few occasions (< 4%) when the counts of the two readers disagreed, discussions between these readers always resulted in a mutually-agreed value for the number of opaque zones.

Figure 2.1. Sectioned otolith of an individual of Acanthopagrus butcheri measuring 262 mm TL from the Wellstead Estuary with seven opaque zones.

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The age of each A. butcheri was determined using 1) the number of opaque zones in its sectioned otolith, 2) the date of capture, 3) the assigned birth date (i.e., the approximate mid-point of the spawning season), and 4) the typical date when newly-formed opaque zones become delineated from the otolith periphery in this species (Sarre and Potter 2000). In the otoliths of some individuals, delineation of the opaque zone at the periphery may occur earlier or later than the assigned date. To account for this, for fish with a narrow translucent edge visible on the outer edge of its otolith, caught within two months prior to the assigned date at which the new opaque zone typically becomes delineated, the number of (delineated) opaque zones was adjusted downwards by a year. For those fish with an opaque periphery (in which case that non-delineated outer opaque zone is excluded from the count of delineated opaque zones) or wide translucent outer margin, caught within two months following that assigned date, the number of opaque zones was adjusted upwards by a year. The age (t) of each A. butcheri was then determined using the following equation:

𝑡 = 𝑧 +𝑚𝑐 + (𝑚𝑑 − 𝑚𝑏) + 1 12 + 𝑑𝑐 365.25 if mc < md 𝑡 = 𝑧 +𝑚𝑐 + (𝑚𝑑 − 𝑚𝑏) − 11 12 + 𝑑𝑐 365.25 if mc ≥ md

where z is the number of opaque zones, mc is the month at capture, dc is the day of the month at capture, md is the month of opaque zone delineation and mb is the birth month.

Sectioned otoliths from 50 individuals of each of the other five species were randomly selected from the otolith collections, which were housed at Murdoch University. Preparation of these sections had followed the same procedure as that used for A. butcheri. These species comprised the sciaenid A. japonicus studied by Farmer et al. (2005), the labrid B. frenchii by Cossington et al. (2010), the serranids E. armatus and E. coioides by Moore et al. (2007) and Pember et al. (2005) respectively,and the glaucosomid G. hebraicum by Hesp et al. (2002).

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Total lengths recorded and ages assigned in the studies for which these fish had originally been collected were accepted for use in this study, as the same procedures as used for

A. butcheri had been employed when recording lengths, ageing, and comparing the ages assigned by two independent readers.

It should be noted that mean monthly marginal increment analyses have validated the annual deposition of opaque zones within the otoliths of all six species, confirming that counts of these zones could be employed for determination of the ages of fish (Sarre and Potter 2000; Hesp et al. 2002; Pember et al. 2005; Moore et al. 2007; Farmer 2008; Cossington et al. 2010).

For individuals of each of the six species, the ‘radius’ of its otolith at the age at which it was caught was measured from its primordium to the outer edge of the otolith along a line perpendicular to the opaque zones (see Appendix, Fig. SA for the location of the radius on sectioned otoliths for each species). Each otolith radius was measured on three separate occasions to the nearest 0.1 µm along the same axis under reflected light, employing the computer imaging package Leica Application Suite v3.6.0 analysis software (Leica Microsystems). Note that the morphologies of the otoliths vary markedly between these species (see Appendix, Fig. SB) but, for each species, the same axis provided the clearest delineation of opaque zones and was thus employed for both ageing and measurement of otolith radii.

To provide data for back-calculation and other analyses, the distance for each

A. butcheri from the primordium of its otolith to the outside edge of the first opaque zone and the increments between the outside edges of successive opaque zones were measured under reflected light to the nearest 0.1 µm. All distances were measured along an axis perpendicular to the opaque zones adjacent to the posterior edge of the sulcus of the otolith.

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To obtain a biological intercept (Campana 1990) for use in the back-calculation component of this study, A. butcheri eggs from the Australian Centre for Applied

Aquaculture Research (ACAAR, Challenger Institute of Technology, Western Australia) were hatched overnight in the laboratory, Murdoch University. The TLs of thirty larvae (two- days old) were measured to the nearest 0.01 mm under transmitted light. The left otolith of each larva was collected and measured under a high-resolution digital microscope camera Leica DFC 425 mounted on a high-performance dissecting microscope Leica MZ7.5 (7.9:1 zoom). The radii of whole otoliths were measured to the nearest 0.1 µm under transmitted light (Fig. 2.2).

Figure 2.2. Left, photograph of the head of a two day-old Acanthopagrus butcheri larva, showing larval otoliths (identified by arrows); top-right, photograph of a whole

Acanthopagrus butcheri larva; bottom-right, extracted larval otolith.