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Journal of Experimental Marine Biology and Ecology 399 (2011) 142–150
Contents lists available at ScienceDirect
Journal of Experimental Marine Biology and Ecology
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j e m b e
Experimental validation of the “relative volume of the pharyngeal lumen (RVPL)” of
free-living nematodes as a biomonitoring index using sediment-associated metals
and/or Diesel Fuel in microcosms
F. Boufahja ⁎, A. Hedfi, J. Amorri, P. Aïssa, E. Mahmoudi, H. Beyrem
University of Carthage, Faculty of Sciences of Bizerte, Laboratory of Environment Biomonitoring, Coastal Ecology and Ecotoxicology Unit, 7021 Zarzouna, Tunisia
a r t i c l e
i n f o
Article history:
Received 27 August 2010
Received in revised form 2 January 2011
Accepted 25 January 2011
Keywords:
Microcosm
Free-living nematodes
Relative volume of the pharyngeal lumen
Biomonitoring
Metals
Diesel Fuel
a b s t r a c t
Since 1981, many numerical indices have been developed for meiobenthic nematodes but later discarded. To
overcome problems and drawbacks in this type of indices, the “relative volume of the pharyngeal lumen
(RVPL)” was first introduced in biological assessments in 2006 and it focused on the assumption that pollution
is commonly expressed in terms of reduced food intake. This index, evaluated by using a morphometric
approach, expresses the sucking potential of the pharynx. A series of microcosm experiments was performed
in order to test the “relative volume of the pharyngeal lumen (RVPL)” as a biomonitoring index in the cases of
8 free-living nematode species (the deposit-feeders, Daptonema normandicum, Desmodora longiseta, and
Leptonemella aphanothecae; the epistrate feeder, Xyala striata; the facultative predators, Oncholaimus
campylocercoides and Oncholaimellus mediterraneus; and the predators Bathylaimus capacosus and
Mesacanthion hirsutum). Each species was exposed to various concentrations of toxicants (nickel, copper,
chromium and Diesel Fuel) used separately or in combinations. Our results showed that RVPL increased with
increasing amount of toxicants, visibly caused by a systematic decrease in body volume with toxicants.
Responses of all examined species suggested an additive toxicological action. Predators (M. hirsutum and
B. capacosus) showed the highest potential of adaptation because of their higher means of RVPL. Observations
during this experiment highlight the importance of pharyngeal pumping and cuticular structure to tolerating
elevated catabolic water loss under stress conditions. Thus the ability to osmoregulate is an important factor
in overcoming food sedimentary toxicants. Based on all these findings, RVPL was found to be an accurate and
precise index of monitoring the effect of metals and Diesel Fuel in marine ecosystems.
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
Free-living nematodes have been widely used to determine the
effects of perturbations in marine systems (Coull and Chandler, 1992;
Austen and McEvoy, 1997; Tita et al., 2002; Vanhove et al., 2004;
Boufahja et al., 2007). They are ideal for experiments because of their
small size coupled with high abundance and diversity (Beyrem et al.,
2010). Using these small and easily maintained organisms allowed us to
reduce the size of the microcosm containers without discernible edge
effects and without sacrificing the animal's indicator potential.
Additionally, most nematode species have short life cycles and therefore
a potential for rapid responses to many toxicants (Guo et al., 2001).
Numerical indices have been used for many years in biomonitoring
programs, for example, the use of the nematode: copepod ratio (Rafaelli
and Mason, 1981) was very popular in the 1970s. In general, nematodes
are more resistant to environmental stress than are copepods. For this
⁎ Corresponding author. Tel.: +216 72 591 906; fax: +216 72 590 566.
E-mail address: [email protected] (F. Boufahja).
0022-0981/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.jembe.2011.01.017
reason, a high ratio indicates pollution, such as oil spills, domestic
sewage, and increasing organic enrichment (Rafaelli and Mason, 1981;
Amjad and Gray, 1983). Soon after, the Trophic Diversity Index (TDI)
was proposed by Heip et al. (1985). Subsequently, simple ratios of
nematodes by trophic group (Wieser, 1953) were employed, the most
recognized is the ratio of non-selective deposit-feeders to epigrowthfeeders (Lambshead, 1986). Later, the application of the Maturity Index
(MI) was developed for free-living nematodes (Bongers et al., 1991).
Numerical indices are popular because they are not subjective and
are easy to calculate. However, many problems limit their usefulness
as indicators of environmental conditions. Numerical indices of
environmental perturbation use taxa abundances or proportions of
feeding groups within a taxon without differentiating life cycle stage
or other weighting for qualitative differences among taxa. The use of
morphometric indices, though vital to demonstrating physiological
and evolutionary responses to environmental perturbations (e.g.,
Pearson, 1948), has been neglected.
It is known that the pharynx of nematodes is a pumping organ that
serves, together with various differentiations of the buccal cavity, the
uptake and transport of food (Hoschitz et al., 2001). As a first step in
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F. Boufahja et al. / Journal of Experimental Marine Biology and Ecology 399 (2011) 142–150
assessing the relationship between environmental quality and
pharyngeal morphometry of nematodes, the “Relative Pharyngeal
Volume” (RPV) was proposed for Paracomesoma dubium (Comesomatidae) (Boufahja et al., 2006). This index, re-called here the
“relative volume of the pharyngeal lumen” (RVPL), gives an idea about
the potential of the pharynx to successfully suck in food. The fact that
there is probably a relationship between pollution and sediment
nutritional quality suggested to us that the morphometrics of this
organ may be of value as an index of environmental perturbation.
Despite this, no work has been published that uses the RVPL in
nematodes as a monitoring tool. The main reason for this neglect is
probably the small size of most meiobenthic nematodes.
The experiments reported here set out to explore the effects of 3
metals (nickel, copper and chromium) and Diesel Fuel on RVPL of
8 nematode species. Toxicity of the chosen metals was previously
proved in the case of the meiobenthic harpacticoid copepod
Amphiascus tenuiremis (Hagopian-Schlekat et al., 2001). On the
other hand, Mahmoudi et al. (2005) have demonstrated experimentally that a nematode assemblage from Ghar El Melh lagoon (NE
Tunisia) was particularly sensitive to Diesel Fuel with effects observed
even at low concentrations.
The objective of the present study was threefold: first, to test the
usefulness of RVPL in assessing pollution; second, to determine the
potential adaptations to environmental perturbations for each species
studied; third, to determine the treatments that are responsible for
the highest differences in RVPL across treatments and controls.
2. Materials and methods
2.1. Collecting site
Natural sediments with their meiobenthos were collected on 25th
January 2006 from Sidi Salem beach (37° 17.385′N 09° 52.289′E) in
Bizerte Bay (Tunisia) (Fig. 1). This sandy beach is a simple ecosystem,
principally driven by the physical forces of waves and tides. The
sedimentary concentrations of the study metals (nickel, copper and
chromium) were lower than the Threshold Effects Level (TEL) of
143
NOAA (1999) (Table 1). Also hydrocarbons were practically absent
(Table 1). On the other hand, results of Beyrem and Aïssa (2000)
showed that the nematode fauna at this site is diverse (species
number = 18–35).
On sampling day, water depth was 1.2 m and salinity was 36.5. The
sediment had a mean grain size of 0.37 ± 0.12 mm, organic matter
content of 0.83 ± 0.2% and was totally composed of coarse size
fractions (99.65 ± 0.24%). Samples of the top 10 cm of sediment were
collected using 10 cm2 hand-cores (Coull and Chandler, 1992). On
return to the laboratory, sediments were homogenized by gentle hand
stirring with a large spatula before they were used for toxicant
sediment spiking or microcosms filling.
2.2. Metal and Diesel Fuel spiking of sediments
Sediment used for metal and Diesel Fuel contamination was first
alternately frozen and thawed three times to defaunate it (Austen et
al., 1994; Gyedu-Ababio and Baird, 2006). Secondly, it was wet sieved
to remove the larger particles (N63 μm).
25 Treatments were used, comprising a control, 3 levels of single
toxicant (Diesel Fuel, nickel, copper or chromium) and 3 levels of 4 chosen
combinations (Table 1). Four replicates of each microcosm were randomly
assigned to each treatment. To produce the Diesel Fuel-spiked sediments,
appropriate doses of Diesel Fuel were added to aliquots of 100 g
(dry weight, dw) in order to obtain final concentrations of 1 mg kg−1
dw, 10 mg kg− 1 dw and 20 mg kg− 1 dw after being mixed with 200 g of
fresh, meiofauna-rich sediment (Table 1). Solutions of nickel chloride
(NiCl2), copper chloride (CuCl2) and chromium chloride (CrCl3) were
made in distilled water. Chloride form of metals was used because it is the
most ubiquitous form in seawater. Then, aliquots of 100 g dw of sediment
were contaminated with appropriate doses of nickel or copper or
chromium in order to obtain final concentrations reported in Table 1
after being mixed with 200 g of fresh sediment. Control samples were the
same as treatment samples except that 100 g of defaunated, uncontaminated sediment replaced the contaminant spiked sediment. Each
microcosm was then gently filled with filtered (1 μm) natural seawater
(Austen and McEvoy, 1997; Mahmoudi et al., 2005) at 36.5 salinity.
Fig. 1. Area of investigation and location of the sampling site.
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F. Boufahja et al. / Journal of Experimental Marine Biology and Ecology 399 (2011) 142–150
Table 1
Targeted and actual concentration of metals (ppm dw ± SD) and diesel (mg kg− 1 dw ± SD) in microcosm sediments at the end of experiment and abundance of nematodes
(individuals ± SD).
Treatment
Targeted
Actual
C
Di(L)
Di(M)
Di(H)
C
Ni(L)
Ni(M)
Ni(H)
C
Cu(L)
Cu(M)
Cu(H)
C
Cr(L)
Cr(M)
Cr(H)
Ni(L) + Di(L)
Ni(M) + Di(L)
Ni(H) + Di(L)
Cu(L) + Di(L)
Cu(M) + Di(L)
Cu(H) + Di(L)
Cr(L) + Di(L)
Cr(M) + Di(L)
Cr(H) + Di(L)
Mix(L)
Mix(M)
Mix(H)
0
1
10
20
0
250
550
900
0
700
1414
2180
0
500
800
1300
250 ppm Ni + 1 mg kg− 1 Di
550 ppm Ni + 1 mg kg− 1 Di
900 ppm Ni + 1 mg kg− 1 Di
700 ppm Cu + 1 mg kg− 1 Di
1414 ppm Cu + 1 mg kg− 1 Di
2180 ppm Cu + 1 mg kg− 1 Di
500 ppm Cr + 1 mg kg− 1 Di
800 ppm Cr + 1 mg kg− 1 Di
1300 ppm Cr + 1 mg kg− 1 Di
250 ppm Ni + 700 ppm Cu + 500 ppm Cr + 1 mg kg− 1 Di
550 ppm Ni + 1414 ppm Cu + 800 ppm Cr + 10 mg kg− 1 Di
900 ppm Ni + 2180 ppm Cu + 1300 ppm Cr + 20 mg kg− 1 Di
0.02 ± 0.00
0.72 ± 0.10
6.24 ± 1.05
13.38 ± 4.00
10.2 ± 3.3
117.8 ± 20.5
396.4 ± 50.9
781.7 ± 70.6
12.1 ± 3.3
594.8 ± 63.2
1307.9 ± 102.6
1922.4 ± 117.0
11.4 ± 1.7
383.7 ± 30.5
662.3 ± 16.9
1188.5 ± 48.2
TEL
15.9
18.7
52.3
Abundance
239 ± 52.5
156.25 ± 21.48
89 ± 15.33
64.50 ± 12.07
239 ± 52.5
123.50 ± 30.45
106.75 ± 24.11
50 ± 9.25
239 ± 52.5
84 ± 8.41
47.75 ± 10.78
26.50 ± 6.40
239 ± 52.5
142.75 ± 18.57
76.25 ± 9.03
52.25 ± 10.68
C = uncontaminated control, Di = Diesel Fuel, Ni = nickel, Cu = copper, Cr = chromium, Mix = mixture of Diesel Fuel, nickel, copper and chromium, L = low contamination,
M = medium contamination, H = high contamination, TEL = threshold effects level of NOAA (1999).
Toxicants were mixed into the sediment with a food blender and the
amended sediment was left to equilibrate for 1 week at 5 °C before
microcosms were assembled.
Morphometry in animals is generally disturbed by pollution before
the animal's death. Therefore, it was essential before RVPL calculations were commenced to realize that targeted doses already cause
perturbations at a numerical level. The accumulative negative effects
of the used Diesel Fuel doses on the abundance of nematodes from
Ghar El Melh lagoon (Tunisia) were previously established by
Mahmoudi et al. (2005) and are here confirmed in the case study of
nematodes from Bizerte Bay, Tunisia (Table 1). The metal doses were
chosen based on the following three assumptions: (1) in anticipation
of the current work, numerous lethal toxicity experiments were
performed using a range of concentrations of metals. Choice of the
used targeted metal doses was based on ratios calculated by dividing
the abundance of nematodes for each couple of treatments. The
retained ratio had to be higher than 1.5 to separate the controls from
the low doses, and higher than 1 to differentiate between two
consecutive doses (Table 1). (2) Approximate concentrations of
copper tested for meiofauna by Austen and McEvoy (1997) were used
in this study. (3) Maximal metal concentrations reported in published
studies were taken into account to decide on the high doses to be
used herein (Moore and Ramamoorthy, 1984 (1337 ppm Cr dw);
Somerfield et al., 1994 (2532 ppm Cu dw); Nicolaidou and Nott, 1998
(889.4 ppm Ni dw)).
All experiments were ended after 30 days (Carman et al., 1995;
Millward et al., 2004) and an aliquot of sediment from each
microcosm was analyzed to determine toxicant concentrations. Hot,
concentrated HNO3 was used to extract metals. A 15 g of dry sediment
(80 °C) was digested by refluxing with 90 ml trace-metal grade HNO3
at 95 °C for 1 h. Following digestion, samples were diluted with
300 ml distilled water. Samples were then shaken, allowed to settle
for 24 h and the supernatant analyzed on a Varian Spectra AA20
atomic-absorption spectrometer with air/acetylene flame and auto-
sampler (Yoshida et al., 2002). Other sediment samples were
preserved in a freezer at −17 °C until total hydrocarbon concentrations were determined by infra-red spectrophotometry (Danovaro
et al., 1995).
2.3. Experimental design
The microcosms in this experiment were based on the original
design of Austen et al. (1994) and Schratzberger and Warwick (1998)
and successfully used by Mahmoudi et al. (2005, 2007), Beyrem et al.
(2007, 2010), Hedfi et al. (2007) and Hermi et al. (2009). Microcosms
(570 ml glass bottle) were flooded with 2–3 cm of filtered water
(40 μm mesh sieve) from the native site in Sidi Salem beach (Fig. 1).
Each microcosm bottle was stoppered with a rubber bung with two
holes, aerated gently using an aquarium air-stone diffuser and finally
run as closed system. Stability of salinity, temperature, dissolved
oxygen and pH was measured daily with a thermo-salinity meter (WTW
LF 196, Weilheim, Germany), an oxymeter (WTW OXI 330/SET,
Weilheim, Germany) and a pH meter (WTW pH 330/SET-1, Weilheim,
Germany), respectively.
At the end of the experiment, sediments were fixed in 4%
neutralized formalin (Mahmoudi et al., 2005).
2.4. Sample processing
Sediments were washed through nested 1 mm mesh sieves to
separate macrofauna from meiofauna (Vitiello and Dinet, 1979). After
washing to remove formalin, meiofauna was separated from material
retained on the 40 μm sieve using a combination of repeated
decantation and then flotation (Wieser, 1960; Vitiello and Dinet,
1979). Samples were preserved in 4% formalin and stained with RoseBengal (0.2 g l−1). All nematodes were picked under a stereo
dissecting microscope and placed in 21% glycerol, evaporated to
anhydrous glycerol, and then mounted on slides for microscopic
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Mesacanthion hirsutum Gerlach, 1953. These species belong to 4
feeding categories as described by Moens and Vincx (1997): depositfeeders (DF), epigrowth-feeders (EF), facultative-predators (FP) and
predators (P).
identification to species level using pictorial keys of Platt and Warwick
(1983, 1988) and Warwick et al. (1998) and descriptions downloaded
from the web site http://nemys.ugent.be/ developed by nematologists
of Ghent University (Belgium). A total of 42 species were inventoried
in all the microcosms. Most species were present in insufficient
numbers (b30 individuals) at least in one of the 3 treatment levels
(low, medium and high) reducing then the statistical rigor of the
analysis. Consequently, the evaluation of RVPL was restricted to 14
treatments and 8 tolerant species based on their lowest number of
specimens collected (at least 30 individuals from the four replicates of
each microcosm): the deposit-feeders, Daptonema normandicum
Lorenzen, 1977, Desmodora longiseta Schuurmans Stekhoven, 1950,
and Leptonemella aphanothecae Luc and De Coninck, 1959; the
epistrate feeder, Xyala striata Cobb, 1920; the facultative predators,
Oncholaimus campylocercoides De Coninck and Schuurmans Stekhoven, 1933 and Oncholaimellus mediterraneus Schuurmans Stekhoven,
1942; and the predators Bathylaimus capacosus Hopper, 1962 and
BV
2.5. Relative volume of the pharyngeal lumen (RVPL)
The relative volume of the pharyngeal lumen (RVPL) expressed in
percentage is calculated by dividing the volume of the pharyngeal
lumen (VPL) by the body volume (BV) (Boufahja et al., 2006). The
body volume (BV) was evaluated in nanoliters (nl) by using the
formula of Warwick and Price (1979): BV = 530 L W2, where L and W
express respectively the total length (in mm) and the maximum
width (in mm). In this work, we considered that the pharyngeal
lumen may have two possible shapes: (1) a straight cylinder
(B. capacosus, D. normandicum, O. campylocercoides, O. mediterraneus,
M. hirsutum and X. striata) or (2) a straight cylinder associated with a
Bathylaimus capacosus
Bathylaimus capacosus
VPL
2,2
BV (nl)
VPL (10-3 nl)
BV (nl)
VPL (10-3 nl)
2,2
1,1
ns ns
ns ns
*** ns
Ni(L)
n = 42
Ni(M)
n = 45
Ni(H)
n = 56
1,1
ns ns
C
n = 33
C
n = 33
Bathylaimus capacosus
BV (nl)
VPL (10-3 nl)
1,1
** ns
Cr(L)
n = 38
*
*** ns
*** ns
*** ns
C
n = 33
Cr(L)+Di(L)
n = 31
Cr(M)+Di(L)
n = 30
ns ns
Cr(H)+Di(L)
n = 32
C
n = 31
BV (nl)
VPL (10-3 nl)
ns
*** **
*** **
Cr(L)
n = 33
Cr(M)
n = 30
**
C
n = 37
Cr(H)
n = 39
4
BV (nl)
VPL (10-3 nl)
*
Cr(M)
n = 32
***
**
Cr(H)
n = 37
4,5
0
0
C
n = 37
**
9
0,6
*
Cr(L)
n = 30
Desmodora longiseta
Daptonema normandicum
1,2
Cr(H)
n = 59
1
0
0
Cr(M)
n = 31
Mesacanthion hirsutum
2
2,2
BV (nl)
VPL (10-3 nl)
**
0
0
BV (nl)
VPL (10-3 nl)
145
*
Cu(L)+Di(L)
n = 42
*** **
*** ***
Cu(M)+Di(L)
n = 41
Cu(H)+Di(L)
n = 44
Xyala striata
2
ns ns
ns ns
** ns
Cr(L)+Di(L)
Cr(M)+Di(L)
Cr(H)+Di(L)
0
C
n = 32
n = 36
n = 33
n = 37
Fig. 2. Graphical summary of means and 95% pooled confidence intervals of body volume (BV) and volume of the pharyngeal lumen (VPL) for 8 nematode species. ns = represents no
statistically discernible difference (p ≥ 0.05) vs. control, * = represents statistically discernible difference (p b 0.05) vs. control, ** = represents statistically discernible difference
(p b 0.01) vs. control, *** = represents statistically discernible difference (p b 0.001) vs. control, n = number of individuals collected.
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F. Boufahja et al. / Journal of Experimental Marine Biology and Ecology 399 (2011) 142–150
Oncholaimus campylocercoides
6,5
ns ns
*** **
Ni(L)
n = 59
Ni(M)
n = 50
0
C
n = 37
Oncholaimus campylocercoides
13
BV (nl)
VPL (10-3 nl)
BV (nl)
VPL (10-3 nl)
13
***
6,5
*
ns
0
C
n = 37
Ni(H)
n = 53
Oncholaimus campylocercoides
13
ns
Ni(L)+Di(L)
n = 34
**
ns
Ni(M)+Di(L)
n = 38
***
ns
Ni(H)+Di(L)
n = 40
Leptonemella aphanothecae
BV (nl)
VPL (10-3 nl)
BV (nl)
VPL (10-3 nl)
4
6,5
** ns
*** ns
*** ns
0
0
C
n = 37
Mix(L)
n = 36
Mix(M)
n = 34
C
n = 44
Mix(H)
n = 34
Leptonemella aphanothecae
2
0
C
n = 44
*** ns
*** ns
*** ns
Cu(L)+Di(L)
n = 64
Cu(M)+Di(L)
n = 37
Cu(H)+Di(L)
n = 52
ns ns
*** ns
Di(L)
n = 31
Di(M)
n = 46
Di(H)
n = 63
2
** ns
Cr(L)+Di(L)
n = 35
0
C
n = 44
*** ns
Cr(M)+Di(L)
n = 30
*** ns
Cr(H)+Di(L)
n = 31
Oncholaimellus mediterraneus
4
BV (nl)
VPL (10-3 nl)
ns ns
Leptonemella aphanothecae
4
BV (nl)
VPL (10-3 nl)
4
BV (nl)
VPL (10-3 nl)
2
2
**
ns
***
***
***
***
0
C
n = 33
Cu (L)
n = 31
Cu (M)
n = 31
Cu (H)
n = 30
Fig. 2 (continued).
posterior ellipsoid (D. longiseta and L. aphanothecae). This ellipsoid is
the equivalent of the pharyngeal bulb lumen. The Volume of the
Cylinder (VC) is calculated in 10−6 nl as follows VC = π LC (WC/2)2 (LC
and WC express in μm the length and the width of the Cylinder,
respectively). That of the Ellipsoid (VE) is calculated in 10− 6 nl by
using the following formula: VE = 4/3 π (WE/2)2 (LE/2) (LE and WE
express in μm the major and the minor axis of the ellipsoid,
respectively).
2.6. Statistical analyses
All data were tested for normality (Kolmogorov–Smirnov test) and
equality of variance (Bartlett test). Data were loge-transformed to
fulfill requirements of parametric analyses. After the data were logtransformed, they were again tested for normality and homogeneity
of variance. Subsequently, means were analyzed using one-way
analyses of variance (ANOVA) across all levels of treatments. A
posteriori multiple-comparisons were performed using Tukey HSD
test. The Z-test was used to test for overall differences between only
two means (n ≥ 30). In all these analyses, statistically discernible
differences were assumed when p b 0.05.
3. Results
3.1. Sediment chemistry
Final sediment concentrations for each single toxicant (nickel,
copper, chromium and Diesel Fuel) at the end of the experiment are
given in Table 1. The toxicant concentrations in all doses were lower
than the targeted values. For the toxicant combinations used, studies
of Mahmoudi et al. (2007) and Beyrem et al. (2007) using free-living
nematodes have already demonstrated that there is no antagonism
among toxicants.
3.2. Relationships between toxicants and nematode variables
The graphical summary of means of body volume and volume of the
pharyngeal lumen of the 8 study nematode species (14 treatments) is
given in Fig. 2. The results of significance testing using the one-way
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147
Table 2
Mean values (% ± SD) of the relative volume of the pharyngeal lumen (RVPL) of nematode species from control and contaminated microcosms and comparisons using Tukey HSD
test. Bold values indicate statistically discernible differences vs. control (p b 0.05).
One-way ANOVA
Treatment
Bathylaimus capacosus (Ni): df = 172; F(3,172) = 6.940; p b 0.001
Bathylaimus capacosus (Cr): df = 157; F(3,157) = 8.080; p b 0.001
Bathylaimus capacosus (Cr + Di(L)): df = 122; F(3,122) = 12.273; p b 0.001
Mesacanthion hirsutum (Cr): df = 126; F(3,126) = 10.647; p b 0.001
Daptonema normandicum (Cr): df = 135; F(3,135) = 48.354; p b 0.001
Desmodora longiseta (Cu + Di(L)): df = 160; F(3,160) = 9.456; p b 0.001
Xyala striata (Cr + Di(L): df = 134; F(3,134) = 11.589; p b 0.001
Oncholaimus campilocercoides (Ni): df = 195; F(3,195) = 6.318; p = 0.001
Oncholaimus campilocercoides (Ni + Di(L)): df = 145; F(3,145) = 6.948; p b 0.001
Oncholaimus campilocercoides (Mix): df = 137; F(3,137) = 29.152; p b 0.001
Leptonemella aphanothecae (Di): df = 180; F(3,180) = 46.056; p b 0.001
Leptonemella aphanothecae (Cu + Di(L)): df = 193; F(3,193) = 71.250; p b 0.001
Leptonemella aphanothecae (Cr + Di(L)): df = 136; F(3,136) = 66.711; p b 0.001
Oncholaimellus mediterraneus (Cu): df = 121; F(3,121) = 29.349; p b 0.001
Control
Low
Medium
Mean
Mean
0.1293 ± 0.0189
0.1293 ± 0.0189
0.1293 ± 0.0189
0.2615 ± 0.0254
0.1121 ± 0.0131
0.0191 ± 0.0011
0.0933 ± 0.0259
0.0292 ± 0.0025
0.0292 ± 0.0025
0.0292 ± 0.0025
0.0197 ± 0.0012
0.0197 ± 0.0012
0.0197 ± 0.0012
0.0592 ± 0.0092
01307 ± 0.0279 0.999 01376 ± 0.0127
0.1277 ± 0.0175 0.999 0.1381 ± 0.0170
0.1483 ± 0.0087 0.003 0.1521 ± 0.0098
0.2807 ± 0.0336 0.542 0.3150 ± 0.0260
0.1309 ± 0.0132 0.008 0.1371 ± 0.0119
0.0211 ± 0.0017 0.002 0.0216 ± 0.0010
0.0917 ± 0.0095 0.999 0.1073 ± 0.0068
0.0308 ± 0.0051 0.787 0.0303 ± 0.0022
0.0321 ± 0.0064 0.728 0.0320 ± 0.0068
0.0333 ± 0.0034 0.039 0.0407 ± 0.0046
0.0205 ± 0.0019 0.171 0.0215 ± 0.0018
0.0236 ± 0.0019 b 0.001 0.0259 ± 0.0015
0.0224 ± 0.0022 b 0.001 0.0249 ± 0.0020
0.0762 ± 0.0059 0.002 0.0846 ± 0.0180
p
Mean
High
p
Mean
p
0.673
0.307
b0.001
0.004
b0.001
b0.001
0.081
0.875
0.775
b0.001
b0.001
b0.001
b0.001
b0.001
0.1625 ± 0.0083
0.1533 ± 0.0149
0.1607 ± 0.0137
0.3423 ± 0.0525
0.1612 ± 0.0195
0.0213 ± 0.0016
0.1292 ± 0.0160
0.0350 ± 0.0026
0.0404 ± 0.0056
0.0427 ± 0.0053
0.0244 ± 0.0016
0.0260 ± 0.0014
0.0257 ± 0.0016
0.1112 ± 0.0150
0.001
0.001
b0.001
b0.001
b0.001
0.001
b0.001
b0.001
b0.001
b0.001
b0.001
b0.001
b0.001
b0.001
Note: see Table 1 for codes.
ANOVA illustrate significant overall differences in the case of body
volume for all treatments and species (Fig. 2). This was only recorded for
6 treatments (O. campylocercoides (nickel), O. mediterraneus (copper), B.
capacosus (chromium), M. hirsutum (chromium), D. normandicum
(chromium), D. longiseta (copper+Diesel Fuel)) in the case of the
volume of the pharyngeal lumen (Fig. 2). This variable, on the other
hand, did not show any statistically discernible variation over the range
of 8 treatments (O. campylocercoides (nickel+ Diesel Fuel and mixture),
L. aphanothecae (Diesel Fuel, copper + Diesel Fuel and chromium
+ Diesel Fuel), B. capacosus (nickel and chromium+ Diesel Fuel) and
X. striata (chromium +Diesel Fuel)).
Results from the multiple comparisons using Tukey HSD test
(Fig. 2 and Table 2) showed at least one significant difference between
nematodes from undisturbed controls and those from treatments. A
statistically discernible increase (Table 2) in mean RVPL was
commonly observed with increasing toxicant concentrations. This
relationship did not stand for body volume and volume of the
pharyngeal lumen; both showed a decline in their mean values with
increasing toxicant concentrations. There was a remarkable decrease
in body volume compared to that of the pharyngeal lumen.
3.3. Responses species and feeding dependencies
The microcosm treatments had statistically discernible and
ecologically significant influence on RVPL for all nematode species
studied (Tukey HSD test, Table 2). Nevertheless, the species responses
fell into two broad groups (Z-test, Table 3): (a) species with high
potential for adaptation, characterized by elevated RVPL (M. hirsutum,
B. capacosus, D. normandicum, X. striata and L. aphanothecae) and (b)
species with less potential for adaptation (O. campylocercoides and
D. longiseta). These results presume that the highest RVPL characterized
predators (M. hirsutum and B. capacosus) and less visibly deposit(D. normandicum and L. aphanothecae) and epigrowth-feeders
(X. striata). Finally, statistically discernible differences (Z-test, Table 3)
were recorded between RVPL mean values of two predators (Chromium: B. capacosusb M. hirsutum) and between two deposit-feeders
copper +Diesel Fuel: D. longiseta b L. aphanothecae).
3.4. Response toxicant-dependent
The analysis of RVPL mean values of 3 nematode species (B. capacosus,
L. aphanothecae and O. campylocercoides) illustrates a clear treatment
effect according to the nature and doses of the toxicant used and shows
generally that the impact of combinations is statistically discernible (Ztest, Table 4) higher than that of single toxicants.
The mean RVPL of B. capacosus in the nickel-treatment was
statistically similar to those observed in the chromium-treatment (Ztest, Table 4). However, L. aphanothecae responded more clearly to the
copper + Diesel Fuel treatment than to chromium + Disel Fuel
treatment.
4. Discussion
There are four cell types in the pharynx: muscle cells, neurons,
structural and glandular cells (Albertson and Thomson, 1976). The
pharyngeal muscle and the hypodermis delimit the body cavity called
Table 3
Comparisons of mean values of the relative volume of the pharyngeal lumen (RVPL) of nematode species by using Z-test (values represent probabilities (p)). Bold values indicate
statistically discernible differences (p b 0.05). Grey zones mark the highest mean values.
Toxicant
Ni
Cr
Cu + Di(L)
Cr + Di(L)
Treatment
Species response comparison
Control
Low
Medium
High
b0.001
0.052
b0.001
b0.001
0.232
b0.001
b0.001
b0.001
b 0.001
0.553
b0.001
b0.001
b0.001
b0.001
b0.001
b0.001
b0.001
0.767
b0.001
b0.001
b0.001
b0.001
b0.001
b0.001
b0.001
0.060
b0.001
b0.001
b0.001
b0.001
b0.001
b0.001
Oncholaimus campilocercoides (FP)
Daptonema normandicum (DF)
Daptonema normandicum (DF)
Bathylaimus Capacosus (P)
Desmodora longiseta (DF)
Leptonemella aphanothecae (DF)
Xyala Striata (EF)
Leptonemella aphanothecae (DF)
Note: see Table 1 for codes. DF = deposit-feeders, EF = epigrowth-feeders, FP = facultative-predators, P = predators.
Bathylaimus capacosus (P)
Bathylaimus capacosus (P)
Mesacanthion hirsutum (P)
Mesacanthion hirsutum (P)
Leptonemella aphanothecae (DF)
Bathylaimus capacosus (P)
Bathylaimus capacosus (P)
Xyala striata (EF)
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F. Boufahja et al. / Journal of Experimental Marine Biology and Ecology 399 (2011) 142–150
Table 4
Comparisons of mean values of relative volume of the pharyngeal lumen (RVPL) of 3 nematode species exposed to different toxicants by using Z-test (values represent probabilities
(p)). Bold values indicate statistically discernible differences (p b 0.05). Grey zones mark the highest mean values.
Species
Bathylaimus capacosus (P)
Leptonemella aphanothecae (DF)
Oncholaimus campylocercoides (FP)
Treatment
Toxicant effect comparison
Low
Medium
High
0.932
0.036
b 0.001
b 0.001
b 0.001
0.021
0.675
0.159
0.492
0.853
0.002
b0.001
b0.001
b0.001
0.034
0.606
b0.001
0.003
0.095
0.679
0.255
b 0.001
0.002
0.554
0.011
b 0.001
0.334
Ni
Ni
Cr
Di
Di
Cr + Di(L)
Ni
Ni
Ni + Di(L)
Cr
Cr + Di(L)
Cr + Di(L)
Cr + Di(L)
Cu + Di(L)
Cu + Di(L)
Ni + Di(L)
Mix
Mix
Note: see Table 1 for codes. DF = deposit-feeders, FP = facultative-predators, P = predators.
the pseudocoelom (Albertson and Thomson, 1976) (Fig. 3). The
hypodermis secretes the body cuticle. Three pharyngeal muscle cells
situated with triradiate symmetry surround the pharyngeal lumen
(Bird and Bird, 1991). Their contractile fibers are radial so that when
they contract, the pharyngeal lumen opens (Raizen and Avery, 1994).
Their apical surface is lined by a cuticle, whereas their basal surface is
lined by a lamina. Between the pharyngeal muscle cells at the apices
of the lumen are three marginal cells. Neurons are embedded in
grooves of pharyngeal muscle in each of the three sectors (Raizen and
Avery, 1994).
4.1. Why do stressed nematodes have higher RVPL?
The pharynx, a tubular organ between the mouth and the intestine, is
suspended in the pseudocoelom. A fluid fills the pseudocoelom and is
under pressure thereby functioning as a hydrostatic skeleton. Nematodes are characterized by eutely (Flemming et al., 2000). Thus, the
reduction in nematode body size is due to reduction cell volume not cell
number. Catabolic water production increases with environmental
perturbation. As a result, the nematodes' internal environment becomes
more hypotonic compared to interstitial seawater. Thus, an out-flux of
water from the body will follow an increase of internal hydrostatic
pressure (Raizen and Avery, 1994; Forster, 1998). The current study
confirmed that stressed nematodes possess high RVPL. It follows that
under stress conditions, the volume decrease is generally less visible for
the pharyngeal lumen than for the entire body. This may depend upon
many factors:
– Pharyngeal contraction and relaxation are caused by depolarization and repolarization of the muscle cell membranes (Byerly and
Masuda, 1979). Avery and Horvitzt (1989) demonstrate that pumping
continued even when the entire pharyngeal nervous system was
killed. It follows that muscle pumping may be qualified as a
spontaneous process. Nevertheless, we suppose that under stress
the nervous system makes pharyngeal pumping more powerful in
order to keep sufficient the magnitude of their pharyngeal contractions, presumably by modulation of depolarization and repolarization
frequency in the muscle cells (Avery and Horvitzt, 1989. On the
whole, it appears that pharyngeal activity is “heart like”.
– Only the basal surface of pharyngeal muscle faces an osmoregulated environment, that of the pseudocoelom (Fig. 3). The apical
membrane faces the lumenal solution which is principally influenced
by food characteristics. Experimental support for the idea of a passive
lumenal membrane in nematodes comes from the work of Byerly and
Masuda (1979), who showed that, in the parasitic nematode Ascaris,
action potentials recorded from the pharyngeal lumen are of the same
polarity as those recorded from apical membrane of the pharyngeal
muscle cells. Thus, it is expected that pseudocoelom lost more water
compared to pharyngeal muscle.
– Chitin is a linear homopolysaccharide serving as a mechanically
strong scaffold material. The anti-parallel arrangement of chitin
molecules contributes significantly to the physicochemical properties
of the cuticle (Merzendorfer and Zimoch, 2003). The body cuticle is
taken internally by the pharyngeal epithelium and used to cover the
lumen of the pharynx (Bird and Bird, 1991). These authors
demonstrate that chitin is absent in the body covering of adult
nematodes but it is detected in their pharyngeal cuticle. The
continuous presence of this substance in the pharyngeal cuticle
during all life stages could be one of the reasons for its lower waterpermeability compared to that of the body cuticle.
– The pharynx has epithelial muscle cells in which myofibrils are
arranged radially (Fig. 3). The myoepithelium encloses the pharyngeal
lumen with monosarcomeric myoepithelial cells on the plains of the
lumen and myofilament-free cells on the apical edges (Bird and Bird,
1991). The basal pharyngeal compartment seems more compact than
the pseudocoelomic compartment. Depending on cell nature and
cytoplasm compactness, the water loss becomes more difficult with
presence of condensed macromolecules.
4.2. Why RVPL is feeding-dependent?
Fig. 3. Schematic of a transverse view of the nematode body (Raizen and Avery, 1994).
The chitinolytic enzymes are produced by many invertebrates,
including nematodes (Coomans et al., 1996; Chatelet et al., 2001). The
marine environment has the greatest resources of chitin (Donderski
and Trzebiatowska, 1999; Majtán et al., 2007) which is produced by
many small-sized sediment-inhabiting organisms. Its main suppliers
are crustaceans, nematodes, flagellates, protozoans, foraminiferans
and diatoms (Donderski and Trzebiatowska, 1999; Majtán et al.,
2007). It is possible that predators (M. hirsutum and B. Capacosus)
consuming prey with chitin in their cuticles present the lowest
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F. Boufahja et al. / Journal of Experimental Marine Biology and Ecology 399 (2011) 142–150
sensitivity to changes in environmental conditions. This may help
explain why these species possess, large mean RVPLs that are
statistically discernible (Z-test, Table 3). The high quantity of chitin
in foods appears to reinforce the rigidity of the pharyngeal cuticle
making easier to resist osmotic stress. The presence of chitin in
diatoms may eventually explain the high RVPL of deposit-feeders (D.
normandicum and L. aphanothecae) and epigrowth-feeders (X. striata).
In addition, deposit-feeders may consume detritus of fresh dead
crustaceans such as copepods, lobsters, shrimps and prawns which
may represent a source of chitin.
4.3. Why RVPL is species-dependent?
No published studies have determined the osmoregulatory potential
of the body cuticle of meiobenthic nematodes or its ability to regulate
body volume. Only Forster (1998) has demonstrated that nematode
species are able, to differing extents, to regulate their water content.
Three factors may distinguish the RVPL differences between species
belonging to the same feeding group and exposed to the same treatment
(Z-test, Table 3): (1) species possessing thick and/or strongly annulated
body cuticles seem to experience a low out-flux water under stress
conditions (Forster, 1998). It is known that the body cuticle of
B. capacosus (a predator) is composed of at least two main layers, a
thin cortical layer and a thicker matrix layer. However, M. hirsutum (also
a predator) has a cuticle with fine transverse striation. Probability values
of multiple comparisons (Tukey HSD test) between means of body
volume of nematodes from control (C) and those exposed to the 3 levels
of chromium (Cr) verify this hypothesis (M. hirsutum: C-Cr(Low) =
0.397, C-Cr(Medium) = 0.003, C-Cr(High) = 0.000; B. capacosus: C-Cr
(Low) = 0.994, C-Cr(Medium)= 0.009, C-Cr(High)= 0.000) and make
clear why M. hirsutum has a greater RVPL than B. capacosus (Z-test,
Table 3). (2) A second feature is the number and length of locomotory
and sensory setae which may contribute efficiently to the osmoregulation by covering of the body. (3) There are also differences among
species in quantity and structure of the monosarcomeres and myofilaments within pharyngeal muscle cells. Differences in RVPL between two
species of deposit-feeders exposed to copper+Diesel Fuel (D. longisetab L.
aphanothecae) seem to demonstrate the effects of these anatomical
differences The decrease in volume of the pharyngeal lumen was only
statistically discernible (Tukey HSD test, Fig. 2) in the case of D. longiseta.
4.4. Is RVPL toxicant-dependent?
Metal–Diesel Fuel combinations were employed to determine if
responses to mixed contaminants were distinct from responses to either
metals or Diesel Fuel alone. Combined responses may be classified as
additive, or synergistic, or antagonistic (Millward et al., 2004). Additive
toxicological effects would be suggested by a statistically discernible (Ztest, Table 4) increase in RVPL mean values in the metals+ Diesel Fuel
treatments compared to metals-only and Diesel Fuel-only treatments.
The addition of metals to Diesel Fuel-contaminated sediment did not
alter the influence of Diesel Fuel alone given their different toxicological
modes of action (Millward et al., 2004). The presence of Diesel Fuel
enhanced the retention of metals in sediments, which may have
important toxicological implications (Millward et al., 2004). These
responses are consistent with previous observations (Carman et al.,
1997; Millward et al., 2001, 2004; Mahmoudi et al., 2007; Beyrem et al.,
2007). Nickel and chromium seem to have comparable toxicities in the
case of B. capacosus. However, L. aphanothecae appears more sensitive to
copper than chromium. A few studies have examined toxicity of metals
on meiobenthic species, but results are difficult to generalize and effects
tend to be taxon-specific (Coull and Chandler, 1992; Mahmoudi et al.,
2007; Beyrem et al., 2007). It is known that metals cause metabolic
dysfunction by, among other things, binding to cytosolic metaloenzymes (Hagopian-Schlekat et al., 2001). If this is the case, hydo- and
lipo-solubilities of these toxicants should control their toxicities.
149
5. Conclusion
Until recently, there has been no attention to morphometric indices in
biomonitoring, whereas much more attention has been paid to indices
based on abundance including some diversity indices. Life cycle changes
in morphometrics limit the usefulness indices based on abundance alone.
In contrast, morphometric indices suggest hypotheses relevant to
evolutionary and molecular domains (e.g., Pearson, 1948).
In the present study, we have experimental validated the “relative
volume of the pharyngeal lumen (RVPL)” of free-living nematodes as
a biomonitoring index using sediment-associated metals and/or
Diesel Fuel in microcosms initially proposed by Boufahja et al.
(2006). In that paper, we addressed the hypothesis that stressed
nematodes are characterized by higher values of the relative volume
of the pharyngeal lumen (RVPL) and so higher sucking potential. This
was qualified as an adaptation to the nutritional value of food in
polluted sediments. In environments polluted by metals and/or diesel
fuel nematode body size shrinks as loss of catabolic water becomes
more pronounced. However, for many reasons, the body volume
appears more affected than that of the pharyngeal lumen.
The experimental approach used here was a powerful tool to test
the suitability of morphometric biomonitoring indices. However, the
forging of closer links between laboratory and field-based research
remains the fundamentally goal of any suggested index.
Dimensions of the entire body or those of given organ, if
considered alone in assessing the effect of pollution, will be
complicated in the field because of differences in age, sex, reproductive phase, food quality and physiological state. Given these
circumstances, ratios normalized by body volume are useful.
Moreover, volumetric ratios may be particularly appropriate when
eutelic and pseudocoelomic characters of nematodes were taken into
account.
Results from the bioassay presented here showed that RVPL may be
used to demonstrate the effects of various metals and diesel fuel. If RVPL
is accepted as an efficient biomonitoring index, minimal sedimentary
concentrations of metals and diesel fuel that are associated with a
statistically discernible and ecologically significant responses for each of
the 8 species studied here may be defined for all 14 tested treatments: B.
capacosus (900 ppm Ni, 1300 ppm Cr, 1 mg kg− 1 Diesel Fuel +500 ppm
Cr), M. hirsutum (800 ppm Cr), D. normandicum (500 ppm Cr), D.
longiseta (1 mg kg− 1 Diesel Fuel + 700 ppm Cu), X. striata (1 mg kg− 1
Diesel Fuel + 1300 ppm Cr), O. campylocercoides (900 ppm Ni, 1 mg kg− 1
Diesel Fuel + 900 ppm Ni, 1 mg kg− 1 Diesel Fuel + 700 ppm Cu+
500 ppm Cr+ 250 ppm Ni), L. aphanothecae (10 mg kg− 1 Diesel Fuel,
1 mg kg− 1 Diesel Fuel + 700 ppm Cu, 1 mg kg− 1 Diesel Fuel +500 ppm
Cr) and O. mediterraneus (700 ppm Cu). Combinations were commonly
associated with the highest RVPL.
This work has tested and validated the RVPL for monitoring the
effects of a limited number of contaminants (nickel, copper,
chromium and Diesel Fuel). Further histological and molecular studies
are now required to test the hypotheses discussed herein and to
generalize the use of RVPL in biomonitoring.
Acknowledgments
The authors would like to thank Prof. Melanie C. Austen (Plymouth
Marine Laboratory, UK) and Prof. Rod N. Millward (Louisiana State
University, USA) for technical support and help with statistical
analyses and Prof. Guy Boucher (National Museum of Natural History,
France) and Prof. Pierre Vitiello (Oceanography Center of Marseille,
France) for species identification and critically reading the manuscript. Thanks go also to Prof. Brian M. Marcotte (Falmouth, USA) for
the revisions he made on the English of this paper. This work was
supported by the Tunisian Ministry of Scientific Research and
Technology (MSRT) [ST].
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