Abstract
The genus Sepiola (Cephalopoda: Sepiolidae) contains 10 known species that occur in the Mediterranean Sea today. All Sepiola species have a light organ that contains at least one of two species of luminous bacteria, Vibrio fischeri and Vibrio logei. The two Vibrio species coexist in at least four Sepiola species (S. affinis, S. intermedia, S. ligulata, and S. robusta), and their concentrations in the light organ depend on changes in certain abiotic factors, including temperature. Strains of V. fischeri grew faster in vitro and in Sepiola juveniles when they were incubated at 26°C. In contrast, strains of V. logei grew faster at 18°C in culture and in Sepiola juveniles. When aposymbiotic S. affinis or S. ligulata juveniles were inoculated with one Vibrio species, all strains of V. fischeri and V. logei were capable of infecting both squid species at the optimum growth temperatures, regardless of the squid host from which the bacteria were initially isolated. However, when two different strains of V. fischeri and V. logei were placed in direct competition with each other at either 18 or 26°C, strains of V. fischeri were present in sepiolid light organs in greater concentrations at 26°C, whereas strains of V. logei were present in greater concentrations at 18°C. In addition to the competition experiments, the ratios of the two bacterial species in adult Sepiola specimens caught throughout the season at various depths differed, and these differences were correlated with the temperature in the surrounding environment. My findings contribute additional data concerning the ecological and environmental factors that affect host-symbiont recognition and may provide insight into the evolution of animal-bacterium specificity.
The existence of symbiotic associations between eukaryotic hosts and microbial partners has long intrigued biologists (1, 8, 9). The diversity and broad range of each host-symbiont pair demonstrate how each partner has coevolved to exploit new ecological niches and to provide a new capability or function for the newly adapted individual or population (10, 25, 34). Although a number of symbiotic partnerships have evolved as parts of adaptations to particular ecological niches, there has always been the question of whether the ecology or the specificity of the partnership drives the evolutionary radiation of the hosts and their symbionts. Do environmental factors contribute to the evolution of independent populations that eventually leads to speciation? If a preference exists between a symbiont and a host, how is specificity maintained in associations in which the partnership is either promiscuous or environmentally transferred?
Studies investigating the nature of symbiotic associations are beginning to answer some of these questions and to provide clues to the underlying mechanisms of host-symbiont cospeciation (11, 15, 16, 26). One example that is shedding light on the evolutionary history of symbiotic associations is the partnership between a group of shallow-water benthic squids (family Sepiolidae) and their luminous bacterial symbionts (genus Vibrio) (22, 23). This system has provided molecular and physiological evidence for the coordinated influence of bacteria on the parallel evolution and specificity of closely related host species (20, 31). Until recently, all luminous bacterium-animal mutualisms were thought to be monotypic; i.e., it was thought that only one species of bacterium was associated with a particular host taxon (27, 33). However, Fidopiastis et al. (12) discovered a new species of luminous bacteria that resides in the light organs of several species of Mediterranean sepiolid squids (genus Sepiola), which results in a two-species consortium (21) (Vibrio fischeri and Vibrio logei). This unique and interesting finding is the first observation of two luminous species of bacteria residing in the light organs of sepiolid squids. What is peculiar about the two-symbiont relationship is that the two Vibrio species differ not only in their 16S ribosomal DNA (rDNA) genotypes but also in some of their physiological characteristics (12). Most notably, the in vitro growth rates of V. logei isolates are higher than the in vitro growth rates of V. fischeri isolates at lower temperatures (18°C), whereas at higher temperatures (26°C) V. fischeri isolates grow faster. Therefore, V. logei symbionts are psychrophilic compared to V. fischeri symbionts found in Sepiola species. There are several Mediterranean Sepiola species that live in the same coastal habitat, and this provides an opportunity to test the present state and nature of the symbiont population. Does the variety of Sepiola species have an effect on the concentration and abundance of Vibrio symbionts in the sepiolid light organs, or do ecological factors (e.g., temperature) have a greater effect on the presence and dominance of either symbiont in sepiolid light organs?
MATERIALS AND METHODS
Collection of specimens of Sepiola species and isolation of Vibrio light organ symbionts.
Specimens of sepiolid squids (Cephalopoda: Sepiolidae) (Table 1) (28) were collected from depths of 20 to 75 m within 10 km of shore near the Laboratoire Arago, Banyuls-sur-Mer, France. The water temperature and depth were recorded at the time of collection in each case. The adult Sepiola specimens were collected during May (S. affinis) and during July and September (S. affinis, S. intermedia, S. ligulata, and S. robusta) (Table 2). The mantle lengths in adult specimens ranged from 10 to 30 mm. All specimens were identified by using the criteria described by Bello (2). Squids were either flash frozen in liquid nitrogen to preserve the bacteria in the light organs or kept alive until dissection and isolation of the light organ symbionts. Once frozen, the bacterial light organ maintains the integrity of the bacteria and can be used later for culturing bacteria in future analyses (12, 29, 33).
TABLE 1.
European Sepiola speciesa
| Species | Habitat | Location | Symbiont | Strains isolated |
|---|---|---|---|---|
| Sepiola affinis Naef, 1912 | Shelf | Mediterranean Sea | V. fischeri | SA1, SA8 |
| V. logei | SA6, SA12 | |||
| Sepiola atlantica d'Orbigny, 1839 | Shelf-edge of slope | Northeast Atlantic Ocean | ? | NAb |
| Sepiola aurantiaca Jatta, 1896 | Outer shelf and upper bathyal | Southern Norway and Mediterranean Sea | ? | NA |
| Sepiola intermedia Naef, 1912 | Shelf | Mediterranean Sea | V. fischeri | SI2, SI4 |
| V. logei | SI7, SI5 | |||
| Sepiola knudseni Adam, 1984 | Inner shelf | Northwest and West Africa | ? | NA |
| Sepiola ligulata Naef, 1912 | Shelf-edge of slope | Mediterranean Sea and Adriatic Sea | V. fischeri | SL2, SL8 |
| V. logei | SL4, SL12 | |||
| Sepiola pfefferi Grimpe, 1921 | Shelf | West Africa and Mediterranean Sea | ? | NA |
| Sepiola robusta Naef, 1912 | Outer shelf | Mediterranean Sea | V. fischeri | SR5, SR10 |
| V. logei | SR1, SR18 | |||
| Sepiola rondoleti Steenstrup, 1856 | Shelf and upper bathyal | Mediterranean Sea | ? | NA |
| Sepiola steenstrupiana Levy, 1912 | Upper sublittoral | Mediterranean Sea | ? | NA |
Adapted from the data of Nesis (28) with permission from the publisher (TFH Publications, Inc.).
NA, not available or collected in this study.
TABLE 2.
Total light organ concentrations of V. fischeri and V. logei in field-caught Sepiola adults
| Squid speciesa | Depth (m) | Temp (°C) | CFU/adult light organb
|
Ratio of V. fischeri to V. logei | Month and yr caught | |
|---|---|---|---|---|---|---|
| V. fischeri | V. logei | |||||
| S. affinis (1) | 15 | 18 | 6.1 × 109 | 2.4 × 109 | 72:28 | May 1999 |
| S. affinis (1) | 20 | 22 | 2.4 × 109 | 6.3 × 108 | 79:21 | July 1995 |
| S. affinis (7) | 10 | 22 | 7.5 × 109 ± 6.6 × 108 | 9.1 × 108 ± 2.1 × 107 | 89:11 | July 1999 |
| S. affinis (2) | 20 | 18 | 5.1 × 108 ± 4.3 × 108 | 3.2 × 108 ± 1.2 × 107 | 61:39 | September 1995 |
| S. intermedia (2) | 75 | 16 | 4.8 × 108 ± 5.4 × 107 | 8.3 × 109 ± 3.4 × 109 | 6:94 | July 1999 |
| S. intermedia (2) | 60 | 12 | 8.4 × 107 ± 4.6 × 102 | 2.1 × 109 ± 1.2 × 108 | 4:96 | September 1995 |
| S. intermedia (1) | 55 | 12 | 1.0 × 109 | 3.8 × 109 | 21:79 | September 1998 |
| S. ligulata (1) | 40 | 16 | 2.1 × 1010 | 7.9 × 1010 | 21:79 | July 1999 |
| S. ligulata (3) | 55 | 10 | 7.1 × 1010 ± 3.2 × 107 | 1.8 × 1011 ± 2.9 × 106 | 28:72 | September 1998 |
| S. robusta (1) | 60 | 12 | 5.1 × 109 | 4.2 × 1010 | 11:89 | July 1995 |
| S. robusta (2) | 60 | 10 | 5.4 × 109 ± 1.5 × 109 | 7.1 × 1010 ± 4.5 × 109 | 7:93 | September 1998 |
The numbers in parentheses are the numbers of adult animals used to calculate bacterial concentrations.
When more than one animal was used to calculate concentrations, the values are means ± standard deviations.
Once animals were collected, individual strains of V. fischeri and V. logei were isolated from adult specimens of S. affinis, S. intermedia, S. ligulata, and S. robusta as previously described (5, 12) (Table 1). For live specimens, each squid was anesthetized, and the light organ was removed and subsequently homogenized in sterile seawater. Aliquots of the homogenate were spread onto seawater-tryptone-yeast extract (SWT) agar medium (5), and duplicates were incubated at 18 and 26°C overnight. Frozen light organs were also homogenized in this manner, and diluted homogenates were spread onto SWT media to determine the concentrations of individual Vibrio species. Both V. fischeri and V. logei strains were isolated from the preparations incubated at 18 and 26°C, and all strains used in the competition experiments were isolated at the same temperature so that the relative growth abilities of the strains were not biased. Once organisms were isolated, golden yellow colonies were observed on all plates, and differences in colony size were observed for some of the isolates.
Chelex DNA isolation methods for the Vibrio symbionts were used to obtain templates for PCR amplification (31). Briefly, DNA from an individual isolate was extracted by placing a single colony in 200 μl of 20 mM Trisbase–0.05 mM EDTA buffer (pH 7.4) containing 5% (wt/vol) Chelex 100 resin (Bio-Rad Laboratories, Richmond, Calif.). The cells were homogenized with an autoclaved pestle to prevent contamination, and the homogenate was incubated at 80°C for 25 min and then boiled for 10 min to denature proteins and lyse the cells. The cell debris and Chelex 100 were pelleted by centrifugation, and the supernatant fluid containing the DNA was used as the bacterial DNA template for PCR. The species identities of individual Vibrio colonies were confirmed by PCR amplification of a region of the 16S rDNA (designated region V1) that differentiates V. fischeri from V. logei (12). The PCR products were visualized by ethidium bromide staining by using a UV illuminator, and size differences were determined by using the amplified bands (V. fischeri, 121 bp; V. logei, 111 bp). Once identified, individual strains were isolated and stored for use in infection and competition studies. The remaining colonies were used for colony lifts for identification of V. fischeri and V. logei strains.
Growth studies.
All of the bacterial strains used in this study were inoculated into 5-ml starter cultures, which were then transferred into flasks containing 50 ml of SWT broth so that the final optical density at 600 nm was approximately 0.01 (5 × 107 cells/ml). The cultures were shaken at 225 rpm and were grown at either 18 or 26°C. Optical density was measured periodically throughout the logarithmic growth phase. Each strain of V. fischeri or V. logei used in this study was isolated from a Sepiola adult obtained from the Mediterranean Sea (Table 1) and was identified by using the methods of Fidopiastis et al. (12) and the methods described above.
Competition and infection experiments.
To determine whether temperature has a direct effect on colonization potential and symbiont composition, several symbiotic strains were analyzed to determine their competitive abilities to infect light organs of juvenile Sepiola squids (S. affinis and S. ligulata) by using a standard colonization method (19, 32). These isolates were used to test whether the species of host determined the symbiotic composition or whether temperature was a significant factor in establishing symbiotic competence. Newly hatched S. affinis or S. ligulata juveniles were placed in vials containing 5 ml of seawater that was inoculated with 103 CFU of either one strain or, in competition experiments, two strains of symbiotically competent bacteria. The bacteria and squids were incubated at 18 and 26°C. After 12 h of incubation, the juvenile squids were transferred to vials containing 5 ml of seawater without symbiotic bacteria. Since bacterial cells from light organ homogenates have 100% plating efficiency (32), the actual extent of colonization could be calculated from the number of CFU arising from aliquots of light organ homogenates that were plated onto seawater nutrient agar medium (5, 6). After 48 h of incubation, juvenile squids were homogenized, and dilutions of the homogenates were plated onto seawater-tryptone agar to determine the number of Vibrio cells resulting from either single-strain infections or two-strain competition infections (19). The relative abundance of each strain (inter- or intraspecies) in a light organ was determined by either visual luminescence of the CFU (30, 31) or by probing for the V1 variable region of the 16S rDNA (12) by using direct colony lifts of the plated light organ homogenates (18). The V1 region probe was labeled by tailing oligonucleotide primers (digoxigenin-dUTP; Boehringer Mannheim) and was hybridized to the colony blots at 58°C overnight. The blots were washed under stringent conditions (15 mM NaCl, 0.1 mM EDTA, 1% sodium dodecyl sulfate; 60°C), and colonies were detected with the fluorescent substrate Vistra ECF (Amersham). All imaging in this study was performed with a Molecular Dynamics model Storm 860 PhosphorImager. The percentages in each competition experiment were calculated for each juvenile squid (30 squids/competition experiment), and the values were averaged and arcsine transformed to test for significance (36).
Determination of symbiont composition in wild Sepiola specimens.
Adult Sepiola specimens were collected at various times throughout the year (Table 2), and the light organ composition of each specimen was examined. All adult specimens were identified by using morphological characteristics, as described by Bello (2). The adult light organs and ink sac were dissected out of the body cavity of each squid and frozen in liquid nitrogen until proper analysis could be completed at the home institution. After freezing, each adult light organ was placed in sterile seawater or SWT medium and homogenized. Serial dilutions were made in order to obtain a reasonable number of CFU to plate onto SWT agar. The light organ homogenates were incubated at both 18 and 26°C to determine whether temperature had a pronounced effect on the relative abundance of each Vibrio species. Individual CFU were isolated from the homogenates, and colony lifts were used for analysis with the V1 region 16S rDNA probe in order to distinguish between V. fischeri and V. logei from individual light organs (as in the competition assays). In addition to the V1 region 16S rDNA probe analysis, phenotypic typing of either V. fischeri or V. logei strains was accomplished by using the protocol described by Nishiguchi et al. (30).
RESULTS
Comparison of populations of V. fischeri and V. logei in adult sepiolids.
An analysis of the adult light organ contents of four Sepiola species revealed that both Vibrio species were present in all of the sepiolid species examined (Table 2). The light organ populations of all S. affinis specimens were primarily composed of V. fischeri, independent of depth or temperature. The concentrations of V. fischeri in S. affinis squids ranged from 98 to 99% of the total light organ isolates. The water temperatures at the time of collection for all of the S. affinis adults ranged from 18 to 22°C, and individuals were collected at depths of 20 m or less (Table 2). For all other species of squids (S. intermedia, S. ligulata, and S. robusta), the majority of the symbiotic bacteria found in the light organs were V. logei (Table 2). The V. logei strains isolated from these species of squids accounted for between 95 and 99% of the total light organ contents during all collection periods (May to September). These squid species were collected at depths ranging from 40 to 75 m, and the temperatures ranged from 10 to 16°C. During the collecting season, the surface temperatures ranged from 24°C at the height of summer to 10°C in the winter months. Although summer sea surface temperatures are much higher than winter surface temperatures, a distinct thermocline was formed between June and September in all years that samples were obtained. At this time, the water temperatures at depths below 25 m ranged from 12 to 16°C.
Growth of V. logei and V. fischeri in vitro.
Table 1 shows all of the host squids and the bacterial strains isolated and used in this study. Each strain of V. fischeri or V. logei was tested for growth at two different temperatures (18 and 26°C) in SWT medium. Due to the relative psychrophily of V. logei (12), the growth constants were expected to be higher for V. logei at colder temperatures (<20°C) and higher for V. fischeri at warmer temperatures (>22°C). The growth constants for all strains of V. fischeri ranged from 0.4 to 0.6 h−1 at 18°C, whereas the growth constants were between 0.8 and 1.3 h−1 for the same strains grown at 26°C (Table 3). The V. logei strains used in this study had optimal growth constants that ranged from 0.9 to 1.4 h−1 at 18°C and from 0.7 to 1.0 h−1 at 26°C in culture. All strains were initially isolated from different species of squid hosts on SWT agar at 18 and 26°C in order to avoid any bias which favored specific temperature-acclimated strains.
TABLE 3.
Growth constants for V. fischeri and V. logei strains isolated from European Sepiola species
| Bacterial strain | Species | Host squid | Growth rate constant at 18°C (h−1) | Growth rate constant at 26°C (h−1) |
|---|---|---|---|---|
| SA1 | V. fischeri | S. affinis | 0.6 | 0.8 |
| SA8 | V. fischeri | S. affinis | 0.6 | 1.0 |
| SA6 | V. logei | S. affinis | 0.9 | 0.7 |
| SA12 | V. logei | S. affinis | 0.8 | 0.7 |
| SL2 | V. fischeri | S. ligulata | 0.4 | 1.3 |
| SL8 | V. fischeri | S. ligulata | 0.5 | 1.3 |
| SL4 | V. logei | S. ligulata | 1.2 | 0.9 |
| SL12 | V. logei | S. ligulata | 1.2 | 0.9 |
| SI2 | V. fischeri | S. intermedia | 0.6 | 1.1 |
| SI4 | V. fischeri | S. intermedia | 0.6 | 1.0 |
| SI5 | V. logei | S. intermedia | 0.9 | 0.7 |
| SI7 | V. logei | S. intermedia | 0.9 | 0.7 |
| SR5 | V. fischeri | S. robusta | 0.4 | 0.9 |
| SR10 | V. fischeri | S. robusta | 0.5 | 0.9 |
| SR1 | V. logei | S. robusta | 1.4 | 1.0 |
| SR18 | V. logei | S. robusta | 1.3 | 0.9 |
Host specificity of Vibrio strains.
In addition to the individual tests of bacterial strains in situ, I performed experiments to determine whether strains isolated from one species of squid were specific to their native host and if host specificity provided a competitive advantage when the native squid species or a nonnative species was infected. No differences in infection competency were observed between S. affinis and S. ligulata juveniles with V. fischeri native strains SA1, SA8, SL2, and SL8 at either 26 or 18°C (Table 4). V. fischeri strains isolated from S. intermedia or S. robusta squids (designated SI and SR strains) did not exhibit any preference for either of the two nonnative juvenile squid hosts at these temperatures (Table 4). Similarly, no significant differences in specificity were observed with S. affinis and S. ligulata juveniles when they were infected with the native strains V. logei SA6, SA12, SL4, and SL12 at 26 or 18°C (Table 5). Again, V. logei strains from S. intermedia and S. robusta (SI5, SI7, SR1, and SR18) did not exhibit any specificity for juveniles of either of the two squid species examined (Table 5).
TABLE 4.
Infection of Sepiola juveniles with V. fischeri strains
| Temp (°C)a | V. fischeri strain | Concn in squid host species (105 CFU/squid) after 48 h
|
|
|---|---|---|---|
| S. affinis | S. ligulata | ||
| 26 | SA1 | 4.1 | 4.5 |
| SA8 | 4.0 | 4.0 | |
| SL2 | 4.1 | 3.9 | |
| SL8 | 3.7 | 3.9 | |
| SI2 | 4.4 | 4.0 | |
| SI4 | 4.2 | 4.1 | |
| SR5 | 3.8 | 4.2 | |
| SR10 | 3.9 | 3.9 | |
| 18 | SA1 | 0.24 | 0.20 |
| SA8 | 0.31 | 0.35 | |
| SL2 | 0.30 | 0.34 | |
| SL8 | 0.31 | 0.34 | |
| SI2 | 0.28 | 0.25 | |
| SI4 | 0.31 | 0.33 | |
| SR5 | 0.19 | 0.22 | |
| SR10 | 0.24 | 0.23 | |
Preparations were incubated at 18 or 26°C.
TABLE 5.
Infection of Sepiola juveniles with V. logei strains
| Temp (°C)a | V. logei strain | Concn in squid host species (105 CFU/squid) after 48 h
|
|
|---|---|---|---|
| S. affinis | S. ligulata | ||
| 26 | SA6 | 0.09 | 0.07 |
| SA12 | 0.09 | 0.09 | |
| SL4 | 0.08 | 0.07 | |
| SL12 | 0.07 | 0.09 | |
| SI5 | 0.07 | 0.07 | |
| SI7 | 0.06 | 0.08 | |
| SR1 | 0.07 | 0.08 | |
| SR18 | 0.07 | 0.06 | |
| 18 | SA6 | 2.2 | 2.1 |
| SA12 | 2.4 | 2.3 | |
| SL4 | 1.8 | 1.7 | |
| SL12 | 1.9 | 1.7 | |
| SI5 | 1.7 | 1.5 | |
| SI7 | 1.6 | 1.9 | |
| SR1 | 2.0 | 1.8 | |
| SR18 | 2.0 | 1.9 | |
Preparations were incubated at 18 or 26°C.
In addition to strain specificity, all V. fischeri and V. logei strains were tested to determine their infectivity at 18 and 26°C (Tables 4 and 5) at the end of a 48-h incubation period. All of the V. fischeri strains inoculated and incubated at 26°C infected both S. affinis and S. ligulata juveniles at concentrations at least 10-fold higher than the concentrations of the strains inoculated and incubated at 18°C (Table 4). No significant differences were observed with the V. fischeri strains examined at the same temperature (Table 4). Conversely, V. logei strains inoculated and incubated at 26°C were present at concentrations that were approximately 10-fold lower than the concentrations of the strains tested at 18°C (Table 5). Again, no differences were observed with V. logei strains tested at the same temperature (18 or 26°C) (Table 5). V. fischeri strains from S. intermedia and S. robusta were also tested at different temperatures with nonnative host squid species (Table 4), and no difference was observed between these strains and the native strains tested at the same temperature (either 18 or 26°C). Similarly, no differences were observed for V. logei strains isolated from S. intermedia or S. robusta when they were measured at the temperatures used for strains obtained from S. affinis or S. ligulata (Table 5).
Competition within and between V. fischeri and V. logei strains in Sepiola juveniles.
Since no intraspecies differences in growth and infection were observed for the V. fischeri or V. logei strains tested, only one representative strain was used for the following competition experiments. V. fischeri native strain SA1 isolated from S. affinis was used in competition experiments with all of the other V. fischeri strains (SL8, SI2, SR5) at 26°C, and no discernable preference was observed among the V. fischeri strains examined (Table 6). Similarly, V. fischeri native strain SL8 from S. ligulata was not preferred over other nonnative strains when it was used in competition experiments performed with S. ligulata juveniles (Table 6). V. logei native strains SA6 and SL12 isolated from S. affinis and S. ligulata, respectively, were also tested at 18°C and exhibited no preference for either squid species during competition with all other V. logei strains used in this study (Table 7). None of the SI and SR strains exhibited specificity for either S. affinis or S. ligulata juveniles when competition experiments were performed at 26 or 18°C.
TABLE 6.
Competition between isolates of V. fischeri in Sepiola juveniles, as determined at 26°Ca
| V. fischeri strains | Ratio of bacterial strains in squid host species
|
|
|---|---|---|
| S. affinis | S. ligulata | |
| SA1 and SL8 | 49:51 | 58:42 |
| SA1 and SI2 | 51:49 | 44:56 |
| SA1 and SR5 | 52:48 | 50:50 |
| SA1 and SA8 | 56:44 | 49:51 |
| SL8 and SR5 | 47:53 | 51:59 |
| SL8 and SI2 | 44:56 | 55:45 |
| SL8 and SL2 | 50:50 | 54:46 |
| SI2 and SR5 | 49:51 | 43:57 |
The results of individual competition experiments are average percentages based on 30 individuals. All percentages were arcsine transformed by methods described by Sokal and Rohlf (36).
TABLE 7.
Competition between isolates of V. logei in Sepiola juveniles, as determined at 18°Ca
| V. logei strains | Ratio of bacterial strains in squid host species
|
|
|---|---|---|
| S. affinis | S. ligulata | |
| SA6 and SL12 | 44:56 | 50:50 |
| SA6 and SI7 | 54:46 | 48:52 |
| SA6 and SR1 | 50:50 | 52:48 |
| SA6 and SA12 | 49:51 | 53:47 |
| SL12 and SR1 | 66:44 | 49:51 |
| SL12 and SI7 | 56:44 | 50:50 |
| SL12 and SL4 | 45:55 | 53:47 |
| SI7 and SR1 | 45:65 | 43:57 |
The results of individual competition experiments are average percentages based on 30 individuals. All percentages were arcsine transformed by methods described by Sokal and Rohlf (36).
In competition experiments performed at 26°C, all juveniles, regardless of species, were infected primarily with V. fischeri strains regardless of the species from which they were isolated (Table 8). The majority of V. fischeri colonies were much larger than the V. logei colonies arising from the homogenized juvenile light organs. The V. fischeri in both S. affinis and S. ligulata juvenile light organs accounted for 78 to 95% of the total light organ population in squids incubated at 26°C (Table 8). Conversely, all light organ competition experiments performed at 18°C resulted in colonization primarily by V. logei isolates from all four squid species sampled (Table 8). In these competition experiments, V. logei symbionts accounted for 77 to 96% of the total light organ population (Table 8). The colonies were primarily the same size, and only colony blots or visibly luminous colony morphology (30) could indicate the difference between V. logei and V. fischeri symbionts.
TABLE 8.
Effect of temperature on the ratio of V. fischeri cells to V. logei cells present in juvenile Sepiola light organs after 48 h
| Competition assay temp (°C) | Bacterial strains
|
Ratio of V. fischeri strain to V. logei strain in squid host speciesa
|
||
|---|---|---|---|---|
| V. fischeri strain | V. logei strain | S. affinis | S. ligulata | |
| 26 | SA1 | SA6 | 85:15 | 91:9 |
| SL8 | SL12 | 89:11 | 95:5 | |
| SI2 | SI7 | 88:12 | 80:20 | |
| SR5 | SR1 | 82:18 | 78:22 | |
| 18 | SA1 | SA6 | 21:79 | 14:86 |
| SL8 | SL12 | 4:96 | 8:92 | |
| SI2 | SI7 | 10:90 | 12:88 | |
| SR5 | SR1 | 15:85 | 23:77 | |
All percentages are averages based on data from 30 juvenile squids for each competition experiment. The percentages were arcsine transformed (36) to test for significance in each competition experiment.
DISCUSSION
Previous studies of luminous bacterium-squid symbioses showed that host light organs were monotypic (22). Until recently, no other species of luminous bacteria were known to exist in members of the Sepiolidae. Recently, Fidopiastis et al. (12) reported that a second Vibrio species is present only in Mediterranean species of sepiolids. This finding suggests that luminous bacterium-squid mutualisms are not species specific but may depend on environmental factors. In this study I investigated the abiotic factors that affect this two-symbiont association and whether temperature influences the degree to which different symbiotic vibrios infect sepiolid squid hosts.
Sepiolid light organ pores are continually open to the surrounding seawater, which potentially provides access for any type of bacterium in the squid habitat. The presence of only V. fischeri or V. logei in the Sepiola light organ shows that there are strong species-specific interactions between these bacteria and their hosts. In the related genus Euprymna, the numbers of bacterial cells are controlled in part by diel venting of approximately 90% of bacteria through the lateral pores of the light organ into the mantle cavity (5, 6). This venting behavior results in a sufficiently high density of symbiotically competent Vibrio cells in the water column to promote colonization of the next generation of juvenile squids (19). Thus, symbiotically competent bacteria that are present in the environment can influence the composition of the squid light organ, particularly if more than one species is present. Strict species recognition does not occur for V. fischeri or V. logei in Mediterranean Sepiola squids, and it appears that one of the more influential factors determining light organ composition is the temperature at which the host is infected and persists. In previous studies to investigate the influence of temperature acclimation in Escherichia coli (3, 24), workers have shown that the genetics of a particular strain has no discernable effect on adaptation to a different environment but does influence the fitness of the strain (4). One may conclude that the sepiolid symbiosis is species specific (V. fischeri and/or V. logei), but the variability in light organ composition and the ability of both symbiont species to infect different host squids at different temperatures indicate how environmental factors can influence the distribution and population dynamics of symbiont colonization.
The degree of relatedness and diversity among numerous symbiotic taxa has traditionally been thought of as a good predictor of cospeciation (17). However, recently, a number of examples have demonstrated that the systematics underlying symbiosis is not as clear as we have traditionally thought (1, 10, 11, 15, 16, 25, 31). Changes in the host association with a particular symbiont may be related to selection pressure on the host's fitness and how the symbiont may or may not affect future generations of host-symbiont pairs (13, 35). Population size and the availability of hosts that can be infected also affect the pairing of host-symbiont associations and possibly the specificity that a mutualism eventually expresses (7, 37). The presence of two species of symbionts that can both infect different host species may be an initial step in establishing a monotypic host-symbiont association. However, how the natural balance between host fitness and symbiont competence is established remains to be determined (14). Because there are several sympatric Sepiola host species living in the area sampled, the possibility of host switching between squids and the establishment of host-symbiont specificity can be studied. In future experiments researchers should examine the degree to which ecological and/or genetic factors control patterns of cospeciation and evolution and how we can better predict which patterns arise from these factors. Whether the symbionts determine new avenues for host evolution and radiation into different ecological habitats in this family of squids is just one of the many questions that should be pursued in future studies.
ACKNOWLEDGMENTS
I thank S. von Boletzky for help with collecting Sepiola adults and egg masses at Laboratoire Arago, Banyuls-sur-mer, France, and for providing insight and knowledge of the Mediterranean Sepiolidae. I also thank J. L. Botsford, D. J. Howard, and J. Randall for reviewing the manuscript prior to submission and M. J. McFall-Ngai and E. G. Ruby of the University of Hawaii for providing insight and supporting my work on the sepiolid-Vibrio symbiosis. I thank C. L. Lickliter of my laboratory at New Mexico State University for providing bacterial growth data.
This work was supported by a summer research grant from the Arts and Sciences research center at New Mexico State University and by the University Research Expeditions Program (UREP), University of California.
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