Effects of Resmethrin and PBO Juvenile Blue Crab (Callinectes sapidus) Foraging

Erin Plachy

Texas A&M University Corpus Christi

6300 Ocean Dr. Corpus Christi, Texas 78412

erin.plachy@spartans.ut.edu

Keywords: Callinectes sapidus, pesticide, resmethrin, Piperonyl butoxide, foraging ability,

mesocosm, brown shrimp

ABSTRACT

The effects of resmethrin and PBO (Piperonyl Butoxide), two common toxicants used to

control mosquito populations, on juvenile blue crab foraging behavior was studied. Resmethrin

and PBO are commonly sprayed together in a 1-to-3 ratio to control mosquito populations.

Juvenile male blue crabs (Callinectes sapidus) and brown shrimp were collected from Corpus

Christi, Texas and kept in tanks for a minimum of 48 hours in a salinity of 18-20 PSU. Before

being placed in mesocosms, crabs were exposed in to control water or 1:3 ppb R-PBO for 12

hours, or 10:30 ppb resmethrin-PBO (R-PBO) for 3 or 12 hours. Shrimp were similarly exposed

to control water. One crab and eight shrimp were placed in each mesocosm, and the number of

shrimp alive at 1, 2, 3, 6, 8, 12, and 24 hours was recorded. The 10:30 ppb R-PBO crabs ate less

shrimp than both the control (p=0.0796) and the 1:3 ppb R-PBO crabs (p=0.00035). The 1:3 ppb

R-PBO crabs ate significantly more than the control ethanol crabs (p=0.0079). Resmethrin

affects the nervous system which reduces a crab’s motor skills, making it difficult to catch prey.

A decrease in predation pressure by blue crabs on prey species could cause a shift in predator-

prey dynamics in estuarine ecosystems.

INTRODUCTION

The blue crab (Callinectes sapidus) is essential to many ecological communities and is a

key predator and prey species in areas along the eastern North America to South America

(Williams 1973). In addition to their ecological importance, blue crabs are also commercially

important; however, crab landings are at their lowest since the late 1960s, indicating a decline in

crab abundance (Sutton and Wagner 2007). The decline is primarily attributed to loss of habitat

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and overfishing (Guillory et al. 2001), but there is a growing concern for insecticide chemicals

entering blue crab habitats through runoff.

Pennington et al. (2001) found that especially after periods of significant rainfall, 93.4%

of all mid-Texas coast estuarine waters contained triazine herbicides from local spraying. Since

blue crabs spend much of their life in estuarine systems, they are likely exposed to runoff

pollution from surrounding human populations/cities. SCOURGE™ is an insecticide often used

for mosquito control and contains the chemicals resmethrin and piperonyl butoxide (Zulkosky et

al. 2005). Resmethrin interferes with the nervous system of insects (Zulkosky et al. 2005) and is

commonly used with Piperonyl butoxide (PBO), a synergist. PBO inhibits an organism from

metabolizing toxins, which amplifies the effect of the resmethrin (WHO 2001; Zulkosky et al.

2005). Several studies have been conducted on the effect of pesticides on blue crabs (Osterburg

et al. 2012; Horst and Walker 1999), but the sublethal effects of resmethrin with PBO (R-PBO)

on juvenile blue crabs are basically unknown.

The objective of this project was to study the sublethal effects of R-PBO as it pertains to

foraging ability of juvenile blue crabs. Since the pesticide’s target, insects, are part of the same

phylum as crabs, there are likely similar side effects from exposure. Because blue crabs are so

important in the estuarine ecosystems and to commercial fisheries in the U.S., it is necessary to

mitigate any additional pressures on the existing population.

MATERIALS AND METHODS

Juvenile male blue crabs and brown shrimp were caught in Corpus Christi Bay and the

Upper Laguna Madre (TX). Animals acclimated to lab conditions for a minimum of 48 hours and

were kept in tanks with untreated salt water (salinity 18-20). Crabs were caught via crabbing

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with raw chicken, and shrimp were caught using a push net or a seine net. Crab size ranged from

35-55 mm, and shrimp size ranged from 35-50 mm. Crabs were required to have both chelipeds

and swimming legs as well as most of their walking legs.

Fourteen-liter plastic tubs equipped with filters, oyster shells, and untreated saltwater

were set up. Twelve hours before the experiment, shrimp and crabs were measured and assigned

to individual bowls or jars with either R-PBO water or control water. Control water included

ethanol because ethanol was present in the pesticide stock solution. Crabs and shrimp were

exposed to 1:3 ppb R-PBO water, 10:30 ppb R-PBO water, or water with ethanol. Since more

than half of the 10:30 ppb R-PBO crabs died overnight, so the effects after 3 hours of exposure

to 10:30 were also studied (Table 1). Shrimp were placed into mesocosm tubs first, and allowed

to hide for 5-10 minutes before one crab was added. The number of shrimp alive in each

mesocosm was recorded at hour 1, 2, 3, 6, 8, 12, 24, 36, 48, 60, and 72 hours, or until the crab

had eaten all the shrimp. Other endpoints recorded included the number of shrimp that were not

hiding, if/where the crab was hiding, and whether or not the crab had clumped the oyster shells.

Table 1. Mesocosm crab and shrimp treatment with corresponding Resmethrin-PBO concentrations and exposure time

Mesocosm Crab Exposure Shrimp Exposure Time Exposed

Control Ethanol 0 0 12 hours

Crab Low Exposure 1:3 ppb R-PBO* 0 12 hours

Shrimp Low Exposure 0 1:3 ppb R-PBO 12 hours

Both Low Exposure 1:3 ppb R-PBO 1:3 ppb R-PBO 12 hours

Crab High Exposure 3-hr 10:30 ppb R-PBO 0 3 hours

Crab High Exposure 12-hr 10:30 ppb R-PBO 0 12 hours

*Resmethrin to PBO ratio is labeled as R-PBO. “0” exposure indicates control ethanol water.

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RESULTS

Statistical tests were complete in JMP and Microsoft Excel (2010). The 10:30 ppb

exposure 12-hour treatment was not used due to lack of replicates. Data was analyzed using a

repeated measures ANOVA (JMP), with pesticide treatment as a fixed factor. Treatment was

nearly significant (p=0.0630), indicating that treatment had an effect on the number of shrimp

alive. Because of the small number of replicates (i.e., n<6 in some cases) in the treatments, this

result should likely be considered significant in order to avoid making a Type II error. Time was

a significant factor (p< 0.0001), and the time-treatment interaction was not significant (p=0.280).

In Excel, a students t-test (assuming equal variances) was completed to compare

treatment levels to each other and to the controls, with a Bonferroni correction. The number that

survived at each time point differed between treatment groups (Figure 1). Crabs exposed to

10:30 ppb for 3 hours tended to eat significantly less shrimp than the control group (p=0.0796)

whereas crabs exposed to 1:3 ppb for 12 hours ate significantly more than the control group

(p=0.0079). In comparison to each other, crabs exposed to 10:30 ppb (3-hour) ate significantly

less than the crabs exposed to 1:3 ppb (12-hours).

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0 1 2 3 6 8 12 240

1

2

3

4

5

6

7

8

9

Average Shrimp Alive: Res. + PBO

Control Ethanol 10:30 ppb 3-hr

1:3 ppb 12-hr

Hour

Num

ber o

f Shr

imp

Aliv

e

Fig 1. Average Shrimp Alive Over Time. Lines show average number of shrimp alive for crabs

exposed to control (green, dashed line), 10:30 ppb resmethrin:PBO (red), or 1:3 ppm

resmethrin:PBO. Error bars are ±SE.

DISCUSSION

Results indicate that that crabs exposed to higher concentrations (10:30 ppb R-PBO)

have suppressed foraging abilities whereas crabs exposed to lower concentrations (1:3 ppb R-

PBO) seem to have slightly enhanced foraging abilities in comparison to the control group.

Though there are virtually no published studies done on crabs with R-PBO, some studies have

investigated the effects of mercury and other heavy metals on other aquatic organisms that

produce similar effects. Heavy metal contamination can reduce feeding and foraging ability in

aquatic organisms, including species such as killifish (Reichmuth et al. 2009; Weis et al. 1999,

2000, 2001, 2011). Weis et al. (2001) concluded that contaminants (e.g., heavy metals) can alter

an organism’s motivation to feed and reduce foraging search effectiveness, and their ability to

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capture prey. These findings are consistent with the 10:30 ppb 3-hr exposure treatment, as crabs

ate fewer shrimp at a slower pace compared to control crabs. Several crabs exposed to R-PBO

were observed with a shrimp in their claw for extended periods of time, indicating that they

might be spending a longer amount of time consuming a single shrimp. Additionally, treatment

crabs were often uncoordinated and where observed swiping/swinging their claws in the

direction of shrimp but were unsuccessful in actually catching shrimp.

For blue crabs specifically, Reichmuth et al. (2009) found that crabs exposed to mercury

were less able to capture active prey such as shrimp, which caused crabs to consume less active

prey like mussels. This indicates that blue crabs, both in this work and ours, may be more

affected by reduced coordination rather than lack of motivation or hunger. Since resmethrin

affects the nervous system, motor skills are greatly reduced, making it more difficult for the

crabs to catch active prey. In the 10:30 ppb R-PBO 12-hr treatment, the crabs that did survive

were observed waving their chelipeds erratically and flipping upside-down as if they had lost all

control of their muscular system; the dead crabs were found the next morning upside-down, with

several autotomized limbs. Though the 10:30 ppb R-PBO 3-hr treatment crabs did not react as

severely as the 10:30 ppb 12-hr treatment crabs, it is likely that they endured similar, but

reduced, side effects.

The implications of pesticide runoff entering estuaries could potentially change the

community structure. Weis et al. (2011) studied several estuarine organisms in a contaminated

estuary and their foraging behaviors. All the species, including blue crabs and killifish, were

negatively affected from exposure, in varying degrees, except for grass shrimp (Palaemonetes

pugio) which had unchanged predator avoidance and was found to have partitioned its energy,

favoring growth and reproduction. The grass shrimp were able to increase their reproduction,

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grow larger, and live longer because of the combination of decreased predation pressure and the

shrimps’ ability to partition resources (Weis et al. 1999, 2011). Exposed to contaminants like

heavy metals and pesticides, blue crabs would not be able to forage as well, having to select less

optimal prey. Their very wide diet variety would be reduced, affecting their life span and

numbers similar to the findings in a study by Weis et al. (1999) on mummichongs exposed to

contaminants. In addition, because blue crabs are such an important predator in estuaries,

predation pressure would be relieved for many prey species who would then be allowed to

increase in numbers. Community structure based on predator-prey interactions would shift if

prey species became more abundant.

The 1:3 ppb R-PBO 12-hr unexpectantly ate significantly more than the control

(p=0.0079), as we expected 1:3 ppb crabs to eat less than the control ethanol group, but more

than the 10:30 ppb 3-hr crabs. This result contradicts those found by the previously discussed

studies with contaminants, though work with this specific toxicant has not been studied. Further

experimentation is needed to better understand why and how exposure might cause in increase in

foraging success and needs. In the future I would repeat the experiment with incremental

increases in concentrations of R-PBO in order to investigate the trend in the number of shrimp

alive over time for each concentration. Also, I would add 10:30 ppb 6-hrs and 8-hrs to see if

there are any changes in results between the 3-hr and 12-hr exposure. If lower exposures of R-

PBO occur in estuaries, the effects in natural systems would be dramatically different than those

predicted in laboratory experiments at higher concentrations. These results indicated that

exposure may cause crabs to increase foraging, which inturn may increase predation pressure on

prey species if these results are indeed consistent.With the increased use of pesticides used to

control mosquitoes in the past few decades, aquatic organisms such as blue crabs will

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increasingly encounter these harmful chemicals; further research is necessary to understand how

pesticides affects these ecologically and commercially important species.

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