The squid Uroteuthis duvauceli is a commercially important fishery species in many coastal regions of the Red Sea. This species was the first recorded Lessepsian immigrant squid species in the Suez Canal and has successfully established in its new environment. In this study, the taxonomic status of this species in the Gulf of Suez, north of the Red Sea, and Suez Canal was elucidated by morphological and genetic analyses. Fourteen morphometric indices were used to distinguish the two populations. ANCOVA revealed that there was a significant separation in morphometric indices between locations (R=0.1, P<0.001). Indices fin length, fin width, arm lengths, tentacle and tentacle club length to mantle length, as well as fin width index to fin length indicated the most difference between the two populations. The morphometric difference between the two populations is most probably due to differences in environmental conditions and resource availability. Genetic analysis based on the mitochondrial cytochrome b (cyt b) gene revealed that the two populations are one species and have note diverged; the intraspecific distance was very low, ranging from 0 to 0.003 in U. duvauceli from the Suez Canal and Gulf of Suez.
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Keywords: morphological studies, genetic analyses, Lessepsian Migration, Uroteuthis duvauceil
Over the past two decades, cephalopods, and squids in particular, have increased in importance as alternative resources to the traditional finfish fishery in different areas in the world (Boyle & Pierce, 1994; Boyle & Rodhouse, 2005). Among the most important squid species in the commercial fisheries are the veined squid Loligo forbesi (Streenstrup, 1856), European squid L. vulgaris (Lamarck, 1798) (Moreno et al., 1994) and Uroteuthis duvauceli (Orbigny, 1848) (Sukramongkol et al., 2007). U. duvauceli is a demersal cephalopod and its range extends from Indian Ocean periphery, including the Red Sea and the Arabian Sea, extending eastwards from Mozambique to the South China Sea and Philippines Sea, northward to Taiwan (Roper et al., 1984). It is abundant both in the Egyptian Red Sea coast and Gulf of Suez. Along these coasts, the commercial catch of U. duvauceli is mainly landed as by-catch of multispecies demersal trawling fisheries. Nevertheless, directed artisanal fishing takes place on a wide scale.
U. duvauceli appeared first in the Suez Canal in 1998 (Personal observation). The commercial fishery was very limited with catches of less than 10 Kg/day in 2006. Then catches increased rapidly to a reported catch of 40 Kg/day Kg. in 2008 (Personal observation). There is no reliable landing data on commercial catches of U. duvauceli in Suez Canal as its landings are incorporated with those of cuttlefish, but there is now an established commercial market. The rapid increase in catches has given rise to serious concerns about the Lessepsian migration. The opening of the Suez Canal in 1869 allowed the migration, termed Lessepsian, of hundreds of Red Sea species that have become established (Por, 1978). Although, squid are highly mobile (Boyle, 1990), little is known about Lessepsian migration of these species. Since the work of Por (1978), no work has been carried out to describe the immigration and establishment of any cephalopod species in Suez Canal. Therefore we set out to document the occurrence and describe how successful the establishment of U. duvauceli was along the Suez Canal.
The aim of the present study was to elucidate the morphometric relationships of U. duvauceli from Suez Canal and Gulf of Suez north of the Red Sea and see if changing of habitats changed morphometry; sex ratio and maturity stages of the two populations were also examined. Mitochondrial DNA was used in this study to determine whether they are two species or one species.
MATERIALS AND METHODS
U. duvauceli were collected in February 2008 from two populations along the coasts of Suez on the Gulf of Suez and the coasts of Ismailia on the Suez Canal (Figure 1). The samples were caught by local fishermen using industrial trawling nets in Gulf of Suez and artisanal beach seine and gill net in Suez Canal. In total, 374 individuals were analyzed, 191 collected from Gulf of Suez and 183 from Suez Canal. All the specimens were stored at ?20 >C immediately after collection.
Fig. 1. Map of Egypt showing the two sampling locations in the Gulf of Suez and in the Suez Canal.
Sex was established by examining the reproductive organs. A three-stage system based mainly on the development of reproductive organs and position of spermatophores or eggs (Boyle & Ngoile, 1993) was used to assess individual maturity: I- immature, II- maturing, III- mature. Tissues from the mantle of five representative specimens were preserved in 95% ethanol until DNA extraction.
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The morphometric and meristic characters analyzed were selected and modified from those recommended by Roper and Voss (1983) and those used in previous studies (Clarke, 1962, 1986; Augustyn & Grant, 1988; Boyle & Ngoile, 1993). Fourteen morphometric characters were recorded (Table 1). Measurements were made on the frozen samples with a ruler or a caliper to the nearest 1 mm. To avoid the effect of size difference, most characters were analyzed in proportion to the dorsal mantle length (DML). A total of 15 indices were calculated and used for analysis (see Table 1 for abbreviations): MC/DML, FL/DML, FW/DML, FW/FL, HL/DML, HW/DML, HW/HL, ALI/DML, ALII/DML, ALIII/DML, ALIV/DML, TL/DML, TCL/DML, FcL/DML, GW/DML. Most of these indices have been used routinely in previous morphometric studies of squids (Haefner, 1964; Bar?n & R>, 2002b). Two meristic characters were also measured; the number of denticles on the largest sucker ring on Arm R3 (NDA3) and on the tentacle club (NDTC).
Table 1. Morphological characters recorded on Uroteuthis duvauceli.
The relationship between dorsal mantle length (DML) and weight were analyzed by the power regression method, W=aDMLb. Analysis of covariance (ANCOVA) was used to test the effects of sex and location on the length>weight relationship.
Data were analyzed using an ANCOVA to investigate the presence of significant differences in morphometric indices between the two locations. The same test was used to investigate the significant difference of morphometric indices between the maturity stages for both sexes. All statistical analysis was performed using the package SYSTAT (2001).
DNA EXTRACTION, PCR AMPLIFICATION AND SEQUENCING
Total Genomic DNA of U. duvauceli samples from the Suez Canal and Gulf of Suez was extracted from a piece of 30 mg of mantle tissue according to the standard CTAB phenol-chloroform protocol described by Roger & Bendic (1988) with slight modifications. The extracted DNA was visualized on agarose gels (Sigma, M>nchen, Germany) at 1% in TBE buffer with 5 ?g/mL of ethidium bromide (Sigma, M>nchen, Germany) under ultraviolet light using a Molecular Imager Gel Doc XR System transiluminator and the software Quantity One v 4.5.2 (Bio-Rad, M>nchen, Germany). The 100-1500 bp DNA ladder (Dominion, MBL, C?rdoba, Spain) was used as molecular weight marker.
The purity and quantity of the DNA obtained was measured at 260 nm with a NanoDrop> 1000 spectrophotometer (Thermo Scientific, Bonn, Germany). The purity of DNA was determined by calculating the ratio of absorbance at 260 nm versus 280 nm.
The primers used were CEF H and CEF as designed by Santaclara et al. (2007). The amplifications were carried out in a final volume of 50 ?L containing 100 ng of DNA template, 5 ?L of 10X buffer, 2 mM MgCl2, 0.4 ?L of 100 mM dNTP, 4 ?L of a 10 ?M solution of each primer, and 1 unit of Taq-polymerase (Bioline, Luckenwalde, Germany). PCR was performed in a Bio-Rad> MyCyclerTM thermocycler. The cycles program was the following: a preheating step of 3 min at 95?C, then 35 cycles of 30 s at 95>C, 1 min at 50>C, 1 min and 30 s at 72>C, and a final extension step of 7 min at 72>C. To ensure the proper working of PCR amplification, PCR products were loaded in agarose gels (Sigma, M>nchen, Germany) at 2% in TBE buffer and 5 ?g/mL of ethidium bromide to allow band detection. The size of the amplified fragments was estimated from a 100 - 1500 bp molecular marker (Dominion-MBL, C?rdoba, Spain).
PCR products were cleaned before the sequencing reaction using the NucleoSpin Extract II (Macherey>Nagel, D>ren, Germany) kit according to the protocol of the manufacturer. PCR products were sequenced on both directions using CEQ 8800 Genetic Analysis System (Beckman Coulter, M>nchen, Germany), with the primers described previously (CEF H and CEF L) and using the CEQ Dye Terminator cycle sequencing Quick Start kit (Beckman Coulter, M>nchen, Germany), according to the recommendations of the manufacturer. Nucleotide sequences obtained were corrected with Chromas 1.45 (Mc Carthy, 1996).
Sequences obtained were aligned with Clustal W (Thompson et al. 1997) available in the BioEdit 7.0 program (Hall 1999) and manually checked. The sequences of U. duvauceli from the Suez Canal and Gulf of Suez were compared. The phylogenetic analysis was carried out with Mega 4.0 (Tamura et al. 2007) using the Tamura-Nei model (Tamura and Nei 1993) to calculate the genetic distances between sequences. Neighbor-Joining algorithm was used as model of evolution; bootstrapping analysis was used to validate the reproducibility of the branching pattern of the tree (Saitou and Nei 1987). Sequences obtained by Santaclara et al (2007) from the same region of other species belonging to Uroteuthis, Loligo, Loliolus, Lolliguncula, and Alloteuthis genera were included in the analysis (EF423052-69; EF4223074-77; EF423104-09; EF423116-23; EF423011-14; EF423128-35; EF423148-51; EF423158-61; EF423110-15).
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There was considerable variation in body size in samples between the two locations. The dorsal mantle length (DML) of squid collected from the Gulf of Suez ranged between 11.0 and 22.7 cm (14.7>2.6, n = 114) in males and between 11.3 and 17.1 cm (13.2 >1.4, n = 77) in female animals. The mantle length range of the Suez Canal squid samples was 8.4 > 26.5 cm (14.4 >4.2, n = 87) in males and 7.5>19.4 cm (12.2 >3.2, n = 96) in females. In the Gulf of Suez sample, males outnumbered females; out of a total 191 collected animals, 114 were males (59.7%) and 77 were females (40.3%). While in the Suez Canal sample, females outnumbered males out of a total 183 collected animals; 87 were males (47.5 %) and 96 were females (52.5%).
ANCOVA indicated highly significant difference (P < 0.001) between the sample collected from the Gulf of Suez and the sample collected from Suez Canal for all variables except for the indices of MC, HL, FCL and GW to dorsal mantle length (DML) and the index of HW to HL of each sex. All the indices for Gulf of Suez population are significantly higher (P < 0.001) than those of Suez Canal population (Table 2). It should be noted that the samples included individuals at different maturity stages (Table 3). In the Gulf of Suez, 89.1% of sampled males were at stage III while 64.9% of sampled females were at stage II. In the Suez Canal sample, 80.5% of sampled males were at stage III and 61.4% of sampled females were at stage II. In
Table 2. Ranges, means and standard deviations of morphometric measurement ratios in Uroteuthis duvauceli,collected from Gulf of Suez and Suez Canal. (See Table 1 for abbreviations).
Table 3 The percentage of Uroteuthis duvauceli samples for different maturity stages in the two localities
view of these differences, the result was also analyzed according to maturity stages, but for females only as more than 80% of the male sample was at stage III. The analysis was performed only for the females in maturity stages II & III, as individuals at stage I were too small to be included in the analysis (n= 4 & 2 for Gulf of Suez and Suez Canal, respectively). Between the two locations, there was no significant difference in all indices (P > 0.001) between stages II and III except for MC index (P < 0.001). MC was significantly higher in stage II (59.0%>2.0 & 58.6%>4.9 for Gulf of Suez and Suez Canal samples, respectively) than in stage III (55.5%>2.1& 49.2%>2.3 for Gulf of Suez and Suez Canal samples, respectively).
There was no significantly different (P > 0.001) for the two measured meristic characters between the squids collected from the Gulf of Suez and those collected from Suez Canal for each sex. The mean denticle number on third arm sucker rings was 8.56>0.85 (range: 8-12) for males collected from Gulf of Suez and 8.56>2.1 (range: 6-12) for males collected from Suez Canal, while the mean was 8.46>0.52 (range: 8-10) for females collected from Gulf of Suez and 8.90>1.2 (range: 6-11) for females collected from Suez Canal. The mean denticle number on tentacle club sucker rings was 18.88>2.1 (range: 11-24) for males collected from Gulf of Suez and 18.0>3.2 (range: 11-28) for males collected from Suez Canal, while the mean was 20.0>3.1 (range: 13-28) for females collected from Gulf of Suez and 19.0>3.2 (range: 10-24) for females collected from Suez Canal.
The length > weight relationship of U. duvauceli was described by the following equations:
W = 0.120 DML 2.32 r2 = 0.96 for Gulf of Suez males
W = 0.67 DML 2.59 r2 = 0.88 for Gulf of Suez females
W = 0.120 DML 2.23 r2 = 0.98 for Suez Canal males
W = 0.084 DML 2.46 r2 = 0.96 for Suez Canal females
All length-weight relationships were highly significant (P < 0.05) with r values greater than 0.80. The regression coefficient (b) of each sex in the two localities is significantly different from 3. ANCOVA revealed that the regression slopes did not differ significantly for both sexes between the two localities (P > 0.05). While the slopes for males and females in the two locations were significantly different (P < 0.05).
The sequences obtained in the present work were deposited in the NCBI database with accession numbers FJ899694 to FJ899704, consecutively (11 sequences). Lolignidae sequences reported by Santaclara et al. (2007) were included in the distance matrix for the construction of the phylogenetic tree and Todarodes sagittatus sequence was added as an out group.
All sequences belonging to the same species were grouped in the same cluster. The tree obtained had a well supported genus-level topology as all the species from the genera Loligo, Lolliguncula, Loliolus, Uroteuthis and Alloteuthis appeared well grouped in clusters. Moreover, the tree topology was in concordance with the phylogenetic and biogeographic studies reported by Anderson (2000a&b) for species belonging to the Loliginidae family using 16S rRNA and COI mitochondrial molecular markers. In the present work, the species were also grouped on basis of their geographic distribution and the following three major clades could be discerned: West Atlantic and East pacific species (L. gahi, L. opalescens, L. pealei, L. diomedeae, L. panamensis and L. bleekeri), East Atlantic species (L. reynaudi, L. vulgaris, L. forbesi, and A. subulata) and Indo-West Pacific species (L. japonica, U. duvaucelli, U. chinensis) (Figure 2). The bootstrap values of branches at level species were 99-100%, reflecting the robustness of the phylogenetic tree.
The genetic distances between the obtained cytochrome b gene sequences reveal that the intraspecific distance mean was 0.002 > 0.000, whereas the interspecific distance was two orders of magnitude higher having a value of 0.134 > 0.009. The mean interspecific distance between species of the same genus was 0.056 > 0.004. These estimates show that the intraspecific distances are very low, ranging from 0 to 0.003 in U. duvauceli from the Suez Canal and Gulf of Suez. Distance matrix scores for the Uroteuthis genus vary between 0.134 and 0.136, as shown in Table 4. When species from different genera are compared with U. duvauceli, these distance scores are higher, for example in the case of Loligo, Loliolus, Lolliguncula, Alloteuthis and Uroteuthis chinensis species where the scores range from 0.134 to 0.197.
Fig. 2. Phylogenetic tree of family Loliginidae showing the relationships among of the species, carried from the alignment of cytochrome b gene.
In the present study, two populations of the squid U. duvauceli were compared morphologically and genetically. Although the two populations were found to be genetically very similar; there was significant separation in most of their morphometric indices. The multivariate analysis of morphological differences shows clear separation between the two population (ANCOVA, R=0.1, P > 0.001). Nine morphometric indices from a total fourteen could be used to effectively distinguish the two populations. ANCOVA indicated that fin length, fin width, arm lengths, tentacle, and tentacle club length to mantle length indices, as well as fin width index to fin length were the indices that contributed most to the difference between two populations. These indices are significantly higher in Gulf of Suez population than in those at Suez Canal. While it is common for the morphometric characters of cephalopods to vary according to geographical location (Boyle & Ngoile, 1993; Carvalho & Nigmatullin, 1998), it has also been found that this variability could be insignificant among distant populations of some loliginid species (Cohen, 1976). In this study, it is unlikely that the difference in morphometric characters of U. duvauceli between two the different localities can be accounted for the differences in maturity stage composition as most samples contained individuals of similar maturity states. Most male individuals from two localities were at stage III, while most female individuals from two localities were at stage II. This inference is further supported by the observation that most of the morphometric indices for the females were not significantly different between stages II and III. Therefore, the observed difference in morphometric characters of two U. duvauceli populations is most probably due to the difference in environmental conditions and resources availability. The present study also indicated the presence of considerable variations in the body structure between the two locations. Population size in Suez Canal was relatively larger than in Gulf of Suez (maximum size 26.5 cm in mantle length for Suez Canal population while 22.7 cm for Gulf of Suez population). Geographic variations in population structure have been reported in other cephalopod species and were usually attributed to the effect of temperature on growth rate (Pierce et al., 1994; Jackson et al., 1997; Forsyth, 2004; Pecl, 2004). In the present study, it is unlikely that size differences can be accounted for the effect of water temperatures on growth rate; there is no large difference in surface seawater temperature between Gulf of Suez and Suez Canal. Summer sea-surface temperatures in the Gulf of Suez reach 30 >C (Mahmoud, 2003) compared to 29 >C for Suez Canal (Madkour et al., 2007). The possible factor to account for this difference could be variation in food abundance. On the other hand, the Gulf of Suez population was observed to have longer tentacles and arms and longer tentacular clubs compared to those from the Suez Canal. Tentacles and arms are specialized for prey capture. It has been suggested that, since tentacles and tentacular clubs are the organs which cephalopods use to capture their prey, individuals with comparatively longer clubs might capture larger prey, therefore allowing them to grow comparatively faster (Mangold-Wirz, 1963; Bello & Motolese, 1983 b). Also, long tentacles and arms may facilitate prey capture if the attack distance is longer (Bello, 1991). Longer tentacles and arms in the Gulf of Suez samples compared to those in Suez Canal are believed to be an advantage in capturing more food for growth. Longer clubs in the Gulf of Suez also might capture larger prey, therefore allowing them to grow comparatively faster. Prey availability has been reported to affect the size structure of some squid species as it influences growth (O'Dor et al., 1980; Brodziak & Macy, 1996). Consequently, it could be hypothesized that the variations in population structure and morphometric characters of the two populations observed in this study might be related to differences in environmental conditions between Gulf of Suez and Suez Canal. The first is very nutrient-limited, it has been called an oligotrophic sea (Baars et al., 1998; Mahmoud, 2003) while the Suez Canal has been called an eutrophic sea (Madkour et al., 2007).
It is possible that the difference in sex ratio between two localities could be due to type of gear rather than to population structure and the small sample sizes need to be taken into consideration. In Suez Canal U. duvauceli were sampled mainly as a by-catch of artisanal beach seine and gill net, whereas in the Gulf of Suez, squid were sampled by industrial trawling techniques.
The calculation of the length-weight relationship showed that the length exponent for weight was less than the expected cubic (b=3) relationship in the two localities, indicating that weight increases more slowly than a corresponding increase in unit length. Meanwhile, the slopes from the regression equations did not differ significantly between the two populations (ANCOVA; P > 0.05), indicating that Gulf of Suez population have the same weight of Suez Canal population at a given length.
Meanwhile, the slopes from the regression equations between the two sexes in the two locations differ significantly (ANCOVA; P < 0.05), indicating that females are heavier (b= 2.59 & 2.46 in Gulf of Suez and Suez Canal populations, respectively) than males (b= 2.32 & 2.23 in Gulf of Suez and Suez Canal populations, respectively) of the same mantle length. This agrees with similar differences observed in other loliginids (Rao, 1988; Coelho et al., 1994; Hendrickson, 2004). The larger weight of female may be related to the fact that the mass of the gonad and accessory reproductive organs constitute a greater proportion of the body mass in females than males at larger sizes, thus for a given length, a female will be heavier than male.
The number of teeth on the arm sucker ring and tentacle club was previously used to distinguish squid species (Gray, 1849; Hoyle, 1885). The number of teeth (6-12) on third arm suckers in the two populations is within the reported ranges (Roper et al., 1984; Riad, 2008). While, the number of 11-28 on tentacle club exceeds the reported ranges which ranges from 14-17 (Roper et al., 1984) and 14-17 (Riad, 2008) for the same species. Meristic characters are highly sensitive to environmental variations during their formation (Barlow, 1961) and teeth number is not a good character for species identification. In the present study the teeth number was a good diagnostic character for U. duvauceli, since there was no significant difference between the two means of teeth number on third arm and tentacle club sucker and their ranges overlap between the two populations.
The mitochondrial cytochrome b (cyt b) gene has been widely used as a molecular marker for genetic identification of a great number of species belonging to different taxa, such as anchovies (Santaclara et al., 2006a), sardines (Jerome et al., 2003), bivalves (Santaclara et al., 2006b; Espi?eira et al., 2009), hakes (Perez & Garcia-Vazquez, 2004), anglerfishes (Espi?eira et al., 2008a), gadoid (Calo-mata et al., 2003) and flatfishes (Espi?eira et al., 2008b). This gene has also been applied for the identification of cephalopod species (Anderson 2000a, 2000b; Santaclara et al., 2007).
Mitochondrial DNA has various advantages in comparison to nuclear: maternal inheritance; hundreds to thousands of copies per cell; absence of recombination; high and constant rate of evolutionary change (Mackie et al., 1999). All these characteristics allow the genetic identification of closely related species, for instance, species belonging to a concrete genus.
The set of primers CEF H and CEF L used in the current study was described by Santaclara et al. (2007). The Neighbor-Joining distance-based phylogenetic tree construction with Tamura-Nei nucleotide substitution model was applied.
The genetic evidence indicates that U. duvauceli from the Suez Canal and Gulf of Suez are one species. DNA sequence divergence in the cyt b gene was compared for U. duvauceli from these two geographic areas. In the mitochondrial sequences insufficient accumulated substitutions were found and for this reason low levels of interspecific polymorphism existed which did not permit the assignment of the samples to two different species. Thus, our phylogenetic analysis data revealed that these stocks cannot be distinguished.
In conclusion, U. duvauceli was not only the first recorded Lessepsian immigrants squid species in Suez Canal but also it has successfully established itself in the environment. The studied morphometric indices provide useful information for distinguishing the two U. duvauceli populations but did not confirm that these two populations are the same species. The difference in growth pattern between the two populations could be of adaptive significance for the resultant changes in environmental conditions. Genetic study of the mitochondrial genes of U. duvauceli in the two localities clearly reveals that they are the same species.
I am extremely thankful to Dr. John Scarpa for his valuable criticism on drafts of this paper. The authors would like to also thank Dr. Rafik Riad (National Institute of Oceanography and Fisheries, Alexandria, Egypt) for assistance with collecting squid samples from Gulf of Suez.