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The rigorous safety assessment conducted on genetically modified crops includes an evaluation of allergenic potential for an associated newly expressed protein (NEP). Since no single method is recognized as a predictor for protein allergenicity, a weight of evidence approach (WOE) has been adopted. In vitro digestion is a part of the WOE approach and is used to evaluate the susceptibility of a NEP to digestion by gastrointestinal proteases. In 2017, the European Food Safety Authority outlined additional digestion conditions and suggested liquid chromatography tandem mass spectrometry (LC-MS/MS) as an analytical method to detect small post-digestion peptides. This technical review paper focuses on the question of whether LC-MS/MS can aid in assessing allergenic potential of in vitro digestion products generated under the newly proposed conditions. After an extensive review, it was determined that LC-MS/MS can detect very small digestion products. However, the method cannot provide relevant information to differentiate whether these products are allergenic or non-allergenic. Therefore, the use of LC-MS/MS for a standard in vitro digestibility assessment provides no improvement in allergenicity prediction. doi: 10.21423/jrs-v09i1wang, Journal of Regulatory Science, Vol. 9 No. 1 (2021): Special Issue on Genetically Modified Organisms

Sponges are highly plastic animals, capable of adapting their morphology, reproduction and recruitment to suit environmental conditions. The intertidal environment is one of extremes and these conditions directly influence the population ecology of the resident organisms. In addition intertidal reefs are also often situated adjacent to coastal developments and may be subject not only to natural but anthropogenic disturbance. As a consequence sponge communities in shallow and intertidal waters are often more dynamic than their sub-tidal counterparts. This thesis examines the population ecology of Cinachyrella australiensis, a Spongia sp. and a newly identified Haliclona (Reniera) rom. from intertidal reefs within Darwin Harbour, Northern Territory, Australia. It also examines the effect of disturbance has upon the growth and recruitment of these sponge populations. Cinachyrella australiensis is slow growing and believed to be very long lived. It had a distinct robust spherical form, flask shaped porocalyces and pronounced tracts of megascleres radiating from the basal portion of the sponge. Within Darwin Harbour however, both spherical and hemi-spherical forms were identified. Spherical forms were from sites with high water flow rates and had high proportions of structural silica and relatively low organic content. Conversely, individuals from sites with lower flow rates had a hemi-spherical form, lower proportions of silica and higher organic content. Spongia sp. was slow growing and is believed to be long lived. This species lacked any silicification and instead had a robust skeleton comprised of anastomising spongin fibres. Spongia sp. was highly plastic with encrusting, massive, vasiform and digitate forms identified. Yet despite this morphological plasticity there were no site related differences identified in this species. Haliclona (Reniera) rom. was a fast growing ephemeral species. It had highly variable tubular, lobate and laterally coalescent forms. The skeleton had regular unispicular tangential isodictyal reticulation, with tracts of longitudinally running oxeas. This species was very fragile and easily damaged by water flow or suspended particulate matter, and was only located at one of the study sites. Both C. australiensis and Spongia sp. produced reproductive material throughout the year with distinct peaks in reproductive activity during November to February. Unfortunately, reproductive periodicity was unable to be determined for Haliclona (Reniera) rom. C. australiensis produced asexual buds that formed as protrusions on the surface of the parent sponge and migrated down the sheltered side of the adult onto the substratum. Haliclona (R.) rom. also reproduced asexually, however produced large numbers of gemmules which persisted on the substratum after the parent tissue had died off. Asexual reproduction may be an adaptation to overcome potential difficulties of sexual fertilisation in intertidal environs. In contrast, Spongia sp. had vivipurous reproduction, producing large numbers of larvae within brood chambers. Vivipary with hermaphroditism may overcome the risks of gamete loss potentially associated with a highly variable habitat. C. australiensis had the greatest number of individuals per m2 in comparison to the other two species despite producing fewer young per individual. The success of C. australiensis may have been due to asexual budding, bud sheltering and occupation of sheltered microhabitats. Asexual reproduction may be regarded as a conservative mechanism directed towards conservation of resources. Budding reduces the dependence upon water borne fertilisation and thus the potential for gamete loss. C. australiensis displayed a distinct small-scale clumped pattern of recruitment, occupying sheltered microhabitats. Individuals recruited in depressions or adjacent to ridges. These microstructures, like the sheltering mechanism, may have provided young with a degree of protection from water flow. Despite the large numbers of larvae produced by Spongia sp. there was limited successful recruitment. Spongia species had an apparent random pattern of distribution. Low numbers of recruits prevented definitive conclusions about microhabitat occupation being made. Haliclona (R.) rom. had no distinct peaks in reproductive activity and like Spongia sp. recruits were randomly distributed. Even after recruitment, population dynamics can be highly variable, particularly in the intertidal region. Much of this variability is due to disturbance events, such as scouring, which can influence growth and recruitment. Disturbance through algae and/ or sponge clearance significantly influenced both growth and recruitment in C. australiensis and Spongia sp. Disturbance also influenced growth rates, disturbed individuals growing slower than their non-disturbed counterparts. However, the same did not apply to recruitment, C. australiensis in disturbed areas had significantly higher recruitment than in non-disturbed areas. The creation of cleared areas of substratum through disturbance had a positive effect on C. australiensis recruitment. The influence of disturbance on Spongia sp. recruitment was not as clear due to the low numbers of recruits. This research has provided new information on the population ecology of three tropical intertidal sponge species. Despite inhabiting the same intertidal location, all three species had very different morphologies, modes of reproduction, patterns in recruitment and growth.