Aquatic Invasions (2014) Volume 9, Issue 1: 47–58
doi: http://dx.doi.org/10.3391/ai.2014.9.1.04
© 2014 The Author(s). Journal compilation © 2014 REABIC
Open Access
Research Article
First evidence of microfungal “extra oomph” in the invasive red swamp crayfish
Procambarus clarkii
Laura Garzoli 1 * , Daniele Paganelli 2 , Marinella Rodolfi 1 , Dario Savini 2 , Mattia Moretto 2 ,
Anna Occhipinti-Ambrogi 2 and Anna Maria Picco 1
1
2
Lab. of Mycology, DSTA-Department of Earth and Environmental Sciences, University of Pavia, Via S. Epifanio 14, 27100 Pavia, Italy
Lab. of Ecology, DSTA-Department of Earth and Environmental Sciences, University of Pavia, Via S. Epifanio 14, 27100 Pavia, Italy
E-mail: laura.garzoli01@gmail.com (LG), daniele.paganelli@unipv.it (DP), marinella.rodolfi@unipv.it (MR),
dario.savini@unipv.it (DS), mattia.moretto01@universitadipavia.it (MM), anna.occhipinti@unipv.it (AOA),
annamaria.picco@unipv.it (AP)
*Corresponding author
Received: 17 July 2013 / Accepted: 10 January 2014 / Published online: 3 February 2014
Handling editor: Chris Chucholl
Abstract
This paper represents the first attempt to study the microfungal flora contained in the digestive system of the most widespread aquatic
invasive invertebrate in the world: Procambarus clarkii (Cambaridae, Decapoda). Understanding its bioinvasion, in terms of ecological risk
and environmental impact, requires a multidisciplinary approach that considers consequences on all levels, from macroscopic to microscopic.
In this study, we investigated both the population dynamics and the dietary habits of the red swamp crayfish captured within a natural
biotope in Northern Italy. The diet was mainly based on plant detritus. The analysis of microfungal flora in the crayfish stomach revealed a
fairly constant composition, unrelated to season and diet. Since most of the fungi were associated with detritus and some species were
particularly frequent, we formulate the hypothesis that the fungi may be selected to decompose plant material in the digestive tract, thus
providing a source of energy to the crayfish. Procambarus clarkii is also shown to be a potential vector of plant diseases as some of the 45
isolated fungal taxa are potentially phytopathogenic.
Key words: population study, diet, microfungi, digestive system, wet woodland biotopes, Northern Italy
Introduction
The red swamp crayfish Procambarus clarkii
(Girard, 1852) is one of the top 27 alien species
of animals to have been introduced into Europe
for aquaculture (Savini et al. 2010), and is
considered to be one of the most dangerous
species in terms of ecological risk and environmental impact (Geiger et al. 2005; Gherardi and
Acquistapace 2007; Tricarico et al. 2010).
Although P. clarkii is a North American native
species (Huner 1988), due to its outstanding
performances in aquaculture productivity, it was
legally introduced into southern Spain in 1973,
and later illegally introduced into most other
European countries between 1970 and 1990 (SoutyGrosset et al. 2006). Many biological traits, such
as the capability to live out of water for long
periods (Gherardi and Barbaresi 2007), a high
reproductive potential (Aquiloni et al. 2010;
Barbaresi and Gherardi 2006; Savini 2007), and
tolerance to environmental pollution (Aquiloni et
al. 2011), are responsible for the rapid spread of
crayfish in the wild. High motility and burrowing
behaviour make this species one of the most
successful fugitives from aquaculture facilities
(Gherardi 2006).
Nowadays, P. clarkii is widespread in Western
Europe, Africa, Asia and North America; the
only continents where the crayfish has never been
recorded are Australia and Antarctica (Gherardi
2006). In Italy, Procambarus clarkii appeared in
the wild for the first time in 1989 in the river
Banna, a tributary of the Po river, after escaping
from an experimental farm (Delmastro 1992); its
distribution currently includes the majority of the
northern and central regions, as well as the
47
L. Garzoli et al.C.
islands (Gherardi et al. 1999a; Chiesa et al. 2006;
Savini and Occhipinti 2008; Scalici et al. 2009;
Aquiloni et al. 2011), and it has also been
recorded on several occasions in southern Italy
(Morpugo et al. 2010).
A comprehensive review of the impact of this
crustacean in wetlands was provided by Geiger
et al. (2005). Lodge et al. (2012) also presented a
significant review on the categories of ecosystem
services potentially affected by nonindigenous
crayfish, including P. clarkii. Overall, there are more
than 40 peer reviewed papers that have proved
and in some cases quantified the environmental
impact of P. clarkii (Savini et al. 2010).
Nevertheless, understanding the consequences
of the introduction of an alien species into a new
environment involves the functioning of the
ecosystem as a whole from macroscopic to
microscopic level, and therefore requires a
multidisciplinary approach that transcends the
boundaries of single area of expertise.
One of the key factors of the success of P. clarkii
is its capability “to make a virtue of necessity”:
crayfish are generally considered to be polytrophic
feeders that can successfully adapt their diet to
food availability (Gutiérrez-Yurrita et al. 1998;
Momot 1995; Correia 2003; Alcorlo et al. 2004).
One of the less considered aspects of P. clarkii
bioinvasion is its interaction with the microbial
community in freshwater ecosystems. As a
consequence of its feeding behaviour, P. clarkii
needs enzymes that can break down different
types of food: this role could be related to
microorganism activity. Fungi and bacteria
associated with leaves can produce cellulosases,
xilanases, pectinases and other enzymes that
aquatic invertebrates may use to digest complex
substrates (Graça 2001). In particular, microfungi
produce a mosaic of heterogeneous substrates on
leaf litter, which are variable in time and space,
and available for shredders (Rossi 1983).
Besides enhancing substrate palatability, fungi
themselves represent a food source and are
known to be a target for aquatic invertebrates.
This double role of microfungi in the feeding
strategies of aquatic invertebrates has been
clearly demonstrated by Graça et al. (1993) for
Asellus aquaticus (Linnaeus, 1758) and Gammarus
pulex Linnaeus, 1758. In the first case, the
crustacean selectively consumed fungal mycelia,
while in the second, leaf material was chosen
depending on fungal colonisation. Although it is
generally assumed that crayfish mainly feed on
conditioned leaves, few authors have clearly
demonstrated this preference in crayfish: only
48
Momot et al. (1978) demonstrated that young
Orconectes virilis did not eat plant material but
rather appeared to scrape it clean off periphytic
growths.
Moreover, symbioses between aquatic invertebrates and their stomach microflora play a
fundamental role in digestive processes. In 1993,
Harris provided a comprehensive synthesis of
available studies on the presence, nature and role
of gut microflora in aquatic invertebrates.
Nevertheless, published data on crayfish gut
microflora and its biochemical activity are scarce
or limited to a few species (Mickënienë and
Šyvokienë 1996).
In the present study, we investigated a small
population of P. clarkii in a natural area of the
Ticino Regional Park (North Italy), mainly
focusing on its diet and its digestive system
fungal microflora. The latter aspect has not been
considered until now, but may facilitate the
success of P. clarkii in a new environment.
Methods
Study area
The Ticino river is one of the main tributaries of
the Po river, flowing in the southern part of
Lombardy (NW Italy), along a valley measuring
110km long and 7km wide. Although this river
plain is characterised by intensive agriculture
and anthropogenic activities, numerous natural
reserves (partial and integral) occupy a large part
of the valley (about 91,000ha). Our study site,
called “I Geraci” (WGS84 45°16'34.46"N, 8°59'
0.14"E), measures 90ha and is situated within the
Ticino Regional Park, one of such natural
reserves (Figure 1).
The Geraci area is located on the hydrological
left side of the Ticino and, due to its proximity to
the river, is often overflooded. This area is a
typical natural biotope of the Southern Po Plain,
surrounded by agricultural fields and small
villages; it is composed of numerous wetlands,
ponds and small rivers, mainly concentrated in
the central part of a dense wood, which is quite
impenetrable and isolated.
The water temperature of the wetlands varied
from 0°C in winter to over 28°C in summer, a
typical range in the Southern Lombardy Plain.
Population abundance of Procambarus clarkii
To investigate the population of P. clarkii, a weekly
sampling program was carried out from June
First evidence of microfungal “extra oomph” in Procambarus clarkii
Figure 1. Geraci: a 90-hectare
natural area (boundaries in a dotted
line) located on the hydrological left
side of the Ticino river within the
Ticino Regional Park (NW Italy).
Circles indicate sampling stations
(T1 to T13). Digital orthophoto of
the studied area derived from the
Lombardy Region Orthophoto 2003
(scale of the frames: 1:40000,
average altitude of flight: 6000m).
2009 to May 2010 using 13 double entrance
crayfish “Trappy Tetra” made of polypropylene
plastic (length 520mm, diam. 210mm and mesh
size 20mm), always set in the same location
within the sampling area (Figure 1). Pig liver
was used as bait to attract crayfish (50g per trap
per week). These traps are highly selective for
crayfish, thus minimising by-catches of fish,
reptiles and small mammals.
For each captured specimen, sex, weight and
total body length (from the tip of the rostrum to
the tip of the telson) were recorded, as suggested
by Scalici and Gherardi (2007); the crayfish
were then frozen at -20°C.
Average monthly catches per unit effort
(AVCPUE) was used as a standardised index of
abundance (Richards and Schnute 1986; Hinton
and Maunder 2003; Savini 2007; Savini and
Occhipinti 2008; Savini et al. 2008).
Diet composition and determination of stomach
microfungal flora
For each sampling month, the food choices and
stomach mycoflora of five crayfish, chosen at
random, were investigated. The stomach content
was analysed following the procedure proposed
by Correia (2003). The percentage of occurrence
of each food category was estimated: detritus,
plants (green plants, seeds, remains of seeds, and
others) and animals.
Crayfish with scars, abrasion or empty stomachs
were excluded from the analysis. This selective
choice was due to the fact that crayfish with
impairments are less active and more stressed
than others (Figiel and Miller 1995; Kraus-Epley
and Moore 2002; Bergman et al. 2003).
Stomachs were excised under a laminar flow
hood to prevent external contamination, then a
smear of stomach content was plated into different
media using a sterile loop: the generic medium
PDA (Potato Dextrose Agar: 200g potato, 20g
dextrose, 15g agar, 1000ml distilled water); the
nutrient medium MEA (Malt Extract Agar: 30g
malt extract, 15g agar, 5g peptone, 1000ml
distilled water); and the specific medium for the
culture diagnosis of Aphanomyces astaci Schikora,
1906, causal agent of the crayfish plague, GOYA
(Glucose Oxolinic Yeast extract Agar: 12g agar,
1g yeast extract, 5g glucose, 10mg Oxolynic
acid, 1000ml distilled water), which is also
favourable for the growth of both fungal parasites
49
L. Garzoli et al.C.
and saprophytes, as reported by Alderman and
Polglase (1986).
Furthermore, the stomach volume was estimated:
a random sample (10 crayfish) of extracted crayfish
stomachs was closed with pincers, filled with water
up to the maximum capacity, and then a
computerised system of image analysis approximated each stomach to a sphere (approximate
volume=2ml).
In order to facilitate the isolation of slow
growing mitosporic fungi, a 1:10 dilution was
performed and plates were incubated at 25°C.
Examination of plates was continued for up to
four weeks to detect the presence of sporulating
fungi.
Initial detection of fungal structures was
carried out by means of stereomicroscope ( 1050) and strains were identified on the basis of
micro-morphological analysis by means of
specific taxonomical keys (Ellis 1971, 1976;
Samson 1974; Hermanides-Nijhof 1977; Pitt 1979;
Domsch et al. 1980; Sutton 1980; Nelson et al. 1983;
Klich 2002; Samson et al. 2004).
Although the aim of this study was to investigate
fungal flora, the amount of both Bacteria and
Actinomycetes (in CFU) for each plate is also
provided.
Lastly, microfungal strains were collected as
pure cultures onto the generic microfungi medium
PDA.
Statistical analyses
All the statistical analyses were performed using
PRIMER 5.0 (Plymouth Routines In Multivariate
Ecological Research, Clarke and Warwick 1994)
and MINITAB 15.0 (Minitab Inc. 2007) software
packages.
A G-test was used to evaluate the sex-ratio
within the population and the diet composition.
A one-way ANOVA was performed to test seasonal
differences in crayfish diet. We run a Tukey post
hoc to identify seasonal differences in crayfish
diet.
Furthermore, the number of specimens captured
in each month of the survey was compared using a
Kruskal-Wallis test, and a General Linear Model
(GLM) was applied in order to highlight
biometric differences.
Differences in microfungal community composition between seasons were tested with an
ANOSIM test based on the Bray-Curtis similarity
index; BIO-ENV analysis was carried out to
correlate stomach content with the recovered
microfungal flora (Clarke and Warwick 1994).
50
Results
Population abundance and structure
A total of 692 P. clarkii specimens were captured
over the one-year survey period: 490 males and
202 females (sex ratio of 2.42 males to 1 female).
The only time that this ratio was not respected
was in late winter (February, March), when the
number of captured females was slightly higher
than males. A G test confirms this statistical
difference between sexes (G=36.93; DF=11;
p=0.0001).
The highest mean CPUE value of P. clarkii
was registered in summer (AVCPUE in July
2009=2.67±2.09), while the lowest was registered in
winter (AVCPUE in February 2010=1.3±0.67)
(Figure 2).
Differences between CPUE per month were
tested by means of a K-Wallis test and results
showed a significant difference (H=22.06; DF=11;
p=0.048).
The overall mean length of specimens was
98.77±11.36mm, the mean length of male specimens
was 97.41±10.94mm, and the mean length of
female specimens was 100.86±11.98mm (Figure
3).
A General Linear Model (GLM) was used to
test biometric differences between the size of
males and females observed during the year. The
results confirmed that there is a significant
statistical difference between months (DF=11;
F=2.9; p<0.05).
Female specimens were significantly larger
than male specimens (DF=1; F=6.9; p< 0.05) and
there was no interaction between variables (DF =
11; F = 1.3; p>0.05).
A size-class distribution analysis over the whole
year showed that the most frequently captured
specimens measured between 90-99mm and 100109mm.
Smaller specimens (size class between 40mm and
49mm) were captured during late winter-early
spring (February-March 2010) (Figure 4).
Stomach content analysis
In the Geraci area, the diet of P. clarkii mainly
consisted of detritus (55.56% of the total) and
plant material (43.22%); animal consumption was
low (1.22%) (G: 2845.48; DF=88; p<0.001).
The highest dietary component was detritus in
spring and summer (76% and 61.5% respectively),
and plants in autumn and winter (72% and 52%
respectively) (G: 58.96; DF=3 p<0.001) (Figure 5).
First evidence of microfungal “extra oomph” in Procambarus clarkii
Figure 2. Monthly Catches Per Unit
Effort (CPUE), a standardised index to
define the abundance of P. clarkii from
June 2009 to May 2010. The whiskers
extend outwards to indicate the lowest
and highest values in the data set
(excluding outliers). Extreme values, or
outliers, are represented as asterisks.
Figure 3. Box-plot of the total body
length of P. clarkii female (white) and
male (grey) specimens collected in the
Geraci area from June 2009 to May
2010. The whiskers extend outwards
to indicate the lowest and highest
values in the data set (excluding
outliers). Extreme values, or outliers,
are represented as asterisks.
The seasonal differences in percentages of
plants found in the stomachs were statistically
significant (1-way ANOVA: DF=3; F=7.94;
p<0.001); the same analysis was performed using
the percentage of detritus as a variable and
differences between seasons were again statistically
significant (DF=3; F=7.14; p<0.005).
Tukey post hoc tests were performed in order
to highlight seasonal differences in crayfish diet.
Autumn significantly differs from spring and
summer in terms of plant and detritus consumption
(Table 1).
Furthermore, in order to highlight possible
differences in diet composition between sexes, a
General Linear Model (GLM) was performed:
results supported the hypothesis that there were
no significant differences between sexes (Detritus:
DF=1, F=1.92, p>0.05; Plant material: DF=1,
F=1.66, p>0.05; Animal: DF=1, F=0.16, p>0.05).
Microfungal stomach flora
A total of 45 taxa were found in the stomachs of
the 45 examined specimens: Table 2 shows the
total frequency (i.e. the percentage of P. clarkii
individuals in which the taxon was observed over
the total number of examined specimens). Microfungal specimens were divided into two categories,
Dematiaceous, i.e. containing melanins in cell
walls, and non-Dematiaceous, according to Ellis
(1971, 1976). The majority of observed taxa (80%)
consisted of non-Dematiaceous fungi, while 20%
consisted of Dematiacea species.
Mycelia sterilia and yeasts were detected with
the highest frequencies (73% and 64% respectively),
followed by Penicillium sp. (62%), Talaromyces
flavus (42%), Trichoderma sp. (33%), Mucor sp.
(22%), Cladosporium sp. (18%), Acremonium sp.
(11%); the remaining taxa were found with a lower
51
L. Garzoli et al.C.
Figure 4. Size-class distribution of P. clarkii male specimens (light grey) and female specimens (dark grey). a) June 2009, b) July 2009, c)
August 2009, d) September 2009, e) October 2009, f) November 2009, g) December 2009, h) January 2010, i) February 2010, j) March
2010, k) April 2010, l) May 2010.
Table 1. Results of Tukey Simultaneous tests. The animal category was not considered as it was inconsistent.
Seasons
autumn vs summer
autumn vs winter
autumn vs spring
summer vs winter
summer vs spring
winter vs spring
autumn vs summer
autumn vs winter
autumn vs spring
summer vs winter
summer vs spring
winter vs spring
52
Seasonal differences in percentages of plants
Tukey test
p value
-3.36
0.0096
-2.00
0.2048
-4.66
0.0002
1.35
0.5357
-1.30
0.5666
-2.65
0.0542
Seasonal differences in percentages of detritus
3.47
0.0070
2.11
0.1673
4.33
0.0006
-1.36
0.5316
0.857
0.8269
2.217
0.1379
Statistical significant
**
NS
***
NS
NS
NS
*
NS
***
NS
NS
NS
First evidence of microfungal “extra oomph” in Procambarus clarkii
Table 2. Frequency of occurrence (%) of microfungal taxa in the stomach samples of P. clarkii in relation to capture seasons. Percentages
are in relation to the total sample (TS); summer 2009 (S09); autumn 2009 (A09); winter 2009/2010 (W09/10); spring 2010 (SP10).
Taxa
TS
S09
A09
W09/10
Sp10
Mycelia sterilia
Yeasts
Penicillium sp.
Talaromyces flavus
Trichoderma sp.
Mucor sp.
Acremonium sp.
Cladosporium sp.
Aspergillus fumigatus Fresen. (1863)
A. brasiliensis Varga, Frisvad & Samson 2007
Aspergillus sp.
Pestalotiopsis guepinii (Desm.) Steyaert (1949)
Rhizopus stolonifer (Ehrenb.) Vuill. 1902
Arthrinium sp.
Cladosporium cladosporioides (Fresen.) G.A. de Vries
Mucor hiemalis Wehmer (1903)
Phoma sp.
Arthrinium phaeospermum (Corda) M.B. Ellis (1965)
Aureobasidium pullulans var. pullulans (de Bary) G. Arnaud 1918
Cladosporium chlorocephalum (Fresen.) E.W. Mason and M.B. Ellis (1953)
Emericellopsis sp.
Khuskia oryzae H.J. Huds. (1963)
Paecilomyces sp.
Penicillium verrucosum Dierckx (1901)
Scopulariopsis sp.
Sordaria fimicola (Roberge ex Desm.) Ces. and De Not. (1863)
Absidia fusca Linnem. (1936)
A. glauca Hagem (1908)
Alternaria alternata (Fr.) Keissl. 1912
Acremonium persicinum (Nicot) W. Gams (1971)
Aspergillus clavatus Desm. (1934)
A. flavus Link (1809)
A. glaucus (L.) Link (1809)
A. versicolor (Vuill.) Tirab. (1908)
Aureobasidium pullulans var. melanogenum Herm.-Nijh (1977)
Cephalotrichum microsporum (Sacc.) P.M. Kirk (1984)
Chaetomium sp.
Coniella sp.
Fusarium sp.
Hemicarpenteles ornatum (Subram.) Arx (1974)
Mortierella turficola Y. Ling (1930)
Mortierella sp.
Oidiodendron flavum Svilv. (1941)
Paecilomyces farinosus (Holmsk.) A.H.S. Br. and G. Sm. (1957)
P. inflatus (Burnside) J.W. Carmich. (1962)
P. lilacinus (Thom) Samson (1974)
Actinomycetes
Bacteria
73
64
62
42
33
22
18
18
11
11
9
9
9
7
7
7
7
4
4
4
4
4
4
4
4
4
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
42
93
70
70
60
90
30
40
30
50
0
10
0
10
10
0
0
0
20
10
10
0
0
0
10
0
0
20
10
0
0
0
0
0
0
0
0
0
10
10
0
0
0
10
0
10
10
10
70
100
70
30
40
20
10
10
10
10
0
40
20
10
10
0
30
0
0
0
10
10
20
0
0
0
20
0
0
0
10
0
0
0
0
0
0
10
0
0
10
0
0
0
0
0
0
0
50
100
70
80
80
20
30
30
10
20
30
0
10
20
10
20
0
10
0
0
0
0
0
10
0
0
0
0
0
0
0
0
10
10
0
0
10
0
0
0
0
0
10
0
0
0
0
0
30
90
80
73
67
40
53
13
20
0
13
0
7
0
7
7
0
13
7
7
0
7
0
7
7
13
0
0
0
7
0
7
0
0
7
7
0
0
0
0
0
7
0
0
7
0
0
0
27
87
frequency than 10%. Bacteria and Actinomycetes were also observed throughout.
An ANOSIM test was performed in order to
highlight differences in the microfungal flora
community between seasons: results suggested
there were no significant differences between
seasons (R=0.207; p: 0.001) and the highest taxa
richness was recorded in spring 2010. Furthermore,
a BIO-ENV analysis correlating stomach content
with its microfungal flora showed a low negative
correlation between variables (p=-0.026).
Discussion
Studies involving multidisciplinary expertise are
of fundamental importance to understand
environmental alterations that can lead to changes in
population dynamic properties. Moreover, they can
be used as indicators of different kinds of
stressors that have never been considered before.
Understanding P. clarkii population dynamics is a
useful way of building effective management
plans to control this dangerous alien species.
53
L. Garzoli et al.C.
Figure 5. Seasonal composition of the P. clarkii diet in the Geraci
area (5 analysed crayfish/month).
Lodge et al. (2012) stated that it is fundamental to
quantify the impact of invasive crayfish through
metrics of direct relevance when making
provisions and regulating ecosystem services,
both cultural and monetised. Therefore, the cost
of one year of systematically trapping P. clarkii
was evaluated in order to better inform
management decisions. The following factors
were taken into consideration: the hourly rate
paid to each operator (€10/hour), bait costs
(€5/kg), trap costs (€15/trap), and transport
mobility expenses. The total budget required to
control the invasive crayfish in a small area such
as Geraci was about €5000: €192 for baits, €300
for traps, €52 for complementary material, €1411
for mobility (car fuel and transfer costs), and
€2880 for two operators.
Intensive systematic trapping, coupled with
other management strategies, such as predatory
fishing, can be effective in controlling the
crayfish population in invaded and closed
ecosystems (Frutiger et al. 1999; Hein et al. 2006),
but it is problematic when used for population
control in an open environment (as in our case
study) where immigration/emigration phenomena
from neighbouring environments affect population
size (Barbaresi and Gherardi 2000). Catches are
also influenced by temperature, crayfish foraging
and reproduction activity, as well as body size
(Correia 2001; Chucholl 2011). Large specimens,
especially males, being more mobile than
juveniles, are more likely to be captured,
whereas juvenile specimens are both “trap-shy”
and small enough to escape.
In our study, the highest number of crayfish
was collected in summer when P. clarkii is more
active, and the lowest number in winter, when
54
the population is mainly composed of juvenile
specimens. Moreover, crayfish collected over the
whole year were always longer than 40mm, thus
post-juvenile specimens. This suggests that
trapping has limited effectiveness in red swamp
population control.
As regards the population structure, we found
a sex ratio of 2.42 males to 1 female. According
to previous studies conducted in central Italy, a
stable population of P. clarkii has a sex ratio of
1:1 (Gherardi et al. 1999a; Scalici and Gherardi
2007). However, as various authors have stated, a
higher proportion of males can depend on the
capture method used, as traps are mainly maleselective (Ackefors 1999; Ilhéu et al. 2003; Savini
2007; Chucholl 2011). Overall, our data confirm
the presence of a stable population of P. clarkii in
the Geraci area.
The highest density of crayfish was registered
in the centre of the Geraci area (T5 to T8). This
area is characterised by small shallow wetlands,
with steep embankments in which P. clarkii
specimens dig their burrows. The bottom of the
ponds is rich in detritus and plant material, in
accordance with our findings on the feeding
habits of P. clarkii.
As food availability is site-specific, knowledge
about the P. clarkii diet can help to understand its
effect on trophic food webs. Our results confirm
that freshwater crayfish are opportunistic and
omnivorous feeders (Lodge and Hill 1994; Momot
1995; Gutiérrez-Yurrita et al. 1998). Correia (2003)
found that P. clarkii mainly feeds on plants during
spring and summer, while it prefers an animal
diet during winter. Our data, however, did not
confirm this trend: stomach content mainly
consisted of plant material during autumn and
winter, and of detritus (i.e. muddy/sand inorganic
matter mixed with decomposed organic matter)
during spring and summer. The nutritional value
of detritus is mainly linked to the microbial layer
surrounding the sediment particles (Hall and
Meyer 1998). The absence of aquatic vegetation
may also have affected the crayfish diet. The
predominance of plant matter in autumn is
probably linked to the large quantity of dead
leaves that fall to the ground and into the water
at the beginning of this season. Thus, our
findings confirm that P. clarkii can shift its diet in
relation to food availability. In our case study,
the animal category was not included in the red
swamp adult diet; animal prey was less
representative in the diet than expected: this
could be explained by the fact that the traps
proved to be adult-selective. In fact, small crayfish
First evidence of microfungal “extra oomph” in Procambarus clarkii
are mainly carnivorous, while larger individuals
are primarily herbivorous; animal-based food is
much more important for young, rapidly growing
juveniles than for adults (Correia 2003).
In recent years, there has been a growing
interest in the relationship between P. clarkii and
environmental microorganisms (Dörr et al. 2011,
2012a,b). Only a few studies are available on
microflora in the digestive tract of P. clarkii
(Gherardi et al. 1999b; Khalil et al. 2009), none of
which focus on fungi.
Microorganisms can enter the crayfish stomach
not only as food: in particular fungi, which can
be highly related to their growing substrates (e.g.
saprotrophs can obtain their nutrients from dead
organisms, while epi- and endophytes can only
survive in association with living plants). As a
consequence, seasonal variations in microbial
communities in the stomach according to diet
were expected. Nevertheless, seasonal differences
in the microfungal community were not consistent,
as the ANOSIM test confirms.
Three partially alternative/complementary
explanations can fit our results:
1) Homogeneity of diet. Fungi are accountable
for recycling components of dead plants. In our
site, detritus is mainly composed of dead plant
fragments. We can hypothesise that microfungal
assemblages linked to plant material are not very
different from those linked to detritus; some of
the very common taxa found in all seasons are
indeed common saprobes, frequently associated
with soil and litter, such as Mucor sp., Rhizophus
stolonifer, Thrichoderma sp., and Talaromyces
flavus. Other detected fungi are linked to plant
hosts/plant material: the widespread taxa
Cladosporium sp. and Penicillium sp., and the less
common Pestalotiopsis guepinii, the causal agent
of the hazelnut twig blight (Türkkan et al. 2011).
This result highlights an aspect related to red
swamp crayfish bioinvasion: a high population
density of P. clarkii could be a vector of
introduction not only of animal pathogens (the
most famous being Aphanomyces astacii) but also
of plant pathogens, whose spores can survive in
the digestive tract of crayfish when they reach
new environments.
2) Procambarus clarkii selection of food
source. It is well known that shredders can
selectively feed on leaves that are colonised by
microorganisms, in particular fungi (Graça et al.
1993). Not only can fungi increase the palatability
of leaves by promoting the transformation of
inedible plant material into edible compounds,
but they are also a better food source than leaves,
in so far as they possess a higher nutritional
value (mostly in terms of nitrogen content)
(Graça 2001). However, although food choice
experiments indicate that most soil animals
prefer fungi with dark pigmented hyphae and/or
conidia (=Dematiacea) (Maraun et al. 2003), our
findings do not support literature data, as the
majority of observations are non-Dematiaceous
fungi. The preference of Dematiaceous fungi by
aquatic invertebrates has been partially related to
the higher nutritional value of melanised hyphae.
Common species of non-Dematiaceous fungi also
possess strong enzymes, such as chitinases in
Trichoderma, Penicillium, Paecilomyces and
Mortierella, which are usually avoided by
arthropods (Maraun et al. 2003). In our study,
Penicillium and Trichoderma were found with
the highest frequency. This could lead to the
third hypothesis.
3) Although less reliable, a fungal selection
within the P. clarkii stomach cannot be excluded
when attempting to explain the homogeneity of
our data-set on fungal assemblages.
Several authors point out the effect of the red
swamp crayfish herbivory (Vasconcelos et al.
2001; Rodriguez et al. 2005; Anastácio et al. 2005;
Savini and Occhipinti 2008). As a consequence, it
needs enzymes that degrade complex polymers
such as cellulose.
Acquired microbial cellulases have been
reported among freshwater invertebrates by
Bärlocher (1982) and Sinsabaugh et al. (1985). In
1964, Yokoe and Yasumasu demonstrated a
considerable cellulasic activity in hepatopancreatic
microsomal fractions (i.e. subcellular fraction of
gland homogenate) of P. clarkii, but this activity
was not related to Gram negative bacilli found in
the stomach of the specimens they analysed.
Gherardi et al. (1999b) also found a cellulosedegrading bacteria in its digestive tract.
Nevertheless, fungal production of endogenous
cellulases has never been considered. Many of
the isolated fungi found in our study are reported
in literature to produce a wide range of hydrolases
that digest polymers such as cellulose, lignin and
chitin, as is the case with Talaromyces flavus, a
species common in soil and in organic substrates.
This species is able to degrade starch and
cellulose and to produce antibiotic metabolites
(such as vermiculine) that are active against
bacteria, streptomycetes and protozoans (Domsch
et al. 1980). Other taxa that are very common
plant parasites and ubiquitous in soil, such as
Alternaria alternata and Fusarium sp., were found
with low frequencies (Domsch et al. 1980).
55
L. Garzoli et al.C.
On the basis of these observations, our
findings could be compatible with the selection
of fungal assemblage in crayfish stomachs.
One of the severe constraints in highlighting
arthropod host/fungi interaction is the difficulty
obtaining axenic culture of gut fungi that can be
used in laboratory infection trials (Kimura et al.
2002). This study is a starting point in this
direction, as the microfungal strains are being
maintained in pure culture.
In conclusion, our data show that the detected
mycoflora is not related to season or diet. We
hypothesised that the lack of differences may be
related to: 1) a relative homogeneity of food
sources, 2) a selection of food source more or
less colonised by fungi, 3) a selection within the
P. clarkii stomach.
The third hypothesis can lead to two main
conclusions: 1) studies on production of enzymes
such as cellulase from endogenous mycoflora
should be encouraged in order to better understand
their role in P. clarkii bioinvasion; 2) the
selection of fungal propagules in the digestive
tract of crayfish and combined propagation of such
microorganisms in excrements deserve particular
attention, since an invasive species that also
behaves as a vector of parasites is a further risk
in terms of “pathogen pollution” (Picco et al. 2011).
Further studies are needed to understand the
functional role of fungi in the digestive system
and their importance in the invasion process.
Acknowledgements
We would like to dedicate this paper to the memory of Francesca
Gherardi, who recently and prematurely passed away. She was a
worldwide renowned leading scientist in the field of biological
invasions and a pioneer in the research on P. clarkii in Italy and
Europe. Some of us have had the privilege of collaborating with
her and will never forget her scientific value, modesty, and ladylike, generous manner. We gratefully thank Charlotte Buckmaster
for revising the English text. We wish to thank Prof. Giuseppe
Bogliani for his precious support to fieldwork logistic. Our
special thanks to Dr. Christoph Chucholl, Dr. Frances Lucy and
two anonymous reviewers for their constructive comments and
suggestions on the manuscript.
References
Ackefors H (1999) The positive effects of established crayfish
introductions in Europe. In: Gherardi F, Holdich DM (eds),
Crayfish in Europe as alien species. How to make the best of
a bad situation. A. A. Balkema, Rotterdam, pp 49–61
Alcorlo P, Geiger W, Otero M (2004) Feeding preferences and
food selection of the red swamp crayfish (Procambarus
clarkii) in habitats differing in food item diversity.
Crustaceana 77(4): 435–453, http://dx.doi.org/10.1163/15685400
41643283
56
Alderman DJ, Polglase JL (1986) Aphanomyces astaci: isolation
and culture. Journal of Fish Diseases 7: 401–405,
http://dx.doi.org/10.1111/j.1365-2761.1984.tb01205.x
Anastácio PM, Parente VS, Correia AM (2005) Crayfish effects
on seeds and seedlings: identification and quantification of
damage. Freshwater Biology 50: 697–704, http://dx.doi.org/
10.1111/j.1365-2427.2005.01343.x
Aquiloni L, Tricarico E, Gherardi F (2010) Crayfish in Italy:
distribution, threats and management. International Aquatic
Research 2: 1–14
Aquiloni L, Martin MP, Gherardi F, Diéguez-Uribeondo J (2011)
The North American crayfish Procambarus clarkii is the
carrier of the oomycete Aphanomyces astaci in Italy.
Biological Invasions 13: 359–367, http://dx.doi.org/10.1007/
s10530-010-9828-2
Barbaresi S, Gherardi F (2000) The invasion of the alien crayfish
Procambarus clarkii in Europe, with particular reference to
Italy. Biological Invasions 2: 259–264, http://dx.doi.org/10.102
3/A:1010009701606
Barbaresi S, Gherardi F (2006) Experimental evidence for homing
in the red swamp crayfish, Procambarus clarkii. Bulletin
Français de la Pêche et de la Pisciculture 380–381: 1145–
1154, http://dx.doi.org/10.1051/kmae:2006017
Bärlocher F (1982) The contribution of fungal enzymes to the
digestion of leaves by Gammarus fossarum Koch (Amphipoda). Oecologia 52: 1–4, http://dx.doi.org/10.1007/BF00349003
Bergman DA, Kozlowski CP, McIntyre JC, Huber R, Daws AG,
Moore PA (2003) Temporal dynamics and communication of
winner–effects in the crayfish, Orconectes rusticus.
Behaviour 140(6): 805–825, http://dx.doi.org/10.1163/156853903
322370689
Chiesa S, Scalici M, Givertin G (2006) Occurrence of
allochthonous freshwater crayfishes in Latium (Central Italy).
Bulletin Français de la Pêche et de la Pisciculture 380–381:
883–902, http://dx.doi.org/10.1051/kmae:2006029
Chucholl C (2011) Population ecology of an alien “warm water”
crayfish (Procambarus clarkii) in a new cold habitat.
Knowledge and Management of Aquatic Ecosystems 401: 29,
http://dx.doi.org/10.1051/kmae/2011053
Clarke KL, Warwick RM (1994) Change in marine communities:
an approach to statistical analysis and interpretation.
Plymouth Marine Laboratory, 176 pp
Correia AM (2001) Seasonal and intraspecific evaluation of
predation by mammals and birds on the introduced red
swamp crayfish Procambarus clarkii (Crustacea, Cambaridae) in a freshwater marsh (Portugal). Journal of Zoology
255: 533–541, http://dx.doi.org/10.1017/S0952836901001625
Correia AM (2003) Food choice by the introduced crayfish
Procambarus clarkii. Annales Zoologici Fennici 40: 517–528
Delmastro GB (1992) Sull'acclimatazione del gambero della Louisiana Procambarus clarkii (Girard, 1852) nelle acque dolci
italiane (Crustacea: Decapoda: Cambaridae). Pianura 4: 5–10
Domsch KH, Gams W, Anderson TH (1980) Compendium of soil
fungi. Academic Press, London, 865 pp
Dörr AJM, Rodolfi M, Scalici M, Elia AC, Garzoli L, Picco AM
(2011) Phoma glomerata, a potential new threat to Italian
inland waters. Journal for Nature Conservation 19(6): 370–
373, http://dx.doi.org/10.1016/j.jnc.2011.06.006
Dörr AJM, Elia AC, Rodolfi M, Garzoli L, Picco AM, D'Amen
M, Scalici M (2012a) A model of co-occurrence: segregation
and aggregation patterns in the mycoflora of the crayfish
Procambarus clarkii in Lake Trasimeno (central Italy).
Journal of Limnology 71(1): 135–143, http://dx.doi.org/10.4081/
jlimnol.2012.e14
Dörr AJM, Rodolfi M, Elia AC, Scalici M, Garzoli L, Picco AM
(2012b) Mycoflora on the cuticle of the invasive crayfish
Procambarus clarkii. Fundamental and Applied Limnology
180 (1): 77–84, http://dx.doi.org/10.1127/1863-9135/2012/0175
First evidence of microfungal “extra oomph” in Procambarus clarkii
Ellis MB (1971) Dematiaceous Hyphomycetes. CAB, Kew,
Surrey, England, 594 pp
Ellis MB (1976) More Dematiaceous Hyphomycetes. CAB, Kew,
Surrey, England, 493 pp
Figiel CR Jr, Miller GL (1995) The frequency of chela autotomy
and its influence on the growth and survival of the crayfish
Procambarus clarkii (Girard, 1852) (Decapoda, Cambaridae). Crustaceana 68(4): 472–483, http://dx.doi.org/10.1163/
156854095X00683
Frutiger A, Borner S, Büsser T, Eggen R, Müller R, Müller S,
Wasmer H R (1999) How to control unwanted populations of
Procambarus clarkii in Central Europe. Freshwater Crayfish
12: 714–726
Geiger W, Alcorlo P, Baltanás A, Montes C (2005) Impact of an
introduced Crustacean on the trophic webs of Mediteranean
wetlands. Biological Invasions 7: 49–73, http://dx.doi.org/10.
1007/s10530-004-9635-8
Gherardi F (2006) Crayfish invading Europe: the case study of
Procambarus clarkii. Marine and Freshwater Behaviour and
Physiology 39: 175–191, http://dx.doi.org/10.1080/10236240600
869702
Gherardi F, Baldaccini GN, Barbaresi S, Ercolini P, De Luise G,
Mazzoni D, Mori M (1999a) The situation in Italy. In:
Gherardi F, Holdich DM (eds) Crayfish in Europe as Alien
species. How to make the best of a bad situation? Balkema,
Rotterdam, pp 107–128
Gherardi F, Acquistapace P, Salvi G, Mastromei G (1999b)
Cellulolytic activity in the hindgut of crayfish. Crustaceana
72: 534–536
Gherardi F, Acquistapace P (2007) Invasive crayfish in Europe:
the impact of Procambarus clarkii on the littoral community
of a Mediterranean lake. Freshwater Biology 52(7): 1249–
1259, http://dx.doi.org/10.1111/j.1365-2427.2007.01760.x
Gherardi F, Barbaresi S (2007) Feeding preferences of the
invading crayfish, Procambarus clarkii. Bulletin Français de
la Pêche et de la La Pisciculture 385: 7–20, http://dx.doi.org/
10.1051/kmae:2007014
Graça MAS (2001) The role of invertebrates on leaf litter
decomposition in streams – a review. International Review of
Hydrobiology 86(4–5): 383–393, http://dx.doi.org/10.1002/15222632(200107)86:4/5<383::AID-IROH383>3.0.CO;2-D
Graça MAS, Maltby L, Calow P (1993) Importance of fungi in
the diet of Gammarus pulex and Asellus aquaticus: feeding
strategies. Oecologia 93(1): 139–144
Gutiérrez-Yurrita PJ, Sancho G, Bravo MÁ, Baltanás Á, Montes
C (1998) Diet of the Red Swamp Crayfish Procambarus
clarkii in Natural Ecosystems of the Doñana National Park
Temporary Fresh-Water Marsh (Spain). Journal of Crustacean Biology 18(1): 120–127, http://dx.doi.org/10.2307/1549526
Hall RO, Meyer JL (1998) The trophic significance of bacteria in
a detritus-based stream food web. Ecology 79: 1995–2012,
http://dx.doi.org/10.1890/0012-9658(1998)079[1995:TTSOBI]2.0.CO;2
Harris JM (1993) The presence, nature, and role of gut microflora
in aquatic invertebrates: a synthesis. Microbial Ecology
25(3): 195–231, http://dx.doi.org/10.1007/BF00171889
Hein CL, Roth BM, Ives AR, Zanden MJV (2006) Fish predation
and trapping for rusty crayfish (Orconectes rusticus) control:
a whole-lake experiment. Canadian Journal of Fisheries and
Aquatic Sciences 63: 383–393, http://dx.doi.org/10.1139/f05-229
Hermanides-Nijhof EJ (1977) Aureobasidium and allied genera.
Studies in Mycology 15: 141–176
Hinton MG, Maunder MN (2003) Methods for standardizing
CPUE and how to select among them. Proceeding of 16th
meeting of the standing committee on Tuna and Billfish.
Mooloolaba, Australia, July 9-16, 2003. Inter-American
Tropical Tuna Commission La Jolla, California USA, p 11
Huner JV (1988) Procambarus in North America and elsewhere.
In: Holdich DM, Lowery RS (eds), Freshwater crayfish:
biology, management and exploitation. Croom Heim,
London, pp 239–261
Ilhéu M, Acquistapace P, Benvenuto C, Gherardi F (2003)
Burrowing of the red swamp crayfish (Procambarus clarkii)
in a temporary stream of south Portugal. Archiv für
Hydrobiologie 157: 535–546, http://dx.doi.org/10.1127/00039136/2003/0157-0535
Khalil RH, El-Banna SA, Mahfouz NB (2009) Study on micro
flora of wild freshwater crayfish (Procambarus clarkii).
Proceedings of the 2nd Global Fisheries and Aquaculture
Research Conference, pp 257–261
Kimura H, Harada K, Hara K, Tamaki A (2002) Enzymatic
approach to fungal association with arthropod guts: a case
study for the crustacean host, Nihonotrypaea harmandi, and
its foregut fungus, Enteromyces callianassae. Marine
Ecology 23(2): 157–183, http://dx.doi.org/10.1046/j.1439-0485.20
02.02778.x
Klich MA (2002) Identification of common Aspergillus species.
Centraalbureau voor Schimmelcultures, Utrecht, 116 pp
Kraus-Epley KE, Moore PA (2002) The effects of antennal
lesions on orientation behaviour of the crayfish Orconectes
rusticus. Chemical Senses 27: 49–55, http://dx.doi.org/10.1093/
chemse/27.1.49
Lodge DM, Hill AM (1994) Factors governing species
composition, population size, and productivity of cool-water
crayfishes. Nordic Journal of Freshwater Research 69: 111–
136
Lodge DM, Deines A, Gherardi F, Yeo DCJ, Arcella T, Baldridge
AK, Barnes MA, Chadderton WL, Feder JL, Gantz CA,
Howard GW, Jerde CL, Peters BW, Peters JA, Sargent LW,
Turner CR, Wittmann ME, Zeng Y (2012) Global
introductions of crayfishes: evaluating the impact of species
invasions on ecosystem services. Annual Review of Ecology
and Systematics 43: 449–472
Maraun M, Martens H, Migge S, Theenhaus A, Scheu S (2003)
Adding to the enigma of soil animal diversity: fungal feeders
and saprophagous soil invertebrates prefer similar food
substrates. European Journal of Soil Biology 39: 85–95,
http://dx.doi.org/10.1016/S1164-5563(03)00006-2
Mickënienë L, Šyvokienë J (1996) Microflora in the digestive
tract of freshwater crayfish. Freshwater Crayfish 11: 441–
449
Minitab Inc., State College, PA, USA (2007) Meet Minitab 15 for
windows, USA
Momot WT (1995) Redefining the role of crayfish in aquatic
ecosystem. Reviews in Fisheries Science 3(1): 33–63,
http://dx.doi.org/10.1080/10641269509388566
Momot WT, Gowing H, Jones PD (1978) The dynamics of
crayfish and their role in ecosystems. American Midland
Naturalist 99: 10–35, http://dx.doi.org/10.2307/2424930
Morpugo M, Aquiloni L, Bertocchi S, Brusconi S, Tricarico E,
Gherardi F (2010) Distribuzione dei gamberi d’acqua dolce
in Italia. Acta Biologica 87: 125–132
Nelson PE, Toussoun TA, Marasas WFO (1983) Fusarium
species: an illustrated manual for identification. The
Pennsylvania State University Press, London, 193 pp
Picco AM, Angelini P, Ciccarone C, Franceschini A, Ragazzi A,
Rodolfi M, Varese GC, Zotti M (2011) Biodiversity of
emerging pathogenic and invasive fungi in plants, animals
and humans in Italy. Plant Biosystems 145(4): 988–996,
http://dx.doi.org/10.1080/11263504.2011.633118
Pitt JI (1979) The genus Penicillium and its teleomorphic state
Eupenicillium and Talaromyces. Academic Press, London,
634 pp
Richards LJ, Schnute JT (1986) An experimental and statistical
approach to the question: is CPUE an index of abundance?
Canadian Journal of Fisheries and Aquatic Sciences 43(6):
1214–1227, http://dx.doi.org/10.1139/f86-151
57
L. Garzoli et al.C.
Rodriguez CF, Becares E, Fernandez-Alaez M, Fernandez-Alaez
C (2005) Loss of diversity and degradation of wetlands as a
result of introducing exotic crayfish. Biological Invasions 7:
75–85, http://dx.doi.org/10.1007/s10530-004-9636-7
Rossi L (1983) Interactions between Invertebrates and Microfungi
in Freshwater. Oikos 44(1): 175–184, http://dx.doi.org/10.2307/
3544059
Samson RA (1974) Paecylomyces and some allied hyphomycetes.
Studies in Mycology 6: 1–119
Samson RA, Hoekstra ES, Frisvad JC (2004) Introduction to
food- and airborne fungi. Centraalbureau voor Schimmelcultures, Netherlands, 389 pp
Savini D (2007) Rinvenimento della specie aliena invasiva
Procambarus clarkii (Atracidea: Cambaridae) nella riserva
naturale “Bosco Siro Negri” (Zerbolò- Pavia). Acta Biologica
83: 33–37
Savini D, Occhipinti-Ambrogi A (2008) Bad Moon Rising: the
Lousiana Swamp Red Crayfish, a threat to the freshwater
ecosystems in Lombardy. Memorie Società Italiana di
Scienze Naturali, Museo Civico di Storia Naturale di Milano
36: 19–20
Savini D, Occhipinti-Ambrogi A, Nicolao J, Perrone M, Garzoli
L, Rodolfi M, Picco AM (2008) Il gambero invasivo
Procambarus clarkii (Girard, 1852) nella lanca della riserva
naturale integrale “Bosco Siro Negri”. Archivio Geobotanico
11(1–2): 49–58
Savini D, Occhipinti-Ambrogi A, Marchini A, Tricarico E,
Gherardi F, Olenin S, Gollasch S (2010) The top 27 alien
animal species intentionally introduced by European
aquaculture and related activities: stocking, sport fishery and
ornamental purposes. Journal of Applied Ichthyology 26: 1–7,
http://dx.doi.org/10.1111/j.1439-0426.2010.01503.x
58
Scalici M, Gherardi F (2007) Structure and dynamics of an
invasive population of the red swamp crayfish (Procambarus
clarkii) in a Mediterranean wetland. Hydrobiologia 583:
309–319, http://dx.doi.org/10.1007/s10750-007-0615-8
Scalici M, Pitzalis M, Gibertini G (2009) Crayfish distribution
updating in central Italy. Knowledge and Management
Aquatic Ecosystem 394–395(6): 1–8, http://dx.doi.org/10.1051/
kmae/2009017
Sinsabaugh RL, Linkins AE, Benfield EF (1985) Cellulose
digestion and assimilation by three leaf-shredding aquatic
insects. Ecology 66(5): 1464–1471, http://dx.doi.org/10.2307/193
8009
Souty-Grosset C, Holdich DM, Noël PY, Reynolds JD, Haffner P
(2006) Atlas of Crayfish in Europe. Publications
Scientifiques du MNHN, Paris, 187 pp
Sutton BC (1980) The Coelomycetes. Commonwealth Mycological Institute, Kew, Surrey, England, 696 pp
Tricarico E, Vilizzi L, Gherardi F, Copp GH (2010) Calibration of
FI-ISK, an invasiveness screening tool for nonnative
freshwater invertebrates. Risk Analysis 30(2): 285–292,
http://dx.doi.org/10.1111/j.1539-6924.2009.01255.x
Türkkan M, Andolfi A, Zonno M, Erper I, Perrone C, Cimmino
A, Vurro M, Evidente A (2011) Phytotoxins produced by
Pestalotiopsis guepinii, the causal agent of hazelnut twig
blight. Phytopathology Mediterranean 50: 154–158
Vasconcelos V, Oliveira S, Teles FO (2001) Impact of a toxic and
a non-toxic strain of Microcystis aeruginosa on the crayfish
Procambarus clarkii. Toxicon 39: 1461–1470, http://dx.doi.org/
10.1016/S0041-0101(01)00105-2
Yokoe Y, Yasumasu I (1964) The distribution of cellulase in
invertebrates. Comparative Biochemistry and Physiology
13: 323–338, http://dx.doi.org/10.1016/0010-406X(64)90027-1