Phosphoglucose isomerase polymorphism in the shrimp Metapenaeus affinis

Three phenotypes of phosphoglucose isomerase (PGI) were detected in abdominal muscle extracts from the shrimp (Metapenaeus affinis) by starch-gel electrophoresis. The observed phenotypes were assumed to be under the control of two allelic genes. This assumption was supported by the observed distribution of phenotypes. There were no significant differences in the distribution of PGI phenotypes among samples of shrimp taken from Basrah waters, Iraq and from Kuwaiti waters, Arab Gulf. The three PGI alleles observed in M. affinis appear to be the same in the two localities.     

Thermal relationships between body temperature and environment conditions set upper distributional limits on oviparous species

We determined the thermal biology of the oviparous Liolaemus boulengeri and the viviparous Liolaemus lineomaculatus populations localised at high and low latitude sites in Patagonia, Argentina. We present data of body temperatures in the field (T_b) and preferred temperature in the laboratory (T_pref), micro-environmental and operative temperatures and the effectiveness of thermoregulation. Liolaemus boulengeri and L. lineomaculatus choose different heat sources for active selection of suitable thermal micro-environments for thermoregulation, and the oviparous L. boulengeri is a more effective thermoregulator (E=0.55) than the viviparous L. lineomaculatus (E=0.43). Even when L. boulengeri is a better thermoregulator and both species show identical timing in the reproductive cycles, there are constraint factors that impose limitations on the southernmost distribution of the oviparous L. boulengeri.     

Evidence for duplication and divergence of the structural gene for phosphoglucoisomerase in diploid species of clarkia

Formal genetic analysis of the mode of inheritance of the electrophoretic phenotypes for phosphoglucoisomerase (PGI) in the annual plants Clarkia rubicunda and C. xantiana showed that these diploid species have two and three genes, respectively, that specify PGI subunits. Electrophoretic examination of seven other diploid species of Clarkia revealed that species assigned to ancestral sections in the current taxonomy have two PGI genes, whereas more specialized species have three PGI genes. Together with evidence that diploid species in two closely related genera have two PGI genes, this suggests the third PGI gene arose within Clarkia. Intergenic heterodimers are formed between polypeptides specified by the third gene and one of the other PGI genes, indicating they have a high degree of structural similarity. The combined genetic, biochemical, and phylogenetic evidence suggests that the third PGI gene resulted from a process of gene duplication. The apparent Michaelis constants (F6P to G6P) of the most common electrophoretic variants of the ancestral gene in C. xantiana and in C. rubicunda are closely similar, but that of the duplicate enzyme is much higher. The intergenic heteromer has an intermediate value. Four alleles have been identified for the duplicate PGI gene in C. xantiana, including a null allele which eliminates the activity of its product. This allele is one of the few examples of a "silenced" duplicate gene. The ancestral and duplicate genes assort independently in C. xantiana. In conjunction with the substantial chromosomal rearrangements that characterize species of Clarkia, this may mean that the duplicate PGI marks a duplicated chromosomal segment that originated from a cross between partially overlapping reciprocal translocations rather than from unequal crossing over.     

用功能生态学方法揭示峨眉髭蟾蝌蚪和棘腹蛙蟾蝌蚪共存的原因

采用功能生态学的研究方法,选取了3个指标(功能丰富度、功能定位和功能生态位的重叠程度)对峨眉山清音阁黑龙江中峨眉髭蟾蝌蚪和棘腹蛙蝌蚪共存的原因进行了解析。结果表明,峨眉髭蟾蝌蚪功能丰富度的实测值远远小于棘腹蛙蝌蚪。采用自助法随机抽样15和51个个体时,峨眉髭蟾蝌蚪的功能丰富度值显著小于棘腹蛙蝌蚪。同时,非参数多元方差分析的结果显示二者的功能定位存在显著差异。此外,二者功能生态位重叠程度的实测值与采用自助法计算所得值均极低。以上结果表明二者的功能生态位存在明显分化,表现为峨眉髭蟾蝌蚪个体较大,开口较小,眼间距较大,尾部发达且多采用背腹弯曲的游泳模式;而棘腹蛙蝌蚪开口较大,眼睛间距较小,尾部较为短小且多采用脊椎弯曲的游泳模式。鉴于功能性状可以反映生物体对生长环境的响应,推测两种蝌蚪主要栖息地的微生境也存在分化。     

Hybridization could be a common phenomenon within the highly diverse lizard genus Liolaemus

Hybridization is likely to occur more often between closely related taxa that have had insufficient time to diverge to the point of reproductive incompatibility; hybridization between deeply divergent lineages is rare. In squamate reptiles, hybridization has been proposed as a possible explanation for the extensive paraphyly observed in mitochondrial gene trees in several species complexes of the South American lizard genus Liolaemus. One of the best‐documented cases is within the L. boulengeri and L. rothi complexes, which diverged ~5.5 million years ago. Here, we describe a comprehensive study for approaching the hybridization hypothesis between these lizard species complexes. We explored the level of gene tree discordance using the novel ‘extra lineage contribution’ statistics (XLC, presented in this study) that quantifies the level of gene tree discordance contribution per individual within a species. We included molecular data (12 nuclear and two mitochondrial genes) from 127 individuals, and results of a coalescent model‐based analysis show that the most likely explanation for the gene tree‐species tree discordance is interspecific hybridization. Our best‐supported hypothesis suggests current and past hybridization between L. rothi (rothi complex) and L. tehuelche (boulengeri complex), and independently between L. rothi and L. boulengeri and L. telsen (boulengeri complex). The hybrid descendants are characterized by intermediate phenotypes between the parental species, but are more similar to L. rothi in body size. We discuss the possible role of hybridization in Liolaemus evolution.     

Population genetic analysis and origin discrimination of snow crab (Chionoecetes opilio) using microsatellite markers

Major habitats for the snow crab Chionoecetes opilio are mostly found within the northwest Atlantic and North Pacific Oceans. However, the East Sea populations of C. opilio, along with its relative the red snow crab (C. japonicas), are two of the most important commercial crustacean species for fisheries on the east coast of the Korean Peninsula. The East Sea populations of C. opilio are facing declining resources due to overfishing and global climate change. Thus, an analysis of population structure is necessary for future management. Five Korean and one Russian group of C. opilio were analyzed using nine microsatellite markers that were recently developed using next-generation sequencing. No linkage disequilibrium was found between any pair of loci, indicating that the markers were independent. The number of alleles per locus varied from 4 to 18 with a mean of 12, and allelic richness per locus ranged from 4.0 to 17.1 across all populations with a mean of 9.7. The Hardy–Weinberg equilibrium test revealed significant deviation in three out of nine loci in some populations after sequential Bonferroni correction and all of them had higher expected heterozygosity than observed heterozygosity. Null alleles were presumed in four loci, which explained the homozygosity in three loci. The pairwise fixation index (F _ST ) values among the five Korean snow crab populations did not differ significantly, but all of the pairwise F _ST values between each of the Korean snow crab populations and the Russian snow crab population differed significantly. An UPGMA dendrogram revealed clear separation of the Russian snow crab population from the Korean snow crab populations. Assignment tests based on the allele distribution discriminated between Korean and Russian origins with 93 % accuracy. Therefore, the snow crab populations around the Korean Peninsula need to be managed separately from the populations in Bering Sea in global scale resource management. Also, this information can be used for identification of snow crab origin which is problematic in worldwide crab trade.     

Oceanographic characteristics of the habitat of benthic fish and invertebrates in the Beaufort Sea

We relate the spatial variability in the distribution of benthic taxa of the Beaufort Sea to oceanographic characteristics of their habitat with the goal of illustrating potential mechanisms linking climate change to Arctic marine communities. Offshore fish of the Beaufort Sea have not been surveyed since 1977 and no synchronous measures of fish distribution and the oceanographic characteristics of their habitat have been made previously. A survey was conducted during August 2008 in the western Beaufort Sea, Alaska. The distribution and abundance of benthic fish and invertebrates were assessed with standard bottom trawl survey methods. Oceanographic data were collected at each trawl station and at several locations between stations. The dominant benthic taxa, Polar cod (Boreogadus saida), eelpouts (Lycodes sp.), and snow crab (Chionoecetes opilio) were associated with cold (<?1.5°C), high salinity (>33) water found offshore of the shelf break, derived from the Chukchi Sea. These waters are expected to be high in secondary productivity, such that we hypothesize that the distribution of fish and crab was driven by conditions favorable for successful foraging. Predictions of the impacts of climate change require an understanding of the mechanisms linking the distribution and abundance of marine organisms to their oceanographic habitat. Our study documents the association of dominant benthic fish and invertebrates of the Beaufort Sea with specific water mass types and is thus a step toward this understanding.     

Spartina alterniflora invasions and effects on crab communities in a western Pacific estuary

We investigated Spartina alterniflora invasions, their relationship with shoreline dynamics and effects on crab communities in the Yellow River estuary, China, where shoreline dynamics have been accelerated due to human-mediated estuarine sediment deposition and sea-level rise. We determined the distribution of Spartina with extensive ground surveys, and quantified shoreline dynamics between 2001 and 2009 by interpreting satellite images. We used pitfall traps to sample crab populations in Spartina-invaded habitats and non-invaded mudflats in 2009 and 2010. Large areas (>0.5 km²) of Spartina plants were found at three locations in the estuary. The seaward limit of Spartina at each location generally coincided with the present shoreline, regardless of historical shoreline advance or retreat. Crab communities in Spartina-invaded habitats significantly differed from those in non-invaded habitats. The total number and biomass of crabs caught per trap were much higher in Spartina-invaded habitats than in non-invaded habitats, however, species richness and Shannon diversity were much lower. These results suggest that Spartina invasions are likely to keep pace with shoreline dynamics accelerated by global change and have significant ecological consequences for crab communities. These issues should be taken into account to improve the use and management of Spartina, especially in rapidly accreting estuaries with large-area mudflats that are important habitats but prone to Spartina invasions.     

Molecular and Biochemical Diversity Among Isolates of Radopholus spp. from Different Areas of the World

Eleven isolates of Radopholus similis from various banana-growing areas around the world and one isolate of R. bridgei from turmeric in Indonesia were compared using DNA and isoenzyme analysis. The polymerase chain reaction (PCR) was used to amplify a fragment of ribosomal DNA (rDNA), comprising the two internal transcribed spacers (ITS) and the 5.8S gene. Restriction fragment length polymorphisms (RFLPs) in this rDNA fragment were used to compare the 10 isolates. The analysis of this rDNA region revealed little variation among the isolates tested. However, data also were obtained by random amplified polymorphic DNA (RAPD) analysis of total DNA, and a hierarchical cluster analysis of these data arranged the R. similis isolates into two clusters. The first cluster consisted of isolates from Nigeria, Cameroon, Queensland, and Costa Rica; the second was comprised of isolates from Guinea, Guadeloupe, the Ivory Coast, Uganda, and Sri Lanka. The isolate of R. bridgei from turmeric in Indonesia appeared to be more divergent. This grouping was consistent with that obtained when phosphate glucose isomerase (PGI) isoenzyme patterns were used to compare the R. similis isolates. The results from both RAPD analysis and PGI isoenzyme studies indicate that two gene pools might exist within the R. similis isolates studied. No correlation could be detected between the genomic diversity as determined by RAPD analysis and either geographic distribution of the isolates or differences in their pathogenicity. The results support the hypothesis that R. similis isolates have been spread with banana-planting material.     

A De Novo Case of Floating Chromosomal Polymorphisms by Translocation in Quasipaa boulengeri (Anura,Dicroglossidae)

Very few natural polymorphisms involving interchromosomal reciprocal translocations are known in amphibians even in vertebrates. In this study, thirty three populations, including 471 individuals of the spiny frog Quasipaa boulengeri, were karyotypically examined using Giemsa stain or FISH. Five different karyomorphs were observed. The observed heteromorphism was autosomal but not sex-related, as the same heteromorphic chromosomes were found both in males and females. Our results indicated that the variant karyotypes resulted from a mutual interchange occurring between chromosomes 1 and 6. The occurrence of a nearly whole-arm translocation between chromosome no. 1 and no. 6 gave rise to a high frequency of alternate segregation and probably resulted in the maintenance of the translocation polymorphisms in a few populations. The translocation polymorphism is explained by different frequencies of segregation modes of the translocation heterozygote during meiosis. Theoretically, nine karyomorphs should be investigated, however, four expected karyotypes were not found. The absent karyomorphs may result from recessive lethal mutations, position effects, duplications and deficiencies. The phylogenetic inference proved that all populations of Q. boulengeri grouped into a monophyletic clade. The mutual translocation likely evolved just once in this species and the dispersal of the one karyomorph (type IV) can explain the chromosomal variations among populations.     

Comparison of Populations of Pratylenchus brachyurus Based on Isozyme Phenotypes

Enzymes from females of five Pratylenchus brachyurus populations and one P. scribneri population were analyzed by isoelectric focusing electrophoresis. Of the 18 enzyme systems investigated, only malate dehydrogenase (MDH), phosphoglucomutase (PGM), and phosphoglucose isomerase (PGI) were detected from all five P. brachyurus populations and P. scribneri. Faint bands were detected for isocitrate dehydrogenase and phosphogluconate dehydrogenase from one P. brachyurus population. Three distinct phenotypic groups were found in the MDH and PGM systems for P. brachyurus populations, but only a single electromorph was detected for PGI. Multiple electromorphs for MDH, PGM, and PGI were detected for P. scribneri; there was no similarity among these patterns with those from P. brachyurus. No phenotypic differences in PGI were observed between females and mixed juveniles of one population of P. brachyurus.     

Evidence for Positive Selection within the PgiC1 Locus in the Grass Festuca ovina

The dimeric metabolic enzyme phosphoglucose isomerase (PGI, EC 5.3.1.9) plays an essential role in energy production. In the grass Festuca ovina, field surveys of enzyme variation suggest that genetic variation at cytosolic PGI (PGIC) may be adaptively important. In the present study, we investigated the molecular basis of the potential adaptive significance of PGIC in F. ovina by analyzing cDNA sequence variation within the PgiC1 gene. Two, complementary, types of selection test both identified PGIC1 codon (amino acid) sites 200 and 173 as candidate targets of positive selection. Both candidate sites involve charge-changing amino acid polymorphisms. On the homology-modeled F. ovina PGIC1 3-D protein structure, the two candidate sites are located on the edge of either the inter-monomer boundary or the inter-domain cleft; examination of the homology-modeled PGIC1 structure suggests that the amino acid changes at the two candidate sites are likely to influence the inter-monomer interaction or the domain-domain packing. Biochemical studies in humans have shown that mutations at several amino acid sites that are located close to the candidate sites in F. ovina, at the inter-monomer boundary or the inter-domain cleft, can significantly change the stability and/or kinetic properties of the PGI enzyme. Molecular evolutionary studies in a wide range of other organisms suggest that PGI amino acid sites with similar locations to those of the candidate sites in F. ovina may be the targets of positive/balancing selection. Candidate sites 200 and 173 are the only sites that appear to discriminate between the two most common PGIC enzyme electromorphs in F. ovina: earlier studies suggest that these electromorphs are implicated in local adaptation to different grassland microhabitats. Our results suggest that PGIC1 sites 200 and 173 are under positive selection in F. ovina.     

Gastropod shells: A dynamic resource that helps shape benthic community structure

Empty gastropod shells are an important resource for many animals in shallow benthic marine communities. Shells provide shelter for hermit crabs, octopuses, and fishes, provide attachment substratum for hermit crab symbionts, and directly or indirectly modify hermit crab predation. Creation of an empty shell due to predation of one gastropod on another and acquisition of that shell by a hermit crab are two key events in the subsequent use of that shell. Shells of different gastropod species and the species of hermit crab acquiring them affect the symbiont complement that attaches to the shell, which in turn may affect future shell use by other symbionts. Certain shell types worn by the hermit crab, Pagurus pollicaris Say, are positively associated with the symbiotic sea anemone, Calliactis tricolor (Lesueur), which protects the hermit crab from predation by the crab, Calappa flammea (Herbst), and possibly from the octopus, Octopus joubini Robson. Shells of other species of gastropods are resistant to being crushed by the spiny lobster, Panulirusargus (Latreille). The inter-and intraspecific interactions centered on the gastropod shell are termed a “habitat web.” The potential of the shell to limit the size and distribution of animal populations demonstrates how this resource helps shape community structure.     

The effects of burrowing of Helice tridens (De Haan) on the soil of a salt-marsh habitat

The physical and chemical effects of the burrowing activity of the mud crab Helice tridens (De Haan) on the soil of a salt-marsh habitat were investigated. Soil-turnover rate caused by burrowing activity was found to be ≈ 3% of the soil from the surface to a depth of 40 cm every day during the summer. The vertical distributions of leaf and stem fragments of the salt-marsh plant Phragmites australis (Trin.) and the vertical distribution of ammonium N concentration in the soil were also investigated. At locations in the marsh where there were many large burrows, numerous leaf and stem fragments were recognized in the soil, while in areas in the marsh containing only a few small burrows these fragments were scanty. The soil depths at which leaf and stem fragments were abundant, corresponded to the depths of the burrows. These results show that mud crabs bury fallen plant fragments in the soil by their burrowing activity. Ammonium N in the soil was also abundant at locations in the marsh where there were many burrows, indicating that organic matter, such as fallen leaves and stems, may be decomposed to inorganic nutrients which are useful to the salt-marsh plants.     

Ecological Assessment of Two Species of Potamonautid Freshwater Crabs from the Eastern Highlands of Zimbabwe,with Implications for Their Conservation

The spatial ecology of freshwater crabs and their conservation status is largely understudied in Africa. An ecological assessment was conducted at 104 localities in 51 rivers and/or streams in the Eastern Highlands of Zimbabwe whereby the distribution and abundances of freshwater crab species were mapped and the possible drivers of the observed trends in population structure explored. In addition, information on crab utilisation as a food resource by local communities was assessed via face to face interviews across the region. Finally, the conservation status of each species was assessed using the IUCN Red List criteria. Only two crab species Potamonautes mutareensis and Potamonautes unispinus were recorded within the region of study. Potamonautes mutareensis was largely restricted to less impacted environments in the high mountainous river system, whereas P. unispinus was found in low laying areas. In stretches of river where both species were found to co-occur, the species were never sampled from the same site, with P. mutareensis occurring in shallower, faster flowing environments and P. unispinus in deeper, slow flowing sites. Interview results revealed that the local communities, particularly in the southern part of the Eastern Highlands around the Chipinge area, had a considerable level of utilisation (55% of households) on the harvesting of crabs for household consumption during the non-agricultural season (May to September). Results from the IUCN Red List assessment indicate that both species should be considered as “Least Concern”. Threats to freshwater crabs in the Eastern Highlands, however, include widespread anthropogenic impacts such as habitat destruction associated with gold and diamond mining, inorganic and organic pollution and possibly exploitation for human consumption. The current study provides important information and insight towards the possible development of a freshwater crab conservation action plan within the region.     

An electrophoretic study of genetic variation in populations of Yarrow's spiny lizard Sceloporus jarrovi (Sauria,Iguanidae)—I. Esterae characterization and polymorphism

• 1.1. Muscle esterase variation in Sceloporus jarrovi, sampled from 25 locations in southeastern Arizona, was investigated employing acrylamide gel electrophoresis.
• 2.2. Three distinct esterase phenotypes were observed, presumably resulting from the expression of two gene loci, Est-1 and Est-2.
• 3.3. Lizards sampled from all 25 locations were found to be monomorphic with respect to esterase encoded at Est-1. Further, lizards sampled from the Santa Rita and Pinaleno Mountains were also found to be monomorphic for esterases encoded at Est-2, whereas those sampled from the Chiricahua and Huachuca Mountains proved to be polymorphic.
• 4.4. Characterization of the esterases utilizing eserine sulfate, diisopropylfluorophosphate, and sulfhydryl-group inhibitors revealed the EST-1 isozyme to be an arylesterase and the EST-2 isozymes to be carboxylesterases.

     

Mangrove sesarmid crab feeding experiments in Peninsular Malaysia

The feeding ecology of mangrove sesarmid crabs in Peninsular Malaysia was investigated by field and laboratory experiments using four mangrove leaf species (Avicennia officinalis, Bruguiera gymnorrhiza, B. parviflora and Rhizophora apiculata) and leaves of different condition (fresh and senescent). Leaves tethered on strings at high (Bruguiera zone) and low (Rhizophora zone) intertidal positions, both upstream (Sungai Pasir) and downstream (Lower Merbok) showed significant amounts of leaf litter removal in 24 h (mean 79±3% initial dry mass). Significantly more B. gymnorrhiza was consumed in Bruguiera zones and significantly less senescent A. officinalis in the upstream Rhizophora zone. In Bruguiera zones, significant numbers of leaves were taken down burrows but there were no preferences for leaf species or condition of leaf taken down burrows at all sites. In 24 h, under laboratory conditions, the sesarmid crabs Sesarma (Perisesarma) eumolpe and S. (Perisesarma) onychophorum were offered with a mangrove species choice of either fresh or senescent leaves. There was no difference in mangrove species taken when the leaves were senescent for both crab species, but when the leaves were fresh, significantly more A. officinalis leaves were consumed by both sesarmid crab species. S. onychophorum ate significantly more B. parviflora than did S. eumolpe. The crab distribution in the field was related to the preferred tree species dominance, indicating that tree species may be important for crab species distribution, or vice versa. The mean rate of leaf consumption was not significantly different between the crab species; S. eumolpe was 29.9±5.9 and S. onychophorum was 35.3±7.2 mg dry mass per wet mass gram of crab in 24 h. Rhizophora spp. were the least preferred species in all feeding experiments, a finding which may have implications for ecosystem functioning in monoculture rehabilitation projects.     

Morphological variation of freshwater crabs Zilchiopsis collastinensis and Trichodactylus borellianus (Decapoda,Trichodactylidae) among localities from the middle Paraná River basin during different hydrological periods

Measures of hydrologic connectivity have been used extensively to describe spatial connections in riverine landscapes. Hydrologic fluctuations constitute an important macrofactor that regulates other environmental variables and can explain the distribution and abundance of organisms. We analysed morphological variations among individuals of two freshwater crab species, Zilchiopsis collastinensis and Trichodactylus borellianus, from localities of the middle Paraná River basin during two phases of the local hydrological regime. Specimens were sampled at sites (localities) of Paraná River, Saladillo Stream, Salado River and Coronda River when water levels were falling and rising. The conductivity, pH, temperature and geographical coordinates were recorded at each site. The dorsal cephalothorax of each crab was represented using 16 landmarks for Zilchiopsis collastinensis and 14 landmarks for Trichodactylus borellianus. The Canonical Variate Analyses showed differences in shape (for both species) among the crabs collected from the Paraná and Salado Rivers during the two hydrologic phases. We did not find a general distribution pattern for shape among the crab localities. During falling water, the shapes of Zilchiopsis collastinensis were not related to latitude-longitude gradient (i.e., showing greater overlap in shape), while during rising water the shapes were ordered along a distributional gradient according to geographical location. Contrary, shapes of Trichodactylus borellianus were related to latitude-longitude during falling water and were not related to distributional gradient during rising water. The cephalothorax shape showed, in general, no statistically significant covariations with environmental variables for either species. These results show that each freshwater crab species, from different localities of the middle Paraná River, remain connected; however, these connections change throughout the hydrologic regime of the floodplain system. This study was useful for delineating how the relation among shapes of crabs of localities varies during two phases of the hydrological regime and for estimating the connections and geographical patterns in the floodplain system.     

Non-Complexed Four Cascade Enzyme Mixture: Simple Purification and Synergetic Co-stabilization

Cell-free biosystems comprised of synthetic enzymatic pathways would be a promising biomanufacturing platform due to several advantages, such as high product yield, fast reaction rate, easy control and access, and so on. However, it was essential to produce (purified) enzymes at low costs and stabilize them for a long time so to decrease biocatalyst costs. We studied the stability of the four recombinant enzyme mixtures, all of which originated from thermophilic microorganisms: triosephosphate isomerase (TIM) from Thermus thermophiles, fructose bisphosphate aldolase (ALD) from Thermotoga maritima, fructose bisphosphatase (FBP) from T. maritima, and phosphoglucose isomerase (PGI) from Clostridium thermocellum. It was found that TIM and ALD were very stable at evaluated temperature so that they were purified by heat precipitation followed by gradient ammonia sulfate precipitation. In contrast, PGI was not stable enough for heat treatment. In addition, the stability of a low concentration PGI was enhanced by more than 25 times in the presence of 20 mg/L bovine serum albumin or the other three enzymes. At a practical enzyme loading of 1000 U/L for each enzyme, the half-life time of free PGI was prolong to 433 h in the presence of the other three enzymes, resulting in a great increase in the total turn-over number of PGI to 6.2×10⁹ mole of product per mole of enzyme. This study clearly suggested that the presence of other proteins had a strong synergetic effect on the stabilization of the thermolabile enzyme PGI due to in vitro macromolecular crowding effect. Also, this result could be used to explain why not all enzymes isolated from thermophilic microorganisms are stable in vitro because of a lack of the macromolecular crowding environment.