Breeding ecology and evidence of reproductive isolation of a widespread stickleback fish (Gasterosteidae) in Nova Scotia, Canada

A stickleback with brilliant white dorsal breeding colours is widely distributed in north-eastern Nova Scotia, Canada, where it often breeds sympatrically with the threespine stickleback, Gasterosteus aculeatus. Breeding males are highly conspicuous and visible at distances of 20 m or more whereas sympatric G. aculeatus are cryptic and difficult to detect even at 2 m. The white stickleback nests only above the substrate in filamentous algae, where G. aculeatus nests only on the substrate. The white stickleback is smaller in size and more terete than G. aculeatus , but it is morphologically similar in having a complete row of lateral plates and similar lateral plate and gill raker numbers. The white stickleback occurs only in environments where there are filamentous algae (which appears to be an obligatory nesting substrate) and where the water is clear, saline and relatively still. Female choice tests in the laboratory show that the white stickleback is reproductively isolated from G. aculeatus , and field observations on natural spawning support this conclusion. We suggest that the bright breeding colouration may have evolved through sexual selection and/or to advertise unprofitability to predators.     

Environmental control of salinity preferences in four sympatric species of sticklebacks: Gasterosteus aculeatus, Gasterosteus wheatlandi, Pungitius pungitius and Apeltes quadracus

Salinity preferences of four sympatric species of sticklebacks ( Gasterosteus aculeatus, Gasterosteus wheatlandi, Pungitius pungitius, Apelles quadracus ) were studied after acclimation to different regimes of photoperiod and temperature. The results indicate that photoperiod and temperature influence salinity preferences of G. aculeatus, G. wheatlandi and A. quadracus but that they are virtually without effect in the case of P. pungitius. Moreover, the overall photoperiod effects on salinity preferences are in accordance with the migratory movements of G. aculeatus and A. quadracus. The effect of temperature was more difficult to analyse because of its unnatural timing. It is clear, however, that temperature is involved in the regulation of salinity preferences of G. aculeatus and perhaps of G. wheatlandi. The relative importance of external factors in the adjustment of salinity preferences differed for the four species. However, the results suggest that for G. aculeatus and A. quadracus salinity preferences are an important component of the orientation mechanisms used during their seasonal migrations.     

Development of an efficient production method for beta-mannosidase by the creation of an overexpression system in Aspergillus aculeatus

AIM: To develop an overexpression system in Aspergillus aculeatus in order to establish an efficient overproduction method of beta-mannosidase (MANB). METHODS AND RESULTS: An overexpression plasmid for the manB gene, encoding A. aculeatus MANB, was constructed and introduced into A. aculeatus cells. The gene was overexpressed under an improved promoter containing 12 copies of Region III cis-elements of Aspergillus oryzae in the transformant, and it secreted 2.56 mg MANB ml(-1) in liquid culture, which obtained a 9.4-fold higher productivity than that achieved in an overexpression system in A. oryzae. Most of the secreted protein in the cultured medium of the transformed A. aculeatus was the overproduced enzyme. CONCLUSIONS: Aspergillus aculeatus with the introduced overexpression plasmid produced 2.56 mg MANB ml(-1) in cultured medium. The improved promoter with A. oryzae Region III functioned in A. aculeatus; thus the strain is an expectant host for recombinant protein productions. SIGNIFICANCE AND IMPACT OF THE STUDY: The overexpression system with the improved promoter in A. aculeatus brought the highest productivity of MANB reported to date. The expression system would be a strong bioindustrial tool for protein production.     

Transformation of Aspergillus aculeatus using the drug resistance gene of Aspergillus oryzae and the pyrG gene of Aspergillus nidulans

Transformation systems for Aspergillus aculeatus has been developed, based on the use of the pyrithiamine resistance gene of Aspergillus oryzae and the orotidine-5'-monophosphate decarboxylase gene (pyrG) of Aspergillus nidulans. An A. aculeatus mutant which can be transformed effectively by the A. nidulans pyrG gene was isolated as a transformation host. This is the first report of transformation of A. aculeatus.     

The isolation and amino acid sequences of echidna (Tachyglossus aculeatus) milk lysozyme I and II.

Comparative studies of monotreme proteins are of particular value in gaining an understanding of the origin of mammals and their interrelationships. The presence of two lysozyme variants, echidna lysozyme I and II, has been confirmed in mature milk samples of Tachyglossus aculeatus multiaculeatus and Tachyglossus aculeatus aculeatus respectively. A simplified procedure is described for their isolation. Their amino acid sequences, the first determined for a monotreme secretory protein, are unusual. They are shown to be c-type lysozymes, each consisting of a single chain of 125 residues (terminating at Cys 125). The only other known c-type lysozyme with this termination is that of pigeon eggwhite. Echidna lysozyme is unique in having no Cys at position 6, but at position 9. It has precisely the residues relevant to the binding of Ca(II), and most of the residues implicated in the galactosyl transferase modifier action of alpha-lactalbumin. However, the weak modifier action previously observed for variant I, prepared by a different method, was not found for the present preparation. The evolutionary significance of the results is discussed.     

Combined molecular and biochemical approach identifies Aspergillus japonicus and Aspergillus aculeatus as two species

We examined nine Aspergillus japonicus isolates and 10 Aspergillus aculeatus isolates by using molecular and biochemical markers, including DNA sequences of the ITS1-5.8S rRNA gene-ITS2 region, restriction fragment length polymorphisms (RFLP), and secondary-metabolite profiles. The DNA sequence of the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene could not be used to distinguish between A. japonicus and A. aculeatus but did show that these two taxa are more closely related to each other than to other species of black aspergilli. Aspergillus niger pyruvate kinase (pkiA) and pectin lyase A (pelA) and Agaricus bisporus 28S rRNA genes, which were used as probes in the RFLP analysis, revealed clear polymorphism between these two taxa. The A. niger pkiA and pelA probes placed six strains in an A. japonicus group and 12 isolates in an A. aculeatus group, which exhibited intraspecific variation when they were probed with the pelA gene. The secondary-metabolite profiles supported division of the isolates into the two species and differed from those of other black aspergilli. The strains classified as A. japonicus produced indole alkaloids and a polar metabolite, while the A. aculeatus isolates produced neoxaline, okaramins, paraherquamidelike compounds, and secalonic acid. A. aculeatus CBS 114.80 showed specific RFLP patterns for all loci examined. The secondary-metabolite profile of strain CBS 114.80 also differed from those of A. japonicus and A. aculeatus. Therefore, this strain probably represents a third taxon. This study provides unambiguous criteria for establishing the taxonomic positions of isolates of black aspergilli, which are important in relation to industrial use and legal protection of these organisms.     

Genetic differentiation between the freshwater subspecies of Gasterosteus aculeatus in Southern California

Samples of Gasterosteus aculeatus microcephalus and the endangered G. aculeatus williamsoni were obtained from downstream and headwaters regions, respectively, of the Santa Clara River system in southern California and electrophoretically examined for the products of 45 loci, six of which were polymorphic. Significant departures from Hardy-Weinberg expectations were evident in genotypic distributions at four of the polymorphic loci indicating a severe restriction of gene flow within the drainage. The genetic distance between these two subspecies was calculated as D = 0.034 ± 0.029, a value comparable to that reported between G. aculeatus microcephalus and G. aculeatus aculeatus from different drainages in northern California.     

Characterization and evolutionary relevance of the sperm nuclear basic proteins from stickleback fish

We have characterized the sperm nuclear basic proteins (SNBPs) of the sticklebacks in the suborder Gasterosteoidei. The complete amino acid sequence of the protamines from Aulorhynchus flavidus, Pungitius pungitius, Gasterosteus aculeatus, (anadromous) and G. wheatlandi, as well as the sequences of the protamines of several species pairs of freshwater G. aculeatus, have been determined. Analysis of the primary structure of these proteins has shown that: a) despite the relatively low amino acid complexity and small molecular mass of these basic proteins, they are very good molecular markers at the generic level. The bootstrap parsimony analysis using their sequences provides a phylogenetic relationship for the old anadromous species of Gasterosteoidei which is identical to that obtained from morphological and behavioral analysis; b) the comparison of the sequences also suggests that protamines from the suborder Gasterosteoidei have most likely evolved from a common gene in the early Acanthopterygii by an extension of the carboxy terminal portion of the molecule; c) protamines are not good markers for recent postglacial freshwater isolates of G. aculeatus. However, in the unique case of Enos Lake (British Columbia), we have been able to detect an additional minor protamine component in the benthic forms of G. aculeatus that is not present in the limnetic forms. Thus, this new protamine must have appeared during the past 12,000 years concomitantly with the speciation of benthics and limnetics in this lake.     

Pollination failure in mediterranean Ruscus aculeatus L.

The reproductive biology of Ruscus aculeatus L. was studied at a mediterranean site during 1996–1999. Field observations showed that reproductive success was very low, because only 3% of the marked flowers developed fruits in 1996 and none in 1997 and 1998. Several experiments were performed to investigate the reasons for reproductive failure in this species. Pollen viability was on average 85%, showing that reproductive failure is not due to poor pollen quality. Two hand-pollination experiments showed that all flowers are potentially able to develop fruits throughout the flowering period, suggesting that no aberration occurs in sporogencsis, fertilization or post-zygote processes in R. aculeatus and that low reproductive success in the field occurs because of lack of pollination. This hypothesis was tested by looking for pollen on the stigmatic surface of randomly sampled flowers, but without success. Moreover, the bodies of insects trapped in the proximity of the female plants were carefully checked for pollen but none was found. The number of pollen grains produced per flower is lower than that reported for wind-pollinated species. Nevertheless, we showed with empirical probes that the possibility of wind pollination cannot be ruled out. The breeding system and reproductive characteristics of R. aculeatus seem to match the traits of the sclerophyllous taxa remnants of the tropical Tertiary flora. Moreover, the climatic fluctuation of the Quaternary and the establishment of the mediterranean climate might have changed the conditions of a good pollination process. At present, the low sexual reproduction of R. aculeatus is due to the lack of pollen transport. Within a population of R. aculeatus , genetic variability is revealed by seeds from the few developed fruits which have been shown to be viable. However, maintenance of the individual plants in the population relies on the resprouting capacity of the species in question.     

刺鳅X染色体DNA文库的构建

刺鳅（Mastacembelus aculeatus）是具有明显X和Y异形性染色体分化的淡水鱼。本实验室通过显微切割（Microdissection）和兼并引物PCR（DOP-PCR）方法,从雌性刺鳅中期染色体分裂相中分离获得X染色体并扩增其DNA,利用T载体和电转化方法,建立了刺鳅X染色体DNA质粒文库。该文库插入片段的平均长度约为500bp,理论上包含X染色体98％以上的序列。当用荧光原位杂交（FISH）来验证文库的专一性时发现,在无竞争性DNA杂交条件下,整个X和Y染色体上都表现出强烈的杂交信号,并且常染色体上也出现一些随机散布信号;当含有竞争性DNA时,常染色体上的信号消失,仅性染色体上异染色质区域保留有较强信号。就此,本文对刺鳅性染色体上的序列类型进行了探讨。     

DNA sex identification in the three-spined stickleback

Two sex linked DNA markers are isolated in the three-spined stickleback Gasterosteus aculeatus and a sex identification test is developed.     

Interspawning interval of wild female three-spined stickleback Gasterosteus aculeatus in Alaska

The interspawning interval, or spawning frequency, of wild three-spined stickleback, Gasterosteus aculeatus , was estimated using histological examination of postovulatory follicles (POF). Females in Alaskan lakes appeared to have as much as a 48 h delay between ovulation and ovoposition, yet the POF method could still be used to estimate the interspawning interval. In two Alaskan lakes the interspawning interval was estimated to range from 2·2 to 7·8 days among individual female G. aculeatus . These estimates were consistent with the range (2·5 to 5 days) of previous estimates among individual females from laboratory observations of spawning G. aculeatus , as well as anecdotal accounts of spawning intervals reported from wild populations in Canada (5–10 days). The interspawning interval of females increased during the course of the spawning season in Alaska, showing that the majority of female spawning activity occurred during the earliest portion of the approximate 6-week reproductive season. The increased interspawning interval appears to be related to a previously reported decrease in body condition in reproductive females during the breeding season. Thus, female G. aculeatus may be unable to sustain the initial rate of reproduction as energy stores that support the rapid growth of vitellogenic oocytes are depleted.     

Cloning and sequencing of beta-mannosidase gene from Aspergillus aculeatus no. F-50

The manB gene, coding for a unique beta-mannosidase (MANB) of Aspergillus aculeatus, was cloned from genomic and cDNA libraries, and sequenced. The gene consists of 2,811 bp encoding a polypeptide of 937 amino acid residues with a molecular mass of 104,214 Da. The A. aculeatus MANB shared amino acid sequence identity with MANB of human (24%), goat (24%), bovine (24%), and Caenorhabditis elegans (22%). When the A. aculeatus MANB was compared with other related enzymes, a Glu residue corresponding to the active site identified by the Escherichia coli beta-galactosidase and the human beta-guclonidase was conserved. This is the first fungal gene that encodes MANB.     

Molecular and phenotypic characterization of Aspergillus japonicus and Aspergillus aculeatus strains with special regard to their mitochondrial DNA polymorphisms

Forty Aspergillus japonicus and A. aculeatus strains, most of them wild-type isolates, were examined using various molecular and phenotypic techniques. The rDNAs proved to be invariable (even strains of the species A. aculeatus exhibited the same restriction profile), while the strains could be classified into seven different mtDNA RFLP groups. Hybridisation data suggest that six of these mtDNA types have certain common restriction sites, while mtDNA type 7, which was exhibited by some A. aculeatus strains, probably has quite different mtDNA organisation and their size was smallest among the strains studied. The RAPD technique and isoenzyme analysis revealed some variabilities within these RFLP groups and strain specific features could also be recognised. Carbon source assimilation spectra were found to be very distinctive for strains of A. japonicus, A. aculeatus and A. niger, providing a useful tool for pre-characterising new wild-type isolates of black Aspergilli. Only a limited correlation was observed between the dendrograms based on genotypic and phenotypic characters.     

Karyotype differentiation between two stickleback species (Gasterosteidae)

The stickleback family (Gasterosteidae) of fish is less than 40 million years old, yet stickleback species have diverged in both diploid chromosome number (2n) and morphology. We used comparative fluorescence in situ hybridization (FISH) on 2 stickleback species, Gasterosteus aculeatus (2n = 42) and Apeltes quadracus (2n = 46), to ascertain the types of chromosome rearrangements that differentiate these species. The A. quadracus karyotype contains more acrocentric and telocentric chromosomes than the G. aculeatus karyotype. By using bacterial artificial chromosome probes from G. aculeatus in our FISH screen, we found that 6 pericentric inversions and 2 chromosome fusions/fissions are responsible for the greater number of acrocentric and telocentric chromosomes in A. quadracus. While most populations of G. aculeatus have an XX/XY sex chromosome system, A. quadracus has a ZZ/ZW sex chromosome system, as previously reported. However, we discovered that a population of A. quadracus from Connecticut lacks heteromorphic sex chromosomes, providing evidence for unexpected sex chromosome diversity in this species.     

棘孢曲霉固态发酵柚皮产柚苷酶的条件优化

【目的】以柚皮为原料,优化棘孢曲霉利用柑橘加工副产物固态发酵柚苷酶的条件。【方法】采用高效液相色谱法检测酶活力,通过单因素试验考察固水比、装样量、接种量、温度对柚苷酶发酵的影响,用正交试验优化发酵条件。【结果】单因素试验结果的显著性分析表明培养基的固水比、装样量和培养温度对柚苷酶产量有显著性影响,而接种量影响不显著;经正交试验确定的优化条件是：固水比1:1 (质量体积比),装样量5 g/250 mL三角瓶,温度为30 °C,接种1 mL孢子悬浮液,发酵8 d。在此优化条件下,柚苷酶酶活力为8.19 IU/g干物质,比初始培养基产柚苷酶活力提高7.38倍。【结论】通过对固水比、装样量和发酵温度进行优化,大幅度提高了棘孢曲霉固态发酵柑橘加工副产物的柚苷酶产量,为柚苷酶的生产提供了一种高产发酵工艺。     

印楝内生真菌棘孢曲霉YM 311498的代谢产物研究

从印楝植物内生真菌棘孢曲霉（Aspergillus aculeatus）YM311498的发酵液提取物中分离得到4个化合物,通过波谱技术分别鉴定为大黄酚（1）、大黄素（2）、琥珀酸（3）、柠檬酸二甲酯（4）,以上化合物均为首次从该种真菌中分离得到。     

Population biology of three-spined sticklebacks, Gasterosteus aculeatus, in Scotland

The age of maturation, longevity and aspects of anti-predator morphology were studied for seven Gasterosteus aculeatus L. populations in Scotland during 1978 and 1979. All seven populations are annual with less than 1% of adults surviving a second year. At the four mainland study sites, breeding adult sticklebacks were larger than at any of the three Hebridean sites. Adult females were larger than adult males at all sites, except Lennox Castle where parasitism may account for the lack of sexual dimorphism. Two of the Hebridean sites, Loch Fada and Loch a Bharpa have spine-deficient G. aculeatus populations which are morphologically unprotected from predation. In the five normally spined populations, some aspects of defensive morphology (index of increase in critical dimension, ventral spine size) are associated with predation risk, while others (lateral plate number) are not. These data are discussed in relation to the current stickleback literature.     

A checklist of parasites recorded on sticklebacks (Actinopterygii: Gasterosteidae) from Poland

Parasites of the three-spined stickleback, Gasterosteus aculeatus (L.), and the nine-spined stickleback, Pungitius pungitius (L.), from brackish and freshwater habitats in Poland are listed. The following list was compiled from published records and own long-term studies. Parasites are listed alphabetically in meaningful groups of parasites with notes about their location on the host and distribution in Poland with references. A total of 51 species (taxa) of parasites were recorded on G. aculeatus (3 Apicomplexa, 1 Microsporidia, 3 Peritrichia, 1 Myxozoa, 7 Digenea, 2 Monogenea, 10 Cestoda, 10 Nematoda, 7 Acanthocephala, 3 Copepoda, 1 Branchiura, 2 Hirudinea, 1 Bivalvia) and 16 parasites on P. pungitius (1 Apicomplexa, 1 Microsporidia, 2 Peritrichia, 4 Digenea, 1 Monogenea, 2 Cestoda, 2 Nematoda, 1 Copepoda, 1 Branchiura, 1 Bivalvia).     

Habitat dependent associations between parasitism and fluctuating asymmetry among endemic stickleback populations

Inconsistencies in the relationship between fluctuating asymmetry (FA) and fitness may be due to selection acting on the degree of trait asymmetry that differs among populations or among traits. We assessed relationships between parasite susceptibility and fluctuating asymmetry in the number of bony lateral plates among 83 populations of freshwater Gasterosteus aculeatus (three spined stickleback) and among lateral plate positions that vary in the selection they experience for symmetry. The correlation between FA and parasite infection was highly variable among samples. Excess of infected asymmetric G. aculeatus increased significantly as the robustness of structural predator defences decreased. This effect was found for one parasite species only (Eustrongylides sp.) and was slightly stronger in females. In addition, there was a trend for there to be an excess of infected females asymmetric in those lateral plates positions that did not experience selection for their symmetry, although the trend only approached significance. These results suggest that selection for trait symmetry can obscure relationships between fitness and individual-wide developmental stability, providing one possible explanation for some of the heterogeneity in FA/fitness relationships seen in the literature. These results are also consistent with previous reports showing that ecological segregation between symmetric and asymmetric G. aculeatus and between sexes can alter the FA/fitness relationship.