Methods

Rearing broad-nosers from the vegetation

By Jennifer C. Girón

Through my years studying entimines in the Neotropical region I've been curious about larval stages but never really describing larvae, even though I know from the literature, particularly from the work by colleagues in Argentina, particularly Adriana Marvaldi, that larvae contain important anatomical characters for taxonomic classification. In fact the monophyly of the Entiminae as a subfamily is supported by features of the larval antenna.

From Marvaldi 1997.

I started trying to rear entimine larvae when I was a master's student in Puerto Rico, about 17 years ago. Here I describe the method I have used, which has been "experimental" in the sense that I have tried it with several taxa. However, it is not experiment-grade since I have not tried multiple adjustments under controlled circumstances to find an optimal way to go about this. This is more an exercise of curiosity and not necessarily a rigorous method.

I figured sharing here might be helpful to those in need of obtaining larvae for any reason, but hopefully to preserve them and describe them eventually, which is what I hope to do one of these days.

I have done this primarily with "large" species (>1 cm; Diaprepes abbreviatus, Naupactus leucoloma (parthenogenic populations in Texas), Epicaerus panamensis and another [unidentified] species of Epicaerus). I also got larvae from Apodrosus argentatus and a likely undescribed species of Geonemini from Puerto Rico in the smaller size range (~7 mm).

Before you embark on this adventure,

Make sure you have time to tend to your growing larvae. Mold is one of the main issues you can get, so you need to stay vigilant that the food source for the larvae doesn't decompose while the larvae are there.
This has been tested in several vegetation entimines, but not in soil-associated species.
Be aware of preservation methods for larvae.
You might want to take note of timing of events.

Dealing with the adults

The first thing to do is, of course, get the adults. Once you collect them in the field, it would be good if you have males and females. Luckily for many entimines there is a simple way to tell them apart: females tend to have a U-shaped fifth abdominal ventrite, whereas in males, the same structure is V-shaped. Of course, there might be exceptions, and then if you only have one sex, there is no way to compare. The other issue, although rare but relatively common in entimines, is that some species are parthenogenic. Bottomline, you should at least get a female (either parthenogenic or mated) for this to work.

What I usually do is:

Find an appropriately-sized container. Make sure you have an appropriate container to keep your adults so that they are unable to escape. Entimines can be really good escape artists if you are not careful. I have lost a few adults in my trials for being over-confident. My top choice here is a simple petri dish with cover, which is heavy enough for the weevils to not be able to lift it (unless the weevils are too big for the dish), but allowing for airflow. Smaller dishes for smaller species would be better, as that would force them to come into contact.

A pair of Epicaerus panamensis (Entiminae: Geonemini) in a small petri dish with a batch of eggs (blue arrow) protected with the extra paper provided.

Provide a layer that can be kept humid and easily replaceable. I used filter paper (a cut piece of coffee filter) in the past, but nowadays I use paper towels, which keep more liquid for longer. Make sure to cut your "substrate layer" to the size of the bottom of your container. You don't want to create creases and folds where the adults can hide from each other.
Caveat here: larvae will crawl in between the layers of the paper towel, so you need to be careful whenever you are replacing it. More on this later. Why is keeping them humid important? I'm not entirely sure. Perhaps my brain is treating them as if they were plants, but so far, I think letting them dry out might not be great, but probably for weevils from dry regions it doesn't really matter. You don't need puddles, use just enough water to make the substrate uniformly moist. Using a plastic pippette will make this easier as you can control where the water goes and how much of it.
Provide some food source. The go-to here is any leafy material you have handy. Entimines are not really picky eaters. Grab a piece of lettuce, or spinach and hope for the best. One thing to consider here is for how long you want to keep the adults. In my experience, you can get a batch of eggs overnight and you can get several egg batches from a single couple.

Compsus caeruleipesCompsus caeruleipes from Perez Zeledon, San Jose, CR on September 26, 2020 at 12:33 PM by Laurent Hesemans

Provide substrate for ovipositing. This is basically some extra paper. Entimines that hang out in the vegetation would fold a feaf of a plant an lay their eggs in between the fold, like the one in the image on the left.
Remove egg batches. I recommend doing this, just so that yo can keep track of how long it takes for larvae to emerge, which might be a good data point for whenever you get to publish your findings. I place the egg batch with its wrapping paper also in a petri dish with wet paper towels. I think keeping these in a humid chamber (with tap water) is more important than with the adults, because the eggs can indeed shrivel and die.

Caring for eggs and larvae

If laid in a batch between layers of paper (or leaves), the eggs will be covered in a sticky substance that will make it difficult to separate the layers in case you want to count how many eggs are in the batch.

For this, humidity and patience will be your friends. Do not attempt to separate the layers when dry, as this will likely rip the eggs apart. Once the sticky substance is softened, I usually peal one of the layers off under the microscope to make sure it peels but does not remove part of the egg case.

An advantage of removing one of the layers is that you can see what's happening inside the egg. You will see the stemmata and the mandibles appear and become evident in about a week, unless the egg laying strategy is not like the one I have been describing.

Some weevils, Apodrosus argentatus for instance, lays eggs one at a time, loose on the substrate. No batch, no sticky substance. The eggs are laid white and darken over time. You may think they are dead, but they are not, so be patient and keep a close look.

For oviposition strategies, check out Anne Howden's 1996 paper!

I like staring at the developing larvae every once in a while. It is mesmerizing to see them moving their mandibles and it for sure makes you wish they were out already, but believe me, you are probably not ready for whenever an entire batch is out and about and crawling!

MVI_0939.MOV

Once you can see mandibles and there is a gap of air between the larva and the egg case, they are nearly ready to come out. It takes around 10-15 days, but it may be less or more. We never know, particularly because not many life cycles have been documented for non-pest species.

As far as I have seen, the first instar is quite active but doesn't necessarily feed right away. Move the larvae to a deeper container. They will manage to get out of shallow containers or get stuck on lids, cloth, and any seals. To transfer them I use a wet (with tap water) fine brush. Counting them helps you keep track of what's going on. You might want to try having each separately, but believe me, even if it's "only 10" of them, it gets time consuming to care for each every day or every second day. I prefer to keep the batch together, but this reduces the accuracy of the track I'm keeping.

The container I have been using is about 4 cm deep. Prepare the container as you did for the adults. This time you want the paper bottom to go over the sides and get stuck to the walls when wet. The larvae will go in every nook and cranny, in between the layers of the paper towel and under the paper towel. It seems that they like the cocooning feeling of the cells created between the layers of paper. Again, keep it humid, but not flooded, as your larvae can potentially drown. Your plastic pipette is your best friend for this.

You might want to consider preserving some first instar larvae as they contain phylogenetically-informative anatomical data. Make sure you sacrifice them in hot (nearly boiling) water before placing them in alcohol for preservation. This way they will not shrivel and you will be able to see all the glorious chaetotaxy when you do your anatomical studies. These are pro-tips provided by Adriana Marvaldi.

As for a food source, I usually put slices (about 3 mm thick) of baby carrots, just in case they feel like eating, but they rarely do at this stage for what I have seen. I use the baby kind because when I used regular carrots, they start degrading very quickly. Baby carrots are easily found in super markets (at least here in the US) and a single bag can last for months if properly stored. Yes, there have been papers with artificial diets, but at least for me, they are not necessarily made with ingredients you would have at home. If you are going on an actual life cycle study with replicates and all, you should probably consider going that route.

Epicaerus AZ larvae 2025.mp4

About a week later, you will start seeing the molts around and from time to time a larva that looks alive but barely moves. These would be probably recently molted and VERY delicate, so be careful if you need to handle them. I also preserve the molts in alcohol but to be honest I haven't looked at them to see if any features are preserved.

When replacing the "substrate paper", particularly with the smaller larvae, make sure to check closely, hopefully under a stereomicroscope, so that you don't leave your precious larvae behind. Also make sure to check any tunnels in the carrot slices.

Adriana recommended growing the larvae on the roots of their host plant and using sterilized, humid soil. I have not tested this. Another potential substrate is perlite. Haven't tested this either. In general, the idea of not being able to see them and checking on them doesn't sound as fun. On the flip side, you would not have to care for them much, besides watering the plant every once in a while. Perhaps growing carrots in a pot would be worth a try?

From here on, keep an eye on your larvae every couple of days and change the carrot slices whenever you see browning or slimy spots. If you start seeing signs of mold, be ready to terminate.

You can preserve the following instars whenever you notice molts. With my most recent attempts I get tired of tending to them before they get to the pupal stage. The only time I managed to get a pupa with this method was with a Diaprepes abbreviatus larva. Only one (out of initial hundreds) and it took over a year. The pupa died before getting an adult, probably because it didn't vave materials to make the cell where they are protected.

Methods

Rearing broad-nosers from the vegetation

By Jennifer C. Girón

Dealing with the adults

Caring for eggs and larvae

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