Cytogenetic proof that the brine shrimp Artemia franciscana (Crustacea, Branchiopoda) is found in Argentina

Artemia brine shrimps are commercially important and they have been extensively studied. This branchiopod crustacean is extensively used in aquaculture and other commercial and applied practices. The genus also awakes an increasing interest worldwide as an experimental model in other basic areas of research such as evolution and cytogenetics. In the present work adult male meiosis and nauplii mitotic cells in two Artemia populations from Argentina (Mar Chiquita and Las Tunas, Córdoba Province) are analyzed and compared to the reference strains A. franciscana (Great Salt Lake, Utah, USA) and A. persimilis (Salinas Grandes de Hidalgo, La Pampa Province, Argentina). The Mar Chiquita population shows the diploid and haploid numbers characteristic of A. franciscana, and a regular male meiosis. In the Las Tunas population diverse diploid and haploid numbers are encountered, and an irregular meiosis is observed in some individuals. Fluorescent DAPI and CMA banding shows bright chromocenters of intermediate size and number in both populations. Cytogenetic and phenotypic data show that the population of Mar Chiquita could be assigned to A. franciscana; the variation found in chromocenter size and number reflects an heterochromatin polytypism already observed in this species. The cytogenetic and morphological traits in Las Tunas population suggest an occasional hybridization between A. franciscana and A. persimilis.     

Review of the biogeography of the genus Artemia (Crustacea, Anostraca)

In this study, we report on the known Artemia habitats worldwide. Recent literature information is incorporated about the taxonomic status of the various populations studied. The genus is composed of di-, tri-, tetra- and pentaploid parthenogenetic populations and of the following bisexual species: A. franciscana franciscana , A. franciscana monica , A. franciscana sp., A. persimilis , A. salina , A. urmiana , A. sinica and A. sp. from Kazakhstan. The problems of characterizing new brine shrimp populations are discussed. In view of the great importance of Artemia as part of the live food chain for the culture of fish and shellfish larvae and the present cyst shortage from the market, the need for commercial exploitation and development of new Artemia sources is now, more than ever, necessary.     

Mechanisms associated with cellular desiccation tolerance of Artemia encysted embryos from locations around the world

Using differential scanning calorimetry we demonstrated the presence of biological glasses and measured the glass transition temperatures (Tg) in dry encysted gastrula embryos (cysts) of the brine shrimp, Artemia, from eleven different locations, two of which provided cysts from parthenogenetic animals. Values for Tg were highest, by far, in Artemia franciscana cysts from the Mekong Delta, Vietnam (VN), these cysts having been produced from previous sequential inoculations into growth ponds of cysts from the San Francisco Bay, California, USA. Tg values for three groups of A. franciscana cysts were significantly higher than those of other cysts (except those of Artemia persimilis) studied here, as well as all other desiccation-tolerant animal systems studied to date. We also measured three stress proteins (hsc70, artemin and p26) in all these cysts as well as the total alcohol soluble carbohydrates (ASC), about 90% of which is the disaccharide trehalose, a known component of biological glasses. We interpret the results in terms of mechanisms involved with desiccation tolerance and, to some extent, with thermal conditions at the sites of cyst collection.     

The post-embryonic development of Artemia persimilis Piccinelli & Prosdocimi

The post-embryonic development of Artemia persimilis Piccinelli & Prosdocimi, reared under laboratory conditions, is described. Larval development is typical of branchiopods. It comprises 17 identifiable instars, which may be grouped into four periods. A comparison is made with Artemia franciscana Kellogg.     

Allozymatic variation in crustacea Artemia franciscana (Anostraca: Artemiidae) of Freat Salt Lake in different experimental conditions

Starch gel electrophoresis was used to analyze the allelic variability of four polymorphic loci (Lap-2, Lap-3, Pgm and Gpi) from a single population of Artemia franciscana (Kellogg, 1906) from the Great Salt Lake (Utah, USA), cultured under eight different experimental conditions. The organisms were cultured to the adult stage under a 2 x 2 x 2 experimental design (22 and 30 degrees C; 30 and 60 ppt salinity; and Dunaliella sp. and Spirulina sp. as food). There were significant differences in allele frequencies at each locus and the mean expected heterozygosity (He) varied from 0.236 to 0.447. Therefore, the hypothesis of no allelic differences among treatments is rejected. With relation to a possible correlation between genetic variability and the phenotypic characteristics, the results show that there is probably a synergic effect between the different salinities and temperatures on the survival of heterozygous organisms in the different loci.     

Identification of a novel DNA methyltransferase 2 from the brine shrimp, Artemia franciscana

DNA methyltransferase 2 (Dnmt2) is a dual-specificity DNA methyltransferase, which contains a weak DNA methyltransferase and novel tRNA methyltransferase activity. However, its biological function is still enigmatic. To elucidate the expression profiles of Dnmt2 in Artemia franciscana, we isolated the gene encoding a Dnmt2 from A. franciscana and named it as AfDnmt2. The cDNA of AfDnmt2 contained a 1140-bp open reading frame that encoded a putative Dnmt2 protein of 379 amino acids exhibiting 32% approximately 39% identities with other known Dnmt2 homologs. This is the first report of a DNA methyltransferase gene in Crustacean. By using semi-quantitative RT-PCR, AfDnmt2 was found to be expressed through all developmental stages and its expression increased during resumption of diapause cysts development. Southern blot analysis indicated the presence of multiple copies of AfDnmt2 genes in A. franciscana.     

Prey selection and efficiency of naïve and experienced juvenile sockeye salmon

Prey selection and growth efficiency of juvenile sockeye salmon Oncorhynchus nerka switched between live prey and pelleted diets were investigated. First feeding sockeye salmon fry were placed into one of three dietary treatments for 7 months prior to assessing potential differences with a growth and a behavioural assay. Dietary treatments were (1) adult Artemia franciscana for 1 month, followed by pelleted feed for an additional 6 months ( Art − BD), (2) pelleted feed from first feeding for 7 months (BD) and (3) adult A. franciscana for 1 month, and a combination of pelleted feed and live adult A. franciscana for 6 months ( Art + BD). Equal numbers from each treatment group were then tagged, pooled into replicate 'common garden' tanks and fed novel live prey items ( Daphnia sp. and mosquito Culex pipiens larvae) for an additional 3 weeks. No significant differences in the growth efficiency of sockeye salmon were found during the 3 week feeding trial on the novel prey items. Additional sockeye salmon from each dietary treatment were used in a behavioural assay to determine if the treatments had an impact on foraging efficiency (prey selection or time to capture prey). No significant differences in prey selection were found among treatment groups in time to capture pellets, A. franciscana or mosquito larvae. Also, no significant differences were found within treatment groups in time to capture different food sources. No substantive benefits in foraging efficiency of sockeye salmon associated with prior exposure to live prey were demonstrated. This suggests that altering existing hatchery practices for juvenile sockeye salmon by offering live food prior to release is unlikely to influence post-hatchery feeding behaviour or increase post-release survival.     

Relative toxicity of nC24 agricultural mineral oil to Tetranychus urticae Koch (Acari: Tetranychidae) and Phytoseiulus persimilis Athias-Henriot (Acari: Phytoseiidae) and its possible relationship to egg ultrastructure

The relative toxicity (LC₅₀ values based on µg oil/cm²) is evaluated of aqueous n C24 agricultural mineral oil (AMO) emulsions to the egg, six-legged nymph (larva), eight-legged protonymph and adult stages of two-spotted mite ( Tetranychus urticae ) and its predator, Phytoseiulus persimilis , on French bean leaf discs, using a Potter spray tower to apply of the oil. The egg of P. persimilis was the least susceptible stage (LC₅₀ 444.84) and its LC₅₀ was significantly higher than all other stages tested of either P. persimilis or T. urticae . The LC₅₀ for adult female T. urticae (LC₅₀ 63.89) was significantly lower than the larva (LC₅₀ 93.86); however, there was no significant difference in response between the protonymph (LC₅₀ 70.44) and the larva, which were both higher than T. urticae eggs (LC₅₀ 17.55). LC₅₀s for P. persimilis larva (LC₅₀ 43.87), protonymph (LC₅₀ 41.55) and adult female (LC₅₀ 53.34) were similar. Scanning electron microscopy showed that the egg surface of T. urticae is usually well covered with fine silk that may trap more oil and increase AMO efficacy. Other possible differences in AMO efficacy between T. urticae and P. persimilis may be due to differences in egg size, egg incubation period, egg surface structure and the presence of vulnerable respiratory cones in T. urticae eggs. Dose of 0.2–0.3% (w/w) is considered to be the most appropriate for n C24 AMOs use against T. urticae in combination with P. persimilis in integrated pest management programs.     

Molecular phylogenetics and asexuality in the brine shrimp Artemia

Explaining cases of long-term persistence of parthenogenesis has proven an arduous task for evolutionary biologists. Interpreting sexual-asexual interactions though has recently advanced owing to methodological design, increased taxon sampling and choice of model organisms. We inferred the phylogeny of Artemia, a halophilic branchiopod genus of sexual and parthenogenetic forms with cosmopolitan distribution, marked geographic patterns and ecological partitioning. Joint analysis of newly derived ITS1 sequences and 16S RFLP markers from global isolates indicates significant interspecific divergence as well as pronounced diversity for parthenogens, matching that of sexual ancestors. Maximum parsimony, maximum likelihood, and Bayesian methods were largely congruent in reconstructing the phylogeny of the genus. Given the current sampling, at least four independent origins of parthenogenesis are deduced. Molecular clock calibrations based on biogeographic landmarks indicate that the lineage leading to A. persimilis diverged from the common ancestor of all Artemia species between 80 and 90 MYA at the time of separation of Africa from South America, whereas parthenogenesis first appeared at least 3 MYA. Common mitochondrial DNA haplotypes delineate A. urmiana and A. tibetiana as possible maternal parents of several clonal lineages. A novel topological placement of A. franciscana as a sister clade to all Asian Artemia and parthenogenetic forms is proposed and also supported by ITS1 length and other existing data.     

智利小植绥螨密度对朱砂叶螨产卵能力的影响

智利小植绥螨是朱砂叶螨的专性捕食者。本研究在室内,研究了智利小植绥螨密度对朱砂叶螨存活及生殖的影响。智利小植绥螨对朱砂叶螨存活干扰结果表明:干扰后24 h各处理朱砂叶螨的死亡率均显著高于对照,且死亡率随干扰密度增大而提高,10头捕食螨/叶碟处理高达76.67%,为对照处理的14.38倍。但是干扰48 h后对叶螨存活影响不大。对朱砂叶螨产卵干扰结果表明:干扰后各时间段朱砂叶螨单雌产卵量均随智利小植绥螨干扰密度的增加而降低。干扰24 h后,除1头捕食螨/叶碟处理与对照差异不显著外,其他各处理均与对照有显著差异。干扰48 h后,10头捕食螨/叶碟处理的叶螨单头产卵量是2.80粒,仅为对照的28.5%。但是干扰96h后,除10头捕食螨/叶碟处理与对照差异显著外,其余各处理与对照均差异不显著。本研究还分析了干扰对96 h内朱砂叶螨单雌总产卵量影响,处理5头/叶碟和10头/叶碟均显著低于对照,并且10头/叶碟显著低于5头/叶碟;1头/叶碟和3头/叶碟与对照相当,但是均显著高于10头/叶碟。结果显示,智利小植绥螨密度对朱砂叶螨有较强的生殖干扰作用,并且捕食螨密度越大,干扰作用越强,后代增殖潜能越小。     

Effects of salinity on growth and survival in five Artemia franciscana (Anostraca: Artemiidae) populations from Mexico Pacific Coast

Salinity is an important factor influencing growth and survival of aquatic organisms such as Artemia, a valuable aquaculture species. This study evaluated the effects of salinity on A. franciscana populations from different water bodies in Mexico's Pacific Coast. With this purpose, five autochthonous bisexual Artemia populations were tested to assess their survival and growth values against salinities of 40, 60, 80, 100 and 120 g/l, under laboratory conditions (25 +/- 2 degrees C; pH 8-10; constant light and aeration). The organisms were fed with 100 mL of rice bran and 2L of Tetraselmis suecica (500 000 cel/ml). The culture experiments were made in 200L plastic tanks, and survival and growth final values were obtained after 21 culture days. Survival and growth curves were determined by a regression analysis (R2). The significant differences between salinities were determined by ANOVA test (p < 0.05). The best survival and growth rates were found at salinities of 100-120 g/l. When the Mexican Artemia populations were cultivated at 40 g/l of salinity, 100% mortality was observed in the juvenile stage. This study determined that survival and growth values of A. franciscana populations increased with salinity. The five A. franciscana populations presented significant differences in their survival rate under various salinity regimes. The studied populations experienced high mortality at salinities under 60 g/l and over 200 g/l, and especially during the metanauplius stage. The present study confirms that growth rates in Mexican A. franciscana populations from Pacific Coast habitats are not inversely proportional to salinity. These A. franciscana populations should be cultured at 100-120 g/l of salinity to obtain better survival and growth rates. This data is useful to improve culture systems in aquaculture biomass production systems.     

Genetics of hybrid male sterility among strains and species in the Drosophila pseudoobscura species group

Taxa in the early stages of speciation may bear intraspecific allelic variation at loci conferring barrier traits in hybrids such as hybrid sterility. Additionally, hybridization may spread alleles that confer barrier traits to other taxa. Historically, few studies examine within- and between-species variation at loci conferring reproductive isolation. Here, we test for allelic variation within Drosophila persimilis and within the Bogota subspecies of D. pseudoobscura at regions previously shown to contribute to hybrid male sterility. We also test whether D. persimilis and the USA subspecies of D. pseudoobscura share an allele conferring hybrid sterility in a D. pseudoobscura bogotana genetic background. All loci conferred similar hybrid sterility effects across all strains studied, although we detected some statistically significant quantitative effect variation among D. persimilis alleles of some hybrid incompatibility QTLs. We also detected allelism between D. persimilis and D. pseudoobscura USA at a second chromosome hybrid sterility QTL. We hypothesize that either the QTL is ancestral in D. persimilis and D. pseudoobscura USA and lost in D. pseudoobscura bogotana, or gene flow transferred the QTL from D. persimilis to D. pseudoobscura USA. We discuss our findings in the context of population features that may contribute to variation in hybrid incompatibilities.     

Disease prevalence and transmission of Microsporidium phytoseiuli infecting the predatory mite, Phytoseiulus persimilis (Acari: Phytoseiidae)

Isolated colonies of the predatory mite, Phytoseiulus persimilis, were used to gain information regarding prevalence and transmission of Microsporidium phytoseiuli. Two colonies of P. persimilis were reared on spider mite (Tetranychus urticae)-infested bean plants in isolated cages. Disease prevalence of predators from Colony 1 remained relatively low (between 0 and 15%) over 57 weeks of observation whereas disease prevalence of predators from Colony 2 increased over 3 months (from 12 to 100%). Disease prevalence among predators from Colony 1 had increased to 100% 2 months after weekly sampling had ceased for this colony and periodic sampling confirmed that disease prevalence among individuals of both colonies remained at 100%. Microsporidian spores were not detected in randomly chosen samples of T. urticae prey mites that were removed and examined biweekly during this period. Although numerous microsporidian spores were observed in smear preparations of fecal pellets examined by light microscopy, spores were not observed on leaf surfaces or predator feces when examined by SEM. The latter appeared as intact aggregates composed of numerous dumbbell-shaped crystals and it is unlikely that spores are liberated from intact fecal pellets onto leaf surfaces. Vertical transmission of M. phytoseiuli was 100%; horizontal transmission was low (14.3%) and occurred only when immature P. persimilis were permitted to develop in contact with infected immature and adult predators. The mean number of eggs produced per mated pair was highest when uninfected females were mated with uninfected males (63.2 eggs per mated pair). Although mean egg production decreased when one or both parents were infected, not all differences were significant. Male predatory mites did not contribute to infection of their progeny. Results suggest that routine examination of P. persimilis for microsporidian spores is essential for the management of M. phytoseiuli within P. persimilis colonies. Low disease prevalence and lack of obvious disease signs or symptoms, as in the case of M. phytoseiuli, increase the probability that these pathogens will escape notice unless individuals are routinely examined for pathogens.     

Cannibalism and interspecific predation in the phytoseiid mites Phytoseiulus persimilis and Neoseiulus californicus: predation rates and effects on reproduction and juvenile development

We examined intra- and interspecific predation of adult females and immature stages of the generalist Neoseiulus californicus and the specialist Phytoseiulus persimilis. Adult females and immatures of both predators exhibited higher predation rates on larvae than on eggs and protonymphs. N. californicus fed more inter- than intraspecifically. Predation on P. persimilis by N. californicus was more severe than vice versa. P. persimilis had higher predation rates on conspecifics than heterospecifics and was more prone to cannibalism than N. californicus. When provided with phytoseiid prey, P. persimilis suffered higher mortality than N. californicus. When held without food, adult females and protonymphs of N. californicus survived longer than the corresponding stages of P. persimilis. N. californicus females were able to sustain oviposition when preying upon P. persimilis, whereas cannibalizing females did not lay eggs. Females of P. persimilis were not able to sustain oviposition, irrespective of con- or heterospecific prey. Immatures of both predators were able to reach adulthood when provided with either con- or heterospecifics. Juvenile development of N. californicus was shorter with heterospecific vs. conspecific larvae; mortality of P. persimilis immatures was less when feeding on conspecific vs. heterospecific larvae. Different behavioral pattern in intra- and interspecific predation are discussed in regard to their feeding types (generalist vs. specialist).     

Development and pathology of two undescribed species of microsporidia infecting the predatory mite, Phytoseiulus persimilis Athias-Henriot

Two undescribed species of microsporidia were found in mass-reared Phytoseiulus persimilis Athias-Henriot from two commercial sources during a routine examination of these predators for pathogens. Both microsporidian species were described from specimens that had been prepared for transmission electron microscopy; live specimens were unavailable for examination. One microsporidium, identified as Species A, was described from two specimens obtained from a commercial insectary in North America. All observed stages of this microsporidium were uninucleate. Rounded-to-ovoid schizonts appeared to develop in direct contact with the cytoplasm of lyrate organ cells (ovarian tissue). Mature spores of Species A were elongate-ovoid and measured 2.88 x 1.21 microm. A polar filament coiled 7 to 10 times in the posterior half of the spore. Sporoblasts and spores were observed in the cytoplasm of cells of numerous tissues and in developing eggs within gravid females. A second species, identified as Species B, was described from five specimens obtained from a commercial source in Israel. All observed stages of this microsporidium were uninucleate. Schizonts of Species B were observed within the cytoplasm of cecal wall cells and within the nuclei of lyrate organ cells. Mature spores were ovoid and measured 2.65 x 1.21 microm. A polar filament coiled 3 to 4 times in the posterior half of the spore. Densely packed ribosomes often concealed the polar filament and other internal spore characteristics. Spores were observed in the cytoplasm of cells of numerous tissues and occasionally within the nuclei of lyrate organ cells. Numerous spores and presporal stages were observed within the ovary and developing eggs. The development and pathology of Species A and B were compared to those of Microsporidium phytoseiuli Bj?ornson, Steiner and Keddie, a microsporidium previously described from P. persimilis obtained from a commercial source in Europe. The occurrence of three species of microsporidia within P. persimilis from three sources raises questions regarding the origin of these pathogens. Because microsporidia may have profound impact on the performance of P. persimilis, consideration must be given to the identification and exclusion of microsporidia from field-collected specimens or from predators that may be shared among commercial sources.     

Sex-specific developmental plasticity of generalist and specialist predatory mites (Acari: Phytoseiidae) in response to food stress

We studied developmental plasticity under food stress in three female-biased size dimorphic predatory mite species, Phytoseiulus persimilis, Neoseiulus californicus, and Amblyseius andersoni. All three species prey on two-spotted spider mites but differ in the degree of adaptation to this prey. Phytoseiulus persimilis is a specialized spider mite predator, N. californicus is a generalist with a preference for spider mites, and A. andersoni is a broad generalist. Immature predators were offered prey patches of varying density and their survival chances, dispersal tendencies, age and size at maturity measured. Amblyseius andersoni dispersed earlier from and had lower survival chances in low density prey patches than N. californicus and P. persimilis. Age at maturity was not affected by prey density in the generalist A. andersoni, whereas both the specialist P. persimilis and the generalist N. californicus accelerated development at low prey densities. Species-specific plasticity in age at maturity reflects opposite survival strategies when confronted with limited prey: to prematurely leave and search for other food (A. andersoni) or to stay and accelerate development (P. persimilis, N. californicus). In all species, size at maturity was more plastic in females than males, indicating that males incur higher fitness costs from deviations from optimal body size.     

Differences in isolated mitochondria are insufficient to account for respiratory depression during diapause in artemia franciscana embryos

In response to cues signifying the approach of winter, adult Artemia franciscana produce encysted embryos that enter diapause. We show that respiration rates of diapause embryos collected from the field (Great Salt Lake, Utah) are reduced up to 92% compared with postdiapause embryos when measured under conditions of normoxia and full hydration. However, mitochondria isolated from diapause embryos exhibit rates of state 3 and state 4 respiration on pyruvate that are equivalent to those from postdiapause embryos with active metabolism; a reduction in these rates (15%-27%) is measured with succinate for two of three collection years. Respiratory control ratios for diapause mitochondria are comparable to or higher than those from postdiapause embryos. The P : O flux ratios are statistically identical. Our calculations suggest that respiration of intact, postdiapause embryos is operating close to the state 3 oxygen fluxes measured for isolated mitochondria. Cytochrome c oxidase (COX) activity is 53% lower in diapause mitochondria during one collection year; the minimal impact of this COX reduction on mitochondrial respiration appears to be due to the 31% excess COX capacity in A. franciscana mitochondria. Transmission electron micrographs of embryos reveal mitochondria that are well differentiated and structurally similar in both states. As inferred from the similar amounts of mitochondrial protein extractable, tissue contents of mitochondria in diapause and postdiapause embryos are equivalent. Thus, metabolic depression during diapause cannot be fully explained by altered properties of isolated mitochondria. Rather, mechanisms for active inhibition or substrate limitation of mitochondrial metabolism in vivo may be operative.     

Intra-specific isotope variations of franciscana dolphin Pontoporia blainvillei regarding biological parameters and distinct environments

The franciscana dolphin Pontoporia blainvillei is the most endangered small endemic cetacean species of the southwestern South Atlantic Ocean. We assessed intra-specific variation in the diet of franciscana regarding biological parameters (sex, total body length, age class and sexual maturity) and between distinct environments (open marine area vs. an area adjacent to an estuary) from the Buenos Aires coast, Argentina, using both stable isotopes and stomach content analysis. We found a significant isotopic enrichment in ¹⁵N in neonates and nursing calves, representative of the lactation period. We found differences in the diet of franciscana between dolphins from the open marine area and dolphins living in waters influenced by the estuary, by both diet estimating methods. The lower δ¹⁵N values in dolphins living in waters adjacent to the estuary could be associated with a higher consumption of small sizes of the striped weakfish (Cynoscion guatucupa), which has lower isotope values than the larger sizes consumed in the open marine area. This trophic differentiation between locations is in agreement with the existence of two genetically distinct populations recently found in the study area. This study reveals geographical variations in the diet of franciscana dolphin, supporting the new delineation of distinct franciscana populations in Argentine waters. Considering the small home range and the limited movement pattern of the species, the population living near the estuary would likely need protection due to its low level of genetic diversity and feeding preferences. Therefore, the preservation and conservation of the mentioned habitat is recommended to ensure population viability of franciscana in the region.     

Examination of Allelic Variation at the Hexokinase Loci of DROSOPHILA PSEUDOOBSCURA and D. PERSIMILIS by Different Methods

Recently a number of electrophoretic techniques have been applied to reveal the presence of additional genetic variation among the electrophoretic mobility classes of the highly polymorphic xanthine dehydrogenase (XDH ) and esterase-5 (est-5) loci. We examined the hexokinase loci of Drosophila pseudoobscura and D. persimilis using a variety of techniques to determine whether further allelic variation could be revealed for these much less polymorphic loci and to analyze the nature of the known variation at the hexokinase-1 (hex-1) locus. The following studies were conducted: 135 strains of the two species from six localities were examined with buffer pH ranging from 5.5 to 10.0; 40 strains of D. pseudoobscura and 9 strains of D. persimilis from Mather were studied using starch gel concentrations ranging from 8.5 to 15.5% and were examined for differences in heat stability and reactivity to the thiol reagent pCMSA; strains were also tested for susceptibility to urea denaturation and differences in relative activities. Major findings of the work are: (1) No additional allelic variation could be detected at any of the hexokinase loci by applying these techniques. The finding of abundant hidden genetic variation in XDH and est-5 does not extend to all enzyme loci. (2) Evidence from studies using pCMSA indicates that the hex-1 alleles 0.6, 0.8, 1.0 and 1.2 of the two species form a series of unit charge steps. Since the 0.94 allele of D. persimilis has mobility intermediate between 0.8 and 1.0, it is argued that routine electrophoretic techniques are sensitive to at least some conservative amino acid substitutions. (3) Strong correlations were found at the hex-1 locus between low allelic frequency, reduced relative activity and reduced stability to heat and urea denaturation. Since the three sibling species, D. pseudoobscura, D. persimilis and D. miranda, all appear to share a common high frequency allele (1.0) at that locus, these findings are taken as evidence that the observed allelic frequencies are a result of directional selection and mutation, rather than any form of balancing selection.