Blue crab hemolymph report, Dec 2014

Quantification and Identification of Bacteria in the Hemolymph of the Puerto Rican Blue Crab, Callinectes sapidus, Collected from the Natural Reserve, Caño Tiburones, and the Waterfront of Islote in

Arecibo.

Research paper submitted for the project class BIOL 3108

Date submitted: 9th of December, 2014

Students: Barnes, Joseph A.

Garrastegui, Emmanuel Pagán, Lillianette

Rodriguez, Génesis N.

Project supervisor: Arbelo, Jose, PhD

University of Puerto Rico at Arecibo: Department of Microbiology

Abstract

During this research project we searched for a correlation between the density and composition of the bacterial flora within the hemolymph of the blue crab, Callinectes sapidus, and the elevated levels of pollution in its habitat. Specimens for the experimental group were collected from the polluted wetlands of Caño Tiburones, while the specimens for the control group were gathered from the waterfront of Islote. Aseptic techniques for hemolymph extraction were designed and implemented, followed by serial dilutions for an estimated cell count. We successfully established the presence of bacteria and, judging by morphological and physiological characteristics, we identified a halotolerant Bacillus spp. and a Micrococcus ssp. within the microflora isolated from the hemolymph. We worked with standard lab techniques for bacterial isolation and biochemical tests and staining for bacterial identification.

1 Bacteria in the Hemolymph of the Callinectes sapidus; UPRA Dec, 2014

Introduction

Caño Tiburones is the largest estuarine wetlands in northern Puerto Rico, and a portion of it as been designated as a natural reserve within the municipality of Arecibo [5]. This reserve lies adjacent to an active landfill, which constitutes a point source of pollution to the nearby areas. The wetland serves as a natural habitat for various types of plant and animal flora, including the blue crab, Callinectes sapidus. The dominance of the C. sapidus has made it a common food source for the indigent who live in the vicinity. The contamination of this wetland by the landfill has brought to our attention the question of how the flora which inhabits the area may have been affected by the new conditions. This experiment was conducted with the purpose of assessing the microbial flora within the C. sapidus in order to determine any correlation that might exist between the microbial density and composition within the hemolymph of the blue crab and the contamination of the host’s environment. The hypothesis in question is the following: Natural habitats with elevated levels of contamination will stimulate the development of higher concentrations of bacteria within the hemolymph of the C. sapidus. The independent variable is the level of contamination in the habitat of the host. The dependent variables are the concentration of bacteria and their composition (foreign versus natural). The null hypothesis is that contamination does not affect bacterial cell concentrations. The alternative hypothesis holds that heavy contamination does alter the levels of bacteria. For the control group we used blue crabs fished from an area with less contamination, while the experimental group consisted of crabs collected from the contaminated wetland. We selected the dockyards at the waterfront of Islote in Arecibo as the site for the control group. Assessing the bacteria within the hemolymph of crabs of the genus Callinectes is not uncommon. Given the commercial importance of C. sapidus as a

widely popular food source in the eastern coastal regions of the U.S., and thus a significant concern for organizations of public health and safety, various studies have been conducted to the end of identifying the natural microflora of the C. sapidus, as well as detecting significant concentrations of pathogenic bacteria and thus prospective health threats [2, 3, and 4]. Research of the microflora of a Callinectes ssp. has also been realized here in Puerto Rico, with a focus of quantifying total bacterial densities and identifying species of bacteria in the hemolymph of C. bocourti collected from eutrophic waters as well as coastal lagoons and estuaries [1]. This earlier study served as the principle basis for the following project, as it demonstrated that crabs in eutrophic waters can sustain bacterial concentrations up to 108 cells mL-1 within the hemolymph [1].

Materials

Sterile syringes, fishing crates, plate count agar (PCA), tryptic soy agar (TSA), tryptic soy broth (TSB), mannitol salt agar (MSA), eosin methylene blue agar, crystal violet agar, bile esculin agar, hydrogen peroxide, lactose broth, 70% ethanol, Gram stain reactants (crystal violet, Gram’s iodine, safranin, 95% ethanol), toluidine blue, citrate/EDTA buffer.

Methods

Phase 1 Collecting crabs; extracting hemolymph; dilution in series: We collected 5 blue crabs (4 males and 1 female) from the wetlands of Caño Tiburones [image 5], and 3 blue crabs (2 males and 1 female) at the waterfront of Islote [image 6]. The specimens were placed in water igloos containing water collected from the respective sites. The specimens were promptly taken to the laboratory of microbiology at UPRA, where we carefully secured their chelas (claws) by wrapping a rubber band around the claw, holding tightly

Students: Barnes, J.A., Garrastegui, E., Supervisor: Arbelo, J. Pagán, L., Rodriguez, G. N.


together the dactyl and propodus [image 1]. We then randomly selected one male from each site, weighed it, and measured the width of the carapace, which we defined as the distance between the lateral spikes located on either side of the carapace. We did the same for the female from each site [table 2]. Using either a sterile 21 or 22 ½ gauge needle, we extracted 1.0 mL of hemolymph by inserting the needle into the body at the base of one of the posterior pereopods (legs), preferably the swimming leg [image 2]. Before inserting the needle, we applied 70% ethanol over the area so as to disinfect the insertion point. Each sample of hemolymph was immediately diluted in series following the procedure delineated in the lab manual [6] [image 3]. The diluted samples were used to prepare pour plates using Plate Count Agar (PCA) as the nutrient medium. After 48 hours of incubation, we counted for colonies using a dark-field colony counter. Preparing bacterial cultures; isolating bacteria: Following the serial dilutions and incubation, we prepared 20 bacterial cultures from the agar plates, each from a distinctive colony. Each bacterial culture was grown in a tryptic soy broth (TSB), incubated for 24 hours. We then prepared a streak plate, using tryptic soy agar (TSA), for each culture and incubated them for 22 hours; from the streak plates we observed 10 distinct types of colonies, and from each we prepared a pure culture using TSA slants. Following 21 hr incubation, we prepared Gram stains for each of the 10 slants to confirm whether or not they were pure cultures, as well as to observe morphology and gram-staining. The 10 bacterial cultures were sub-cultured periodically to maintain a fresh stock culture. Differential media; biochemical tests; identification: We attempted to grow selected bacterial cultures on differential media on the basis of observed morphology and gram-staining. The differential media we used are the

following: mannitol salt agar, bile esculin, crystal violet, and eosin methylene blue. We performed the following biochemical tests: catalase test and carbohydrate (lactose) fermentation (standard test for detecting coliform bacteria). We also tested for endospore-formation in the case of the gram (+) rod-shaped bacteria we isolated.

Phase 2 Collecting crabs; extracting hemolymph; dilution in series: We fished 5 blue crabs from the waterside of Islote. We collected the specimens in a water igloo containing water gathered from the same site. Securing their claws in the same manner of phase 1, we selected three crabs, all females, and extracted 1.0 ml of hemolymph from each. We used sterile 21 gauge needles with 3ml-syringes; prior to extraction we aseptically transferred 0.5 ml of sterile citrate/EDTA buffer into each syringe. The citrate/EDTA buffer was prepared in the lab using the substances published by an earlier experiment with Callinectes ssp. crabs [1] [table 4]. The needle insertion point was also disinfected with 70% ethanol before extraction. Each sample of hemolymph/buffer was promptly diluted in series following the procedure delineated in the lab manual [1]. PCA was used to prepare the pour plates and after 45 hours of incubation, we counted for colonies using a dark-field colony counter.

Results and discussion

Phase 1 We were unable to determine the cell concentration for any of the 4 hemolymph samples extracted and diluted. The samples from the female crabs generated only 6 colonies, numbers far below the permissible counting range. The samples from the male crabs also generated colony counts outside the acceptable counting range, however the male from Caño Tiburones (specimen 1) produced colony numbers that exceeded the counting range in the plates with dilutions of 10-6 and 10-7 [table 3]. The serial dilutions




for specimen 1 generated higher colony counts in the pour plates with higher dilution factors, an outcome contrary to the natural assumption that colony count on the agar plate should be inversely proportional to the dilution factor. The coagulation of the hemolymph outside of the crab body might account for this unexpected result. It is possible that due to coagulation, the bacterial cells were mostly clumped together, thus the first dilution would generated sparse but very large growths, each arising from an agglomeration of bacterial cells rather than from a single cell. After the second dilution and vigorous shaking, however, the cell clumps were loosened and distributed more evenly across the solution, and thus produced numerous, smaller colonies, each arising from a single cell. Of the 10 pure cultures which we isolated, 4 of them (cultures 1, 2, 3 and 10) appear to belong to a strain of a Bacillus ssp. in accordance to Bergey’s Manual of Determinative Bacteriology flow chart [7], wherein large, gram (+), rod-shape, spore-forming, not strictly anaerobic bacteria are classified as members of the genus Bacillus [image 8]. This strain has demonstrated the unique ability of growing on media with a high salt concentration. All four cultures grew on mannitol salt agar (MSA) with a salt concentration of 7.5% [table 1], and were able to ferment the mannitol sugar as well. It had been possible that the culture was a mixed culture, with a strain of staphylococcus hidden among the more prominent bacillus rods; however, we removed these doubts by carrying out a simple stain for the cultures that grew on the MSA. The observed morphology of the bacteria was no different from what we had seen earlier, they remained bacillus rods, thus we ruled out the possibility of contamination by staphylococcus. The presence of a halotolerant Bacillus ssp. can be attributed to the fact that the natural habitat of the host blue crab is in salt water from the ocean. The bacteria which live naturally in its host, or invaded it, are adapted to the marine environment, having

halotolerance as a requisite characteristic necessary for survival. 2 of the 10 pure cultures isolated (cultures 4 and 8) were gram (+), catalase positive, coccus-shaped bacteria capable of growth on MSA [image 7]. They did not ferment mannitol, thus ruling out S. aureus as a possible identity. However, culture 4 had a yellow pigment, which is characteristic of Micrococcus ssp., according to Bergey’s Manual of Determinative Bacteriology [7]. Culture 7 displayed streptococcus morphology and was negative for catalase. However, it failed to grow on bile esculin medium, and no blood agar was available to carry out further tests. Culture 5, 6, and 9 demonstrated small-rod shape cells, and their Gram stains were inconclusive despite repeated attempts. Their growth on crystal violet suggests that they are gram (--), however, only culture 6 grew on eosin methylene blue agar, which differential medium is inhibitory towards gram (+); thus, although culture 6 may be classified as gram (--), cultures 5 and 9 remain inconclusive. All three cultures were tested for lactose fermentation and were ruled out as possible coliform bacteria; culture 6 fermented lactose without production of gas, while cultures 5 and 9 were unable to ferment lactose [table 1].

Phase 2 As table 5 demonstrates, the pour plates generated hardly any growth, far too little for any viable cell count. To account for this absence of colony growth, it is important to note significant differences between phase 1 and 2. Whereas in phase 1, we tested the hemolymph of 2 males and 2 females from 2 different sites, in phase 2 we only tested females from one site. It is particularly evident that the males in phase 1 produced the greatest number of colonies, particularly the male from Caño Tiburones [table 3], whereas the two females generating only a few colonies more than those tested in phase 2. A final distinction is that the specimens tested in phase 2 were conspicuously smaller in size compared to those in phase 1.


Tables and Images

Table 1 Morphological and Physiological Properties

Culture Sex/Site

Cellular morpho-

logy

Gram

Catalase

Endo-spore forma-tion

Mannitol Salt Agar 7.5% NaCl

Eosin methy-lene blue

Lac-tose Fermenta-tion gr/gas

Crystal violet

Bile Escu-lin

1

M – CT

Strepto-bacillus

+

+

Growth; Mannitol fermentation

2

M – CT

Strepto-bacillus

+

+


3

M – CT

Strepto-bacillus

+

+


4

F – I

Coccus

+

+

Growth; No fermentation

5

F – I

Small-short

bacillus

N/A

No growth

--/--

Pale pink

6

F – CT

Small-short

bacillus

N/A

Tan color

--/+

Tan color

7

F – CT

Strepto-coccus

+

--

--

8

F – I

Staphylococcus

+

+

Growth; No fermentation

9

M – I

Small-short

bacillus

N/A

No growth

--/--

Soft violet

10

M – I

Strepto-bacillus

+

+


M / male; F / female; CT / Caño Tiburones; I / Islote; + / positive; -- / negative N/A – non applicable: Gram stains for cultures 5, 6, and 9 were inconclusive despite repeated attempts.



Table 2 Specimen Characteristics

Specimen (Callinectes

sapidus)

Site

Sex

Carapace width

Mass

1 Caño Tiburones Male 13.0 cm 168.0 g 2 Caño Tiburones Female 14.5 cm 180.0 g 3 Islote Female 13.8 cm 156.0 g 4 Islote Male 13.6 cm 232.0 g

Table 3

Phase 1: Colony count for series dilution with dilution factors of 4 through 7 Specimen 10-4 10-5 10-6 10-7

1 11 8 TNTC TNTC 2 1 0 0 1 3 1 1 0 2 4 1 0 9 3

Table 4

Ingredients for citrate/EDTA buffer Substances Molar concentration Calculated mass Measured mass NaCl ………………... D-glucose …………... Trisodium citrate ........ Citric acid ……….….. EDTA ………………. (Ethylenediaminetetraacetic acid)

……... 0.14 M ……... 0.10 M ……... 0.030 M ……... 0.026 M ……... 0.010 M

……… 3.2726 g ……… 7.2072 g ……… 3.0967 g ……… 1.9983 g ……… 1.4890 g

……. 3.27262 g ……. 7.19840 g ……. 3.09650 g ……. 1.99146 g ……. 1.48931 g

All substances were dissolved in 400 ml of distilled water; the solution was sterilized in the autoclave at 121 °C at 15 psi.

Table 5

Phase 2 colony counts for serial dilutions with dilution factors of 4 through 7 Specimen 10-4 10-5 10-6 10-7

1 1 0 0 0 2 1 0 1 0 3 0 0 0 0

All specimens were collected from the Islote site; all were female; all were noticeably younger and smaller than the earlier specimens collected during phase 1.




Images Photos taken during phase 1 and 2

Image 1 Image 2

Image 3 Image 4

Image 5 Image 6

Image 7 Image 8


It should also be brought to attention the fact that the nutrient media (i.e. PCA, TSA) we used in growing the bacteria may not have all the requisite nutrients necessary for sustaining growth for the bacteria associated with the blue crab hemolymph. In the work by Corwell, Wicks and Tubiash [2], bacterial counts for the hemolymph of collected specimens were higher when using MSYE growth media, which contains the major ionic constituents of seawater, than when using SMA, the Standard Methods Agar which is comparable to the TSA and PCA commonly used here. In other words, our choice of growth media may be inadequate to maintain the growth of bacteria collected from the hemolympth, and thus our plate counts may be biased against more fastidious marine microorganisms.

Students: Barnes, J.A., Garrastegui, E., Supervisor: Arbelo, J.

Conclusions

The tests and results here are not enough to prove or disprove our hypothesis. Sample numbers were too small and furthermore the testing procedure itself is experimental and under development. We have, however, demonstrated that the hemolympth of the blue crab is not aseptic and can hold a notable density of bacterial cells [table 3], and that the bacterial flora can be diverse [table 1]. Furthermore, the specimen with the highest density of bacteria in the hemolymph was isolated from Caño Tiburones. It is recommended that we perfect the procedure for counting cells in the hemolymph with further testing. For instance, we can attempt to grow E. coli on growth media supplemented with the citrate/EDTA buffer, and compare its growth to a control group, E. coli on growth media not supplemented with the buffer. This simple test would determine whether or not the buffer in any way inhibits cell growth. Another recommended step would be to more carefully control any confounding factors, such as differences in the weight, age, sex, size and health of the specimens.

Future research on this project bares the promise of demonstrating to others the harmful impact that pollution can have on the native flora of the wetlands of Caño Tiburones, and the potential health risks it signifies towards those humans who fish and consume blue crabs which inhabit the same contaminated waters.

References

[1] Rivera, A., Santiago, K., Torres, J., Sastre M.P., Fuentes Rivera, F., 1999. Bacteria associated with hemolymph in the crab Callinectes bocourti in Puerto Rico. Bulletin Marine Science, Vol. 64, No. 3, p. 543 – 548. [2] Colwell, R.R., Sizemore, R.K., Tubiash, H.S., Lovelace, T.E., 1974. Bacterial flora of the hemolymph of the blue crab, Callinectes sapidus: numerical taxonomy. Applied Microbiology, Vol. 29, No. 3, p. 393-399. [3] Colwell, R.R., Tubiash, H.S., Wicks, T.C., 1975. A comparative study of the bacterial flora of the hemolymph of Callinectes sapidus. Marine Fisheries Review, Vol. 37, Nos. 5-6 [4] Givens, C.E., Burnett, K.G., Burnett, L.E., Hollibaugh, J.T., 2013. Microbial communities of the carapace, gut, and hemolympth of the Atlantic blue crab, Callinectes sapidus. Spinger-Verlag Berlin Heidelberg, DOI 10.1007/s00227-013-2275-8. [5] Estado libre asociado de Puerto Rico, Departamento de recursos naturales y ambientales (2007). Hojas de nuestro ambiente: reserva natural caño tiburones. San Juan, Puerto Rico. [6] Cappuccino, J.G., Sherman, N., (2011). Microbiology: a laboratory manual. Pearson Benjamin Cummings, San Francisco, United States of America. [7] Bergey’s manual of determinative bacteriology: identification flow charts. Accessed 30, Nov 2014. http://www.uiweb.uidaho.edu/micro_biology/250/IDFlowcharts.pdf

Pagán, L., Rodriguez, G. N.

http://www.uiweb.uidaho.edu/micro_biology/250/IDFlowcharts.pdf

http://www.uiweb.uidaho.edu/micro_biology/250/IDFlowcharts.pdf

Documents

Blue crab hemolymph report, Dec 2014