The embryo of the grass shrimp, Palaemonetes pugio, is surrounded during development by a protective extracellular coat designated as the embryonic coat (EC). At hatching, this EC is composed of four embryonic envelopes (EE), each of which is composed of multiple layers. The outermost layer of the EC, the outer investment coat (OIC), is derived primarily, if not completely, from pleopods of the female. The first envelope (EE1) forms as a bilayered envelope, EE1a and EE1b, immediately after oviposition. The OIC becomes closely associated with EE1 and remains in close contact with EE1 until hatching occurs. An additional layer, EE1c, is added to the inner side of EE1 between 3 and 5 d after oviposition. Three more embryonic envelopes, EE2, EE3, and EE4, are formed between the embryo and EE1 by 7 d after oviposition. Formation of embryonic envelopes continues until 10 d after oviposition; by this time each envelope is morphologically distinct in composition, with "outer" and "inner" sides clearly identifiable. All but the innermost embryonic envelope (EE4) are shed by the embryo about 6 h before hatching. Permeability of the EC during the 12-d incubation period is found to decrease between 0 and 5 d after oviposition, and then increase until hatching. Fluorescently labeled lectins react positively with the OIC, indicating the presence of glucose and N-acetylglucosamine residues. Thus, the palaemonid EC is a dynamic structure throughout embryonic development.

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Abstract. The current report discusses the process in which a methods comparison study in the National Animal Health Laboratory Network is performed. Specific details are provided for designing and analyzing studies intended to evaluate analytical sensitivity, efficiency, analytical specificity, cross-contamination, repeatability, operator variability, and to compare the performance of methods using diagnostic samples. As an example, a case study is presented comparing the performance of a candidate reverse transcription polymerase chain reaction (RT-PCR) chemistry to the current RT-PCR chemistry in use when the assay was originally validated. The present study revealed that, for all of the validation factors evaluated, the candidate method performed at least as well and generally better than the current method. The candidate method was, therefore, deemed fit for the original intended purpose of the current method and rendered acceptable for use. A discussion of the case study is intended to further motivate consideration of the study designs chosen.