2. I. Background
II. Results from 2014 and 2015 VT Fish and Wildlife
surveys
III. Histology of normal vs affected skin
IV. Preliminary results from most recent USGS
survey (2015)
V. Potential risk factors
4. Beginning in 2013
“Black tar-like substance” on skin surface
Multiple locations:
South Bay, Hospital Cove (main lake) – shallow sites
Black River
Willoughby River
Halls Creek
19. Reports began in 2013
Prevalence of melanomas confirmed
Began study on potential risk factors
Combination of UVB/sunlight radiation and
contaminant exposure
What’s next?
20. Kathy Spring, Darlene Bowling, Pam
Whittington, USGS, for histology technical
support
Jud Kratzer and Len Gerardi, VT Fish and
Wildlife, for fish collections
Editor's Notes
This weekend I’ll be giving a talk at the AFS meeting, so this is a practice run for you guys. My topic is on melanomas found on BBH from Lake Memphremagog in Vermont.
To give you an outline of my presentation, I’ll start with some background and give you a sense of why this is important. Then, I’ll discuss the results from prevalence surveys taken in 2014 and 2015 by the VTFWS. Also, so you understand the severity of the melanomas, I’ll show you normal BBH skin and compare the histology to what we see in melanomas. I’ll talk about the most recent survey in 2015 that was completed by both VTFWS and USGS and we’ll talk about some potential risk factors.
Here is Lake Memphremagog where the observed epizootic is occurring. It is a natural lake (almost 1800 sq. km.) within the boundaries of VT and Quebec. Over here is South Bay, a 470 acre shallow bay in the southern part of the lake on the VT side; and this is where most of the sampling for this project took place.
Reports began in 2013. Observations from anglers of raised black skin pigmentation and a black tar-like substance on the skin surface of BBH in the lake raised concern of potential contamination. The lesions were reported in multiple locations, including South Bay and Hospital cove which are within the main lake as well as Black River, Willoughby River and Halls Creek.
These pictures are examples of what the lesions look like. You can see some of them are significantly raised while others are only slightly raised. You can also see that the lesions are in different locations on the fish, versus any one spot in particular, and they range in size.
Some more examples demonstrate again that the lesions are not just on the head or the body, but all over; even inside and around the mouth. These pictures also give you a good idea of the size ranging from small like the top left picture to almost covering half the fish like the top right picture.
To determine the prevalence of the lesions in the lake, VT Fish and Wildlife conducted an initial survey in Hospital Cove. This was in June of 2014. They found 26% of the 73 adult BBH collected were affected. They collected 7 tissue samples and sent them to Dr. Vicki Blazer at USGS for histological analysis and initial examination of the lesions demonstrated melanoma rather than the more common hyperplastic lesions (which Kelsey and Vicki will talk about next?)
The lesions from the initial survey in 2014 were observed in fish 225 mm and longer as you can see by the red bars on the graph. Age data was not available, but based on length these are likely to be bullhead 3yo and above.
One year later in 2015, VT Fish and Wildlife conducted a follow-up survey. This time it was in South Bay. They found the prevalence to still be higher than usual; 28% of the 75 individuals collected were affected with the melanomas. So how do we know these are melanomas?
Looking at the skin histologically, we can tell if there is melanoma. This is normal BBH skin and it is very organized. Normal bullhead skin has an epidermis with many alarm or club cells, a BM that contains most of the melanocytes or melanophores (some are in the epidermis but they are small and compact), and then below the BM is a thick layer of dense connective tissue before you get to the muscle.
Melanomas histologically are much more disorganized compared to normal bullhead skin. Notice the progression you see starting with the top left picture and getting worse as you look to the right and then below, with this one being the worst. In this one you can still see alarm or club cells, then you move to the next one where all the alarm cells have been lost, but you can still see some structure, then you move below to this one that has completely lost structure and eventually the skin turns like this one where mostly all normal cells are lost.
Looking at them more closely, this was the first one. These are the alarm or club cells, the structure is still there, and the melanocytes are just beginning to penetrate down into the dermis and some into the muscle.
Here, you can see the club cells in the epidermis have been lost and there are a lot more neoplastic cells invading down into the hypodermis and muscle. Also note that there is still some normal structure. Here is the epidermis, the BM and then below is the connective tissue.
In the most progressive stages, the tissue will completely lose its normal structure and there will be even more neoplastic cells. In the tissue on the right (in the boxed region) is where all the normal cells have been lost and replaced with neoplastic cells.
So, in an effort to determine what factors could be contributing to the melanomas, this past summer (along with pHd candidate Cassidy Hahn and Dr. Blazer of USGS as well as Tom Jones from VT Fish and Wildlife), we went up to VT to sample. We sampled 50 total fish; 20 normal-appearing and 20 with melanomas from the main lake as well as 10 normal-appearing individuals from a nearby reference lake. We sampled for contaminant analysis, molecular/transcriptome analysis, in situ hybridization, histology and virology. We sampled pieces of skin, liver and fillet for contaminant analysis. Pieces of skin were sampled and preserved in RNAlater for molecular analysis and in PAXgene for in situ hybridization. Pieces of organs were sampled and preserved in Zfix for histological analysis and bacterial cultures as well as tissues were sampled for virology analysis for the Fish and Wildlife service Wild Fish Survey.
During our field sampling, we observed 3 fish (out of 20) with very invasive melanomas invading from the skin down into the muscle. This is somewhat atypical for melanomas that have been seen before in BBH. Particularly, this degree of invasiveness is something we haven’t seen before.
We also observed metastasis (muh-tas-tuh-sis) in some fish (during our field sampling) where the melanocytes actually moved or migrated to another tissue through the blood. Out of 20 affected fish, 2 had black areas in the gills, 1 had black areas in the ovary and 1 had black areas in the intestine.
So what could the epizootic be associated with? There are a few risk factors we think could potentially be correlated with the presence of melanoma. First, UVB radiation and sunlight are potential factors because they are major risk factors for melanomas in mammalian spp. so there could be a possible association with climate change, water clarity, or water level. Contaminants are also a concern because of melanin’s role as a free-radical scavenger. Although melanomas in other fish have not shown a lot of evidence for contaminants (most have been correlated with UVB radiation), we cannot rule this out because melanocytes do happen to be hormonally regulated and there has been a lot of evidence to suggest contaminants as a factor for endocrine disruption so this could be part of the explanation. Some combination of radiation and contaminants seems to us to be the most likely scenario, but at this point we still can’t rule out other things like heredity or infectious agents. Heredity is a potential risk factor based on UVB and chemical carcinogenesis studies done with the platyfish-swordtail hybrid; but we don’t think the cause could be hereditary for 2 reasons; one, fish surveys have been conducted in the area for many years and there has been no evidence of melanomas reported before this time and two, currently only a discrete population in the lake is being affected. We also think infection is unlikely because we currently don’t know of any melanomas that are caused by infectious agents.
To sum everything up, reports of the melanomas began in 2013 and anglers were reporting black skin pigmentation and a black tar-like substance on the skin surface of BBHs in the lake. VT Fish and Wildlife conducted 2 surveys to determine prevalence and sent tissue samples to Dr. Blazer for confirmation of melanomas rather than the more common hyperplastic lesions. Once this was established, we went up to VT to work with Tom Jones of the Fish and Wildlife service to sample fish for a study on potential risk factors. And now based on our preliminary data and observations, the take away is that we hypothesize that the melanomas are most likely associated with a combination of radiation exposure and contamination. So what’s next? We want to work out the skin transcriptome because we hope the molecular analysis will provide more insight into the cause. We already have all the samples in PAXgene and RNAlater so once we get that completed we’ll have more of an idea of what we need to do after that.
What contaminants do you think could be in the water? Do you know what type of landscape is surrounding the lake? Any agriculture?
Surrounding the lake is mostly forested area, no big factories or farms, they are somewhat concerned about runoff but don’t know what or where the runoff is from, at this point we really don’t know what specific contaminants could be involved but they are doing sediment and skin contaminant analysis so hopefully those results will give us a better idea.
they did notice that the reports started coming in after a major storm event. In May of 2013 there was very heavy rainfall and flooding associated with hurricane/tropical storm Irene.
