Gill histology of Pandalus danae under acute salinity shock

By Melina G. Wettstein

ABSTRACT

Thalassinidean shrimp, commonly known as mud and ghost shrimp, are disrupting Washington’s proftable Pacifc oyster aquaculture industry by disturbing sediment. As the sediment loosens, the oysters sink, destroying the bed. In an attempt to replace harsh chemicals as pest control, a brine solution to osmotically eliminate these shrimp has been researched. Shrimp, like many other invertebrates, are osmoconformers and cannot control their internal pressure, so extreme changes in salinity cause acute death. Unfortunately, when this high salinity water is pulled back to the ocean at high tide, subtidal invertebrates, like the coonstripe shrimp Pandalus danae may be impacted. Here we show the death rates of P. danae in varying salinities, and gill dehydration due to osmotic pressure through histology. Mortality rates were high for all salinity treatments. All the gills processed through histology showed intense dehydration. Lethal exposure time varied by salinity treatment, but the amount of dehydration did not. Our results show that runoff from the brine treatments to kill mud shrimp may impact P. danae and other osmoconformers in the subtidal. This study provides information about collateral ecological impacts on other organisms in the brine control method. This study only showed the impacts of acute salinity, and did not take into account the currents or geography of the subtidal in a real ecosystem, which must be further studied.

INTRODUCTION

Washington State aquaculture is incredibly important to the economy, contributing $184 million in 2010 (Washington Sea Grant 2015). Unfortunately for the industry, oyster beds are full of pests, specifcally thalassinidean shrimp like mud and ghost shrimp (Biffarius arenosus). Burrowing shrimp have an indirect negative effect on survival of oysters by bioturbation and sediment destabilization (Feldman 2000). The oysters need a solid substrate to attach to and grow on, which is loosened by the mud shrimp as they burrow. It is not uncommon for aquaculturists to pull sinking oysters from sediment in infested beds (Smith 1996). Suspended sediments from shrimp digging negatively impact oyster’s filter feeding mechanisms as well (Feldman 2000). Through a special permit, oyster farmers typically use carbaryl, a pesticide that is the most effective method for removing shrimp from oyster beds (Feldman 2000). On the downside, newly settled subadult Dungeness crabs (Cancer magister) are directly exposed to the acutely toxic chemical spray, causing mass mortality. For this reason, oyster farmers have been pushed away from traditional pest control methods (Feldman 2000).

In saltwater systems, organisms must either expend energy to maintain their internal salt concentrations, or they must be able to tolerate some variation as the environment changes around them (Shumway 1977). Crustaceans are typically osmoconformers, meaning their osmolarity, or internal salt concentration, is at the same salinity as the water they inhabit (Foster 2010). Changing salinity is dangerous for osmoconformers because they lack the internal mechanisms to balance internal water and salt concentrations, so water diffuses freely in or out of their cells as needed to achieve equilibrium. Any changes cause stress on the proteins, enzymes, and internal processes of the organism. Rapid changes in salinity can lead to either internal damage in a hyperosmotic system or dehydration in a hypoosmotic system. Because gills are optimized for gas exchange, they have an extremely large surface area and thus high water transport (Freir 2008). This means that in a high salinity shock, this organ is likely the point of failure, especially for crustaceans with gills that are always exposed. In the aquarium industry, reef-keepers often use rapid salinity change to shock or kill crustacean parasites or kill them before introducing new corals into their tanks, as many crustaceans are prone to salinity shock (Lougher 2016). Therefore, applying, high salinity water over oyster beds at low tide could be used for crustacean pest removal.

Researchers and aquaculturists are testing 60 to 80 practical salinity unit (psu) brine solution in a new venture to rid oyster beds of thalassianian shrimp without harsh chemicals—a targeted osmotic attack (unpublished). The oysters can close up to survive, but the shrimp are left exposed to the brine. In an acute high salinity treatment, the targeted shrimp would be unable to keep its cells, specifcally in the gills, hydrated and would die from salt shock. Though the targeted species is eliminated in this treatment, the high salinity water pulse may suddenly food the subtidal zone during high tide. Because most invertebrates are osmoconformers, they are all susceptible to the brine treatment. The treatment that killed the ghost shrimp could be the downfall of many other crustaceans in the subtidal.

The goal of this experiment was two-fold. First, using the dock or coonstripe shrimp Pandalus danae, we can test the tolerance of common Pacifc Northwest shrimp species to the brine treatment being washed back into the ocean. Though not directly found near oyster beds, P. danae is a model organism for the study because it is widely distributed throughout the Puget Sound, and has morphology similar to many other crustaceans. Then, we can use histology to observe the osmotic issues within the physiology of the shrimp when exposed to the high salinity. Histology examines thin slices of tissue on a microscopic level, and so the osmotic changes in structure post-exposure can be observed. Using histology to determine an osmotic failure has not been done before. Through subjecting shrimp to varying salinities then comparing the gills of all the shrimp, we can test the damage done by the high salinity treatment to other crustaceans.

METHODS

Collection: P. danae of sizes ranging from 5 to 10 cm were collected with a net from the dock at Friday Harbor Labs, San Juan Island, Washington both during night lighting and during the day. They were then stored in a fow-through system for a few days to a few weeks until needed, being fed freeze dried shrimp every few days.

Experimental set up: Starting with a base of natural sea water, the salinity was adjusted to 35 psu (control), 45 psu, 55 psu, and 65 psu, shrimp were randomly assigned 500 mL solution of the treatment condition (n = 20 per treatment). One shrimp each was added to each container. Signs of movement were checked every 5 minutes for four hours, the rough length of a tidal cycle, when the brine solution would mix with sea water. If the shrimp was entirely unresponsive and no moving limbs could be seen, that organism was considered dead. The gills were removed and placed in salinity adjusted 10% formalin solution. At the end of four hours, all shrimp were measured for length from tip of rostrum to tip of tail, stretched out into a straight line.

Histology: The extracted gills were prepared for histological analysis. After being fixed in formalin for 24 hours, the gills underwent a dehydration step in ethanol. Gills were placed in a 50%, 70%, 90%, and 95% ethanol-seawater solution at the experimental salinity. After an hour in 100% ethanol, the gills then went through two one-hour xylene baths to displace the ethanol until the tissue was clear. Tissue was then placed in liquid paraffin at 59°C and put in a 65°C oven for an hour. The paraffin was changed twice more with hour long intervals between before being embedded into a block with the largest gill surface area down. After setting overnight, the tissue was sliced on the microtome to a thickness of 8 microns. The tissues underwent a standard Eosin and Hematoxylin stain. Gill histology photos were taken with an INFINITY5-5 camera on a Nikon compound microscope.

Statistics: ANOVA, followed by a Tukey’s HSD post hoc test, was calculated, comparing time of death for each treatment to determine any statistical difference. ANOVA was also calculated to ensure no size bias. Averages and standard deviation, as well as ANOVA and Tukey’s HSD values were calculated in R.

RESULTS

At the end of 4 hours in the salinity treated water, all shrimp in the 55 psu and 65 psu conditions had died. The average time of death for the shrimp in 65 psu water was 29.5 ± 12.7 minutes and the average time of death was 37 ± 12.0 minutes for the 55 psu shrimp. These two conditions were statistically the same (P = 0.905). The time of death varied in the 45 psu condition, with one dying as early as 20 minutes into the experiment and some surviving the entire trial by lying on their sides, only to perish post-experiment. The shrimp for the 45 psu treatment had an average death time of 186.65 ± 67.7 minutes. All of the control shrimp survived the trial. The differences between the 35 psu treatment, the 45 psu treatment, and the 55 psu/65 psu treatments are all statistically significant (P ≤ 0.00004) (Figure 1).

After the trial, the 45 psu shrimp that survived the 4 hours were not able to recover from the treatment. Though some shrimp survived the treatment, they were unable to recover and died. Post-treatment, in the 35 psu control treatment, one shrimp was lying on its side but remained alive, though on its side, beyond the treatment for hours, before dying and subsequently being eaten by another shrimp. This is likely not caused by the treatment, given that it was a control.

All the shrimp in each treatment were the same average size. The average size shrimp among all the treatment groups was 7.48 ± 1.15 cm (Figure 2). Shrimp size was not statistically different across treatment groups (ANOVA, P = 0.89). There was no correlation between shrimp size and how rapidly that shrimp perished (P = 0.45). Histology on the gills revealed the osmotic issues when the shrimp were exposed to the varying salinities. The control gills are normal, with an even width throughout the entire gill (Figure 3A). The treatment condition gills— from 45, 55, and 65 psu—all appear relatively similar and completely dehydrated (Figure 3). For all treatments, the main length of the gill is much thinner, but the tip is still infated. The 45 and 55 psu gills are comparable in their dehydration. The 65 psu gills appear slightly more dehydrated than the other salinity treatments, and are signifcantly shrunken when compared to the control gill.

FIGURE 1: Boxplot showing time of death for Pandalus danae for all treatments. The box for the 35 psu treatment is a line because all of the shrimp survived the four hours. The box for the 45 psu treatment has a very large range, yet none of the data is an outlier. The 55 psu treatment and 65 psu treatment both have very low averages. The overlap between the two times of death is clear. Both treatments have a shrimp that survived later as an outlier, enforcing the idea that the shrimp are unable to survive in these salinities.

FIGURE 2: Boxplot showing the average lengths of Pandalus danae in each salinity treatment. The average for each treatment was between 7 and 8 cm. There is not a significant difference in the sizes, given by the overlap in boxes.

FIGURE 3: Gill histology of the Pandalus danae.(A) 100x magnifcation of shrimp gills from the 35 psu, control treatment. They are fully hydrated. (B) 100x magnifcation of shrimp gills from the 45 psu treatment. (C) 100x magnifcation of the shrimp gills from the 55 psu treatment. (D) 100x magnifcation of the shrimp gills from the 65 psu treatment. The gills are dehydrated and thin until the very tip.

DISCUSSION

As confirmed through the histological study, the shrimp died from gill tissue dehydration and destruction. All gill tissue except the very tip was completely dehydrated and presumed to no longer work for gas exchange. This was consistent through all treatments, despite the varying salinities. This indicates that the complete dehydration of the gills determines time of death, and the salinity impacts how quickly the dehydration occurs. If the dehydration was partial or incomplete, the 45 psu gills would appear in a state between the standard 35 psu gills and the completely dehydrated 65 psu gills. Though some studies have shown other organs as points of stress in prolonged high salinity environments, the gills are the frst organ to fail in shrimp. This result is unsurprising because of the extremely high exposed surface area for the gills compared to any other organ.

Due to the impermeable crustacean carapace, the only major potential for crustacean body water loss is through exposed tissue. Though this is an advantage from an osmotic point of view, as the rest of the shrimp is not subject to dehydration at such a large rate, it is a struggle from an oxygen transport perspective. Crustacean gills are very important because the exoskeleton limits any cutaneous gas exchange (Bridges 1980). Thus, failure within the gills explains why death occurred so rapidly, within 15 minutes for multiple shrimp in the 55 and 65 psu treatments. This relationship between cutaneous gas exchange and protection from the osmotic pressure would be another element to determining the high salinity runoff impact for a larger variety of invertebrates beyond crustaceans. Each invertebrate morphology is dramatically different, but these differences could be a source of future research into the impacts of brine water from oyster beds.

Due to osmotic failure, P. danae dies rapidly when exposed to high salinity. In the context of high salinity treatment on oyster beds, the salt runoff would infuence the subtidal organisms. As the proposed treatment of 80 psu brine washes into the general water mass, the highly dense salt water may sink below the normal sea water and suffocate the organisms. This is especially impactful for osmoconformers. While freshwater decapods have the ability to osmoregulate within their tissues, saltwater crabs and shrimp never developed this trait (Foster 2010). In addition, crustaceans tend to do better with gradual salinity increases and do not have a high salinity tolerance (Lougher 2016). This study tested an acute salinity shock, similar to the conditions of the brine treatment, rather than the gradual acclimation needed for survival. Though there is no research of how quickly this salt brine would rush through and stay the subtidal, the large salt input would lead to acute salinity shock with little time for acclimation. The brine treatment mixing with the incoming tide means the subtidal organisms are not subjected to a pure treatment, but the salinity would be within the range of our experimental treatments. The extent of this mixing, and the range of subtidal impacts depends on the bathemetry and the tidal patterns of the area. This is an area for future studies to evaluate the effect of increased salinity. Even in water 10 psu higher than the control, the shrimp struggled and ultimately died, even when returned to normal seawater. Though able to maintain some bodily processes, they were unable to recover from the intense damage the dehydration caused. The brine solution is outside their ability to acclimate.

Before any large-scale implementation of oyster bed control with brine solution can be implemented, further studies are needed to discern how extent of treatment and tidal mixing would dissipate the pesticide treatment and impact invertebrates, especially vulnerable crustaceans. If a way of removing pests is necessary, the brine treatment and runoff may be the ‘lesser of two evils’ when compared to such a toxic chemical. Juvenile and larval Dungeness crab death is a major issue in using carbaryl, as low population numbers have been directly correlated to oyster beds treated with carbaryl (Feldman 2000). Though the crabs have been proven to do poorly in low salinities, both as juveniles (Reed 1969) and adults (Curtis 2010), very little has been observed for their response to high salinity shock. It is uncertain whether the brine solution would be any better for their populations than the chemical alternatives. If the acute salinity shock is limited when the brine is washed into the subtidal, the ecological impacts could be lesser than the chemicals currently in use. Through a future ecological study, the impact of both chemicals and brine solution should be weighed, both in the oyster beds, and in the subtidal.