​Rearing Notes 8/4/17-8/13/17:

We are now attempting to breed our mourning cloaks (Nymphalis antiopa). They originated from an egg cluster collected on willow (Salix) along the Ohlone Greenway Trail (Berkeley, California), June 29, 2017.

Many more dozens of antiopas swarmed our tub again today, and this time there are a lot more of them are female. Most of the males have all eclosed, and maybe a few dozen more females are still left to eclose. The tub is soaked in their bright red meconium. We took a few more out to photograph as well.

We have not released any of the butterflies that have eclosed so far and have been keeping them all in the tub. Since a good majority of them are already eclosed, we decided to give breeding them a shot. We have no experience breeding these before or any similar nymphalids, but it is possible and has been done before by some. The general idea is to just get them all in a large screen cage around the host and food sources in full sun and wait patiently, and hopefully they mate naturally and lay eggs.

For our set-up, we have a large self-constructed wooden screen cage around a small potted willow tree we propagated back in the spring as the host and a live milkweed plant rooted to the ground as a nectar source. Whether or not the butterflies will actually drink from the milkweed flowers, we do not know yet, but so far they don't seem interested. In the wild, antiopas seldom visit nectar flowers and instead prefer tree sap and rotten fruit. If it turns out that they will suck from the milkweed in captivity, then obviously it is the simplest way to provide a food source. However, it may not be the case, so we will most likely need to provide them with some of their preferred food. Others have used bananas and sugar water which would be quite simple to provide them with.  

We have put several dozens of the butterflies into the cage and could have put more, but overcrowding will likely make it harder for them to mate. So far, they don't do much at all, just resting wings closed along the sides of the cage just like they do in the tub. Or, when the sun is over them, some will open their wings and bask. Only when disturbed do they flap around for a bit before returning to resting. 

If after several weeks they do not mate or lay, then perhaps it is because they are hibernating, as the overwintering stage is the adult. In that case, they will pull over until probably next March or April and perhaps then they will breed. In our area, we usually stop seeing antiopas flying past July, and they only seldom have another brood at this time. However, our butterflies were reared indoors as larvae and received a much longer photoperiod than in the wild, so possibly that might've tricked them into not going into hibernation as adults. This is our first time rearing this species so we simply do not know. We'll just wait and see what happens.

Well, as expected - since they pupated all together, they're now eclosing all together. Probably several dozens more butterflies emerged today, the vast majority of which were males (apparent since we separated the sexes into two piles, and most of the empty pupal shells were in the male pile), probably since their smaller size allows for faster development. Like other nymphalids we have seen, they excrete quite a lot of meconium, and it is bright red and watery. 

In the tub, the butterflies very calmly rest in lose groups along the sides. They are very tame and don't move much at all. Only when taken out and disturbed do they flap their wings, but if your gentle, they'll only fly a few foot or so before landing and falling "asleep" again, even when outside. In the sun, the black ventral side of the wings (seen when folded) have a blue sheen to them. The calmness of the butterflies made it relatively easy to get a bunch of shots of them, though it was slightly trickier to get them to open their wings for more them a split second at a time. We also got some more shots of the pharate pupae and newly eclosed butterflies. 

Enjoy them...

After an alarmingly short 9-11 days since pupation, several of our antiopas eclosed today. The butterflies emerged both from the incubator and in the containers in the room, so it seems the temperature hardly made a difference in developmental speed if any at all. In fact, most of the remaining pupae all look quite close to eclosion. For these pupae, we got a large tub lined with paper towels and spread them on the bottom for eclosion.

The adult butterflies are very large for nymphalids and are not sexually dimorphic, though the females are slightly larger than males (and were more massive as pupae). They fold their wings at rest which, ventrally, are black with a white boarder, making them extremely cryptic on dark, bark-like backgrounds. On the other hand, the dorsal side when opened is very stunning, having a reddish brown ground color with a cream boarder and a row of blue spots on black. The wing shapes are also quite unique, being irregularly scalloped on the margins. The body is short and rounded - typical nymphalid shape, and is very hairy.

​Unfortunately, we just now realized that if had acted sooner, we could have cold-shocked the fresh pupae in the refrigerator to induce stunning aberrants with the cream boarder enlarged and consuming the black portion with blue spots. All the remaining pupae are already quite developed and are thus too old to be cold shocked.

Shockingly, of the remaining pupae, a tachnid parasitoid exited a reddish undeveloped one and pupated which is extremely confusing to us since the larvae were all reared from egg indoors and the container was almost always closed - could it be possible that there had been a tachnid flying around in the house and somehow parasitized some of the larvae without us noticing while we were changing them? We really can't figure this one out. But, hopefully, of the remaining pupae, not too many more are parasitized.

​Authors
Alan Liang

After all our mourning cloak (Nymphalis antiopa) larvae pupated, we sexed the pupae.

Now that all of our antiopas have pupated, we removed all the pupae from their substrates (branches, lid) and collected them in plastic containers. We could have left them hanging and have them eclose in their natural position which would likely result in less crippled butterflies, but collecting the pupae grants us the rare opportunity to sex and collect data from them.

Sexing Nymphalis antiopa pupae is quite straightforward and similar to sexing any Lepidoptera pupa in general - just inspect the sex marking at the ventral rear of the abdomen. As in other Lepidoptera species, the females have a long, distinct slit cutting through the center of the fourth and fifth abdominal segment past the wings. The male's sex marking, on the other hand, is contained only in the fifth segment, and is a small hole rather than a slit. The photos below show these markings. 

We were able to reliable sex the vast majority of our 157 pupae, though some of them were difficult due to deformities on the abdomen. We ended with 71 females and 84 males, with 2 that we were unable to determine due to severe deformities near the sex marking. The sex ratio is not quite even which is quite unusual for such a large sample size, so we may have misassigned some females as males. However, we double checked and it didn't appear that we had made any mistakes so we probably could have only misassigned a few at most. This underrepresentation of females could suggest that there were some unusual factors that increased female mortality, so just to verify whether or not the sex ratio is truly unreasonable or not, we decided to take the statistical approach.

The proportion of females is 0.458 and males is 0.542. A one-proportion z-test showed that the 0.458 female proportion is not significantly less than the expected 0.5 even with a sample size 0f 155 (z=1.044; p=0.148). So, looks like we simply got a bit unlucky with the low proportion of females, though it is not unreasonable extreme. 

After we had finished sexing the pupae, our next task was to weigh and collect the mass data to compare the male and females masses. See Nymphalis antiopa Pupa Mass Data.

We collected the mass data of our mourning cloak (Nymphalis antiopa) pupae.

​After collecting and sexing all of our antiopa pupae and sorting them into two containers for each sex (see Sexing Nymphalis antiopa Pupae), we proceeded to weigh them to collect mass data. We were most interested in seeing the difference in mean mass between the sexes.

After collecting the data, we did some basic statistics. Below are the summary stats and histograms, one stacked for both sexes plus one for each sex.

Summary Satistics for Nymphalis antiopa Pupa Mass Data
                      Mean                    Std Dev                  Median       Min      Max      N  
Male           0.7880952381      0.06476047044     0.8               0.6          0.9       84
Female       0.8957746479     0.08006536565      0.9               0.7          1.1         71
All               0.8374193548      0.08983996384      0.8               0.6          1.1        155

Looking at the graphs first, the stacked male and female graph appears to be mostly unimodal and symmetric but slightly skewed right, with no outliers. The female graph is unimodal and very symmetric, almost a perfect normal curve. The male graph, on the other hand, is a little bit unusual, being slightly skewed left with a very high concentration in the center, o.8.  

The female masses are indeed higher the male's, having a mean of 0.896 g to the male's 0.788 g, over a decigram higher. A confidence interval for the difference in means is 0.085 to 0.131 (C=0.95), so clearly the difference is real. A two-sample t-test for the difference in means between the sexes also shows that the female mean mass is statistically significantly higher than the males (t = 9.2555; df = 153; p<0.0001). 


We also can't help but notice how shockingly little variation there is for the mass of both sexes, especially the male's, with standard deviations of just 0.0648 and 0.080 for males and females respectively. From what we've seen with most other larvae we have reared, the size variation is almost always very high, probably due to a plethora of random factors other than genetics while rearing - some larvae will just grow and grow while others hardly do at all, resulting in large and tiny pupae from the same rearings. It is usually only when larvae are thinned out to low densities, or if they are highly gregarious that pupae size tend to be more consistent.

​With the antiopas here, the pupae are very consistent in size and as larvae, they were extremely gregarious all the way up to pupation (they pupated in rows on the same branches), and they all grew at the same rate, all molting to the successive larval instars in near perfect synchrony. This suggests that competition between solitary larvae is a considerably large factor in their performance when reared in high densities. 

Rearing notes 7/23/17-7/27/17:

Rearing notes 7/17/17-7/23/17:

Rearing notes 7/14/17-7/16/17:

Rearing notes 7/9/17-7/13/17: