Using capture fishery-derived fish oil and fishmeal in aquafeeds is unsustainable. This study mimicked semi-intensive shrimp ponds, including primary producers, in mesocosm tanks. Fatty acid mass balances were computed to distinguish between diet-based and primary production-based LC-PUFA contributions to shrimp (Litopenaeus vannamei) production and meat quality. Performance and body fatty acid composition were compared of shrimp fed a commercial diet containing fish oil and fishmeal (control), with a fishmeal- and fish oil-free diet (low LC-PUFA diet: LOW). Six mesocosms were each stocked with 60 juvenile shrimp and randomly assigned to the two diets. After an 8-week grow-out period, biomass production, survival and proximate body composition were similar between diets. Control shrimp contained twice as much LC-PUFA and omega-3 fatty acids than LOW shrimp. Large quantitative losses (85%) were found in both treatments of the LC-PUFA-precursors alpha-linolenic acid (ALA) and linoleic acid (LA) that were being used as energy source by the shrimp instead for LC-PUFA synthesis. Whereas losses were also observed for eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in the control group, there was a gain for these components in the LOW tanks. LOW shrimp sourced at least 32% of their total EPA gain and 15% of their total DHA gain from the algal-based food web. This quantitative analysis of the fate of major dietary fatty acids strongly suggests that the pond's primary production can provide shrimp additional LC-PUFA. Finding a balance between LC-PUFA contribution through formulated feed and natural production seems possible and deserves further research.