Testing Artificial Diets for Papilio zelicaon

We tested an artificial diet for rearing anise swallowtail (Papilio zelicaon) larvae. The formulation of the diet is discussed.

---

Our goal this Summer is to try to establish working colonies of as many swallowtails species as possible for a research project. However, in order to mass rear larvae, it would require a lot of host plant. The trouble with machaon-group species (e.g. Papilio zelicaon, P. polyxenes, P. machaon, etc. ) is that their umbel host plants are scarce in Ithaca. It is not necessarily that there are not enough plants growing; the species that grow here that is also problematic. For one, the three main naturalized species are golden alexanders (Zizia aurea), Queen Anne's lace (Daucus carota), and wild parsnip (Pastinaca sativa) each are only available for a short part of the year. Golden alexanders grow early in the season, bloom for a month and disappear soon after the Summer actually begins; Queen Anne's lace only grow in the latter half of the season; and wild parsnip also grows early but leaves become unuseable once it starts blooming in June. Another problem is that none of the species grow large enough or are anywhere near leafy enough to be practical. What would be ideal is if there was an abundant supply of sweet fennel (Foeniculum vulgare), which can grow up to six feet tall with dense foliage and does not divert resources from leaves to bulb like florence fennel. Larvae also perform better on sweet fennel than on any of their native hosts, at least for P. zelicaon and P. polyxenes, in our experience. In California, naturalized sweet fennel (Foeniculum vulgare) from Europe can be found growing wild in just about any municipal area, which made it easy back when we reared P. zelicaon in the Bay Area. In Ithaca, fennel can only be found in gardens or as an occasional escapee. 

Since we have access to greenhouse resources, we plan to grow our own fennel soon, but that will take some time even given that authentic wild fennel germinates and grows remarkably fast. Even then, there is only so much that can be done in a greenhouse. What would be truly revolutionary is if we could formulate an artificial diet using fennel base as feeding stimulant that would produce a large amount of food per unit of fresh fennel weight to feed machaon-group swallowtails. We are already very familiar with preparing plantago (Plantogo) -based artificial diet to rear Junonia coenia, so we figured that we could try this. Although the ingredients and their ratios for artificial diets optimal for rearing different species differ, they are fundamentally similar (the same basic ingredients are universal in lepidopteran diets and different species still share relatively similar nutritional requirements), so we would expect that a reasonable modification of existing diets of other species might work for the machaon-group swallowtails. 

The ingredients and their ratios for the diet we ended up preparing were decided based on artificial diets tested for Junonia coenia and Papilio xuthus. It included the following: dry ingredients: 10 g wheat germ, 5 g casein, and 2.0 g wessin salt; boiled ingredients: 100 mL distilled water, 4.0 g chopped fresh fennel (often, recipes call for dry leaf powder, but we did not have the time or equipment to prepare this), 3 g agar; and vitamins/inhibitors: 2.0 g pre-made vitamin mix, 0.3 g sorbic acid, 0.2 methyl parabens. Supplementary ingredients that were included in the Junonia diet such as yeast, sucrose, linseed oil, and other vitamins/inhibitors were left out, for now, to keep things simple for future testing and because they were not included in the xuthus diet. Also, in this initial testing phase, we were more focused on getting the larvae to initiate feeding on the diet at all before worrying about the diet's nutritional content. We tested a chunk of this diet on five newborn larvae of P. zelicaon on May 23.
​
After a day, the larvae may have eaten this diet, but it was difficult to tell. The larvae were still alive, which may not have been the case if they did not eat, and may have been slightly larger than newborn larvae. However, it was hard to identify any frass. Larvae eat less and produce less frass on diet since it is more nutrient dense than most foliage and the frass would be a similar color to the diet. In any case, if the larvae did eat, we were not sure if they actually ate the "artificial" components of diet or if they could have survived simply by nibbling on the pieces of boiled fennel leaf. 

In any case, we thought the the diet we prepared could have been far too dry. Not only is enough water content crucial for larval survival and overall performance, the dryness of the food can make it hard for larvae to bite into it, depending on what the larvae are normally adapted to feed on. From past experience, we know that swallowtail larvae tend to prefer tender, young shoots of their host and struggle with biting into mature foliage in early instars. Therefore, we decided to re-make the diet with increased water. In addition, we were still concerned that the larvae may not have been stimulated to eat the diet. Since fennel is the one ingredient that we can increase or decrease without affecting the diet's nutritional profile, we decided to double the amount of fennel. (Though, ideally, it would be best to minimize the fennel needed since the purpose of the diet is to amplify the live plant resource.) The new diet contained a higher ratio of fennel (2.5x), water (2x), and agar (1.33x) to make it softer and increase the concentration of fennel feeding stimulants and water content. The new diet was noticeably softer, but in hindsight, since refrigerator storage will naturally dry the diet over time, it probably would not have hurt to add even more water. We offered this diet to three newborn larvae, shown below. 

A day later, there was frass and the larvae did seem to have grown noticeably, though perhaps not as much as their siblings feeding on fennel foliage. ​Thus, the changes we made to the ratio of ingredients may have helped in stimulating them to feed, but they may not have a very good growth rate on the diet.

Since the larvae have initiated feeding on the diet, the next problem to address would be whether they can actually complete their life cycle on the diet. This is complicated because larvae have specific nutritional requirements that, if not met, can drastically impact general performance as well as their ability to molt, pupate, eclose properly, reproduce, etc.. Dietary can play an important role in hormone balance and proper eclosion since pupae burn fat to survive. Linseed oil, an ingredient we did not include, is a source of certain fatty acids, and reportedly helps with getting adults to eclose properly in some studies. The carbohydrate to protein ratio is also particularly important. Carbohydrates stored from the larval stage are necessary for the egg production of most species (though for species that feed on nectar, it is less important), but sufficient nitrogen likely plays a critical role in overall survival. We could have supplemented the diet with sucrose, which is often used in artificial diets to boost carbohydrate ratio, but we chose not to for this reason. So even if the larvae do not die, we will have to see these larvae through for at least one full generation (up until we obtain viable eggs from them) to assess how well it works. 

---

​Brian Liang
Brian Liang is a student at Cornell University in Ithaca, New York pursuing an undergraduate degree in entomology. He is co-owner and a main contributor of the Liang Insects blog, insects articles, and site design.

---