Culture-Based Strategies for Reduction of Protease Activity in Filtrates from Aspergillus niger NRRL-3

Summary While Aspergillus strains are also being considered as potential hosts for production of extracellular heterologous proteins, the proteases produced by the host are highly problematic in that they typically modify and degrade the recombinant proteins. Culture-based approaches for minimization of protease activity in culture supernatants of Aspergillus niger NRRL-3 included reduction or elimination of peptide nitrogen in the medium, preferential use of a defined salts medium rather than a non-peptide nitrogen medium containing yeast-nitrogen base, supplementation of the medium with carboxymethylcellulose and cultivation at pH 6.5 rather than 7.5. In general, increased proteolytic activity was observed after maximum biomass was observed and biomass was declining suggesting the majority of protease activity was released by cell lysis. Carboxymethylcellulose shifted mycelial morphology from pelleted to filamentous. Mycelium lysis in the centre of pellets, with resultant release of intracellular proteases, would explain why filamentous cultures exhibited much lower proteolytic activity than pelleted cultures.     

High levels of multiple paternity in a spermcast mating freshwater mussel

Multiple paternity is an important characteristic of the genetic mating system and common across a wide range of taxa. Multiple paternity can increase within‐population genotypic diversity, allowing selection to act on a wider spectre of genotypes, and potentially increasing effective population size. While the genetic mating system has been studied in many species with active mating behavior, little is known about multiple paternity in sessile species releasing gametes into the water. In freshwater mussels, males release sperm into the water, while eggs are retained and fertilized inside the female (spermcast mating). Mature parasitic glochidia are released into the water and attach to the gills of fish where they are encapsulated until settling in the bottom substrate. We used 15 microsatellite markers to detect multiple paternity in a wild population of the freshwater pearl mussel (Margaritifera margaritifera). We found multiple paternity in all clutches for which more than two offspring were genotyped, and numbers of sires were extremely high. Thirty‐two sires had contributed to the largest clutch (43 offspring sampled). This study provides the first evidence of multiple paternity in the freshwater pearl mussel, a species that has experienced dramatic declines across Europe. Previous studies on other species of freshwater mussels have detected much lower numbers of sires. Multiple paternity in freshwater pearl mussels may be central for maintaining genetic variability in small and fragmented populations and for their potential to recover after habitat restoration and may also be important in the evolutionary arms race with their fish host with a much shorter generation time.     

Competitive habitat utilization in the damselfly,Mnais nawai (Zygoptera: Calopterygidae) coexisting with a related species,Mnais pruinosa

Reproductive behaviors related to habitat utilization were studied in males of the damsefly,Mnais nawai, which has two male forms, territorial orange-winged males (nawai) and non-territorial pale-orange-winged males (sahoi), at the upper part of a mountain stream where they partiallycoexist with a related species,Mnais pruinosa, which also has two male forms, territorial orange-winged males (esakii) and non-territorial hyaline-winged males (strigata). These two species showed parapatric distribution; the lower part of the stream was occupied byM. nawai, and the upper part byM. pruinosa. In the present study, cross-matings occurred between bothMnais species, although normal intraspecific matings occurred more frequently than cross-matings. Territorial males of both species copulated with conspecific females that entered their territory and guarded the ovipositing females, probably to avoid sperm displacement resulting from subsequent copulations. Severe competition for oviposition sites by territorial males even occurred between the two species. On the other hand, non-territorial males of both species have alternative mating strategies (including several tactics such as sneaking, takeover and interception). The possible benefits from conflict among territorial males of both species is discussed.     

Evolutionary genetics and ecology of sperm-dependent parthenogenesis

Sperm-dependent (or pseudogamous) forms of parthenogenetic reproduction occur in a wide variety of animals. Inheritance is typically clonal and matroclinous (of female descent), but sperm are needed to initiate normal development. As opposed to true parthenogenesis (i.e., sperm-independent reproduction), pseudogamous parthenogenetic lineages must coexist with a ‘sperm donor’— e.g., males from a conspecific sexual lineage, conspecific hermaphrodites, or males from a closely related sexual species. Such sperm donors do not contribute genetically to the next generation. The parasitic nature of sperm-dependent parthenogenesis raises numerous ecological and evolutionary questions. How do they arise? What factors help stabilize coexistence between the pseudogamous parthenogens and their sperm donors (i.e., ‘sexual hosts’)? Why do males waste sperm on the asexual females? Why does true parthenogenesis not evolve in pseudogamous lineages and free them from their dependency on sperm donors? Does pseudogamous parthenogenesis provide compensatory benefits that outweigh the constraints of sperm-dependence? Herein, we consider some genetic, ecological, and geographical consequences of sperm-dependent parthenogenesis in animals.     

A Moricandia arvensis– based cytoplasmic male sterility and fertility restoration system in Brassica juncea

 A cytoplasmic male-sterility system has been developed in mustard (Brassica juncea) following repeated backcrossings of the somatic hybrid Moricandia arvensis (2n=28, MM)+B. juncea (2n=36, AABB), carrying mitochondria and chloroplasts from M. arvensis, to Brassica juncea. Cytoplasmic male-sterile (CMS) plants are similar to normal B. juncea; however, the leaves exhibit severe chlorosis resulting in delayed flowering. Flowers are normal with slender, non-dehiscent anthers and excellent nectaries. CMS plants show regular meiosis with pollen degeneration occurring during microsporogenesis. Female fertility was normal. Genetic information for fertility restoration was introgressed following the development of a M. arvensis monosomic addition line on CMS B. juncea. The additional chromosome paired allosyndetically with one of the B. juncea bivalents and allowed introgression. The putative restorer plant also exhibited severe chlorosis similar to CMS plants but possessed 89% and 73% pollen and seed fertility, respectively, which subsequently increased to 96% and 87% in the selfed progeny. The progeny of the cross of CMS line with the restorer line MJR-15, segregated into 1 fertile : 1 sterile. The CMS (Moricandia) B. juncea, the restorer (MJR-15), and fertility restored F₁ plants possess similar cytoplasmic organellar genomes as revealed by ‘Southern’ analysis. Received: 17 September 1997 / Accepted: 18 February 1998     

Floral display size and spatial distribution of potential mates affect pollen deposition and female reproductive success in distylous Pulmonaria officinalis (Boraginaceae)

In animal‐pollinated plants, both the spatial distribution of flowering individuals and the number of flowers that an individual displays affect pollen deposition rates and female reproductive success. Heterostylous species are likely to be particularly sensitive to the contingencies of spatial distribution, as they are reproductively subdivided into distinct mating groups, which usually exhibit self‐ and intra‐morph incompatibility and differ in floral morphology. In this paper, we explore the joint effects of both spatial distribution of potential mates and floral display size on morph‐specific pollen deposition rates and seed set patterns in two natural populations of Pulmonaria officinalis, a distylous species with a weak self‐incompatibility system. Both total stigmatic pollen load and the proportion of legitimate pollen decreased with increasing spatial isolation. Legitimate (intermorph) pollen transfer was, however, asymmetric and decreased more rapidly with decreasing proximity to a compatible legitimate mating partner in the S‐morph than in the L‐morph. Total stigmatic pollen loads per flower increased with increasing floral display size, indicating that large plants are disproportionately more visited than smaller individuals. However, because legitimate pollen deposition decreased with increasing floral display size, these results also suggest that larger numbers of flowers increase the degree of geitonogamous pollination. In both the L‐ and S‐morph, seed set significantly decreased with increasing isolation from a legitimate mating partner, but in the L‐morph seed set was less dependent on the spatial distribution of the S‐morph. In addition, seed set significantly increased with floral display size in the L‐morph, but not in the S‐morph. These findings indicate that the spatial distribution of potential mates and variation in floral display size may cause morph‐specific differences in pollen deposition rates and female reproductive success.     

Little effect of delayed mating on fecundity or fertility of female fungus gnats Lycoriella ingenua

The effect of delayed female mating for the mushroom fungus gnat Lycoriella ingenua is investigated. We examine the effect of delaying female mating on the fertility and egg viability of female flies that have a mating delay of 0–5 days after emergence. Male fly age is held constant. Female age does not impact male acceptance and most flies copulate within seconds of pairing. We find that female flies experiencing mating delays of 0–4 days after emergence lay a similar number of eggs onto artificial substrates. Females that experience a mating delay of 5 days lay 54% fewer eggs than those that mate on day 0 (day of emergence). There is no effect of mating delay on the percentage of larvae that emerge. The results of the present study indicate that mating delays have little effect on the fertility or fecundity of the mushroom fungus pest L. ingenua.     

No mate preference associated with the supergene controlling social organization in Alpine silver ants

Disassortative mating is a powerful mechanism stabilizing polymorphisms at sex chromosomes and other supergenes. The Alpine silver ant, Formica selysi, has two forms of social organization—single‐queen and multiple‐queen colonies—determined by alternate haplotypes at a large supergene. Here, we explore whether mate preference contributes to the maintenance of the genetic polymorphism at the social supergene. With mate choice experiments, we found that females and males mated randomly with respect to social form. Moreover, queens were able to produce offspring irrespective of whether they had mated with a male from the same or the alternative social form. Yet, females originating from single‐queen colonies were more fertile, suggesting that they may be more successful at independent colony founding. We conclude that the pattern of asymmetric assortative mating documented from mature F. selysi colonies in the field is not caused by mate preferences or major genetic incompatibilities between social forms. More generally, we found no evidence that disassortative mate preference contributes to the maintenance of polymorphism at this supergene controlling ant social organization.     

Phenotypic selection in hawkmoth-pollinated Platanthera bifolia: targets and fitness surfaces

Abstract.— The present study explored phenotypic selection on phenological and morphological reproductive traits in hawkmoth-pollinated Platanthera bifolia (Orchidaceae), a Eurasian perennial herb displaying bisexual, long-spurred flowers. The work was carried out during three flowering seasons (1993–1995) in a Swedish population. Fitness was estimated as the number of pollinia removed (male fitness) and fruits produced (female fitness). Targets and patterns of selection were compared between years and sex functions by the use of multiple linear regression (including correlational selection estimates, i.e., of combination of traits), analysis of covariance, and projection pursuit regression (PPR). Results from the nonparametric surface-fitting-method PPR showed that selection was mostly linear, thus justifying the use of the parametric methods. In all study years, male and female fitness were highest in plants with many flowers. This reflects that flower number sets an upper limit to fitness and that a large inflorescence attracts more pollinators. In 1994, the summer was dry and the average spur length of P. bifolia was shorter than in the other years. In this year, male and female fitness were positively related to spur length, apparently because the spur of short-spurred plants was somewhat too short relative to the tongue length of the local pollinator for optimal pollen export and import. Additionally, the dry weather in 1994 caused a tendency for correlational selection, which was not found in the other years of study. Among small individuals (apparently more sensitive to drought than large ones), early-flowering plants had higher male and female fitness. The results show that patterns of selection may vary both between years and between sex functions in perennial hermaphroditic plants. The present study is one of the first to consider correlational selection in plants, which probably is of common occurrence and deserves to be investigated more.     

Linking larval nutrition to adult reproductive traits in the European corn borer Ostrinia nubilalis

Throughout an organism's lifetime, resources are strategically allocated to many different functions, including reproduction. Reproduction can be costly for both sexes; females produce nutrient‐rich eggs, whereas males of many species produce large and complex ejaculates. In capital breeding insects, nutrients are mainly acquired during the larval period, yet allocation decisions impact the reproductive fitness of adults. The present study examines the effect of larval dietary nitrogen on both male and female reproductive traits in the European corn borer moth Ostrinia nubilalis Hübner, whose adults do not feed and whose males transfer a large, nitrogen‐rich spermatophore. One day post‐eclosion, O. nubilalis adults reared on one of three different diets (3.0%, 1.6%, or 1.1% nitrogen) are mated and two experiments are undertaken: one to measure nitrogen and carbon content of male ejaculates, and the other to determine female fecundity and fertility. Although male larval diet does not alter the percentage nitrogen content of adult somatic tissue, males reared on the higher nitrogen diet (3.0%) produce spermatophores with increased nitrogen relative to somatic nitrogen. Furthermore, females raised on the 3.0% nitrogen diet receive spermatophores with lower carbon : nitrogen ratios and thus more nitrogen. Overall, females lay more eggs as their larval dietary nitrogen increases, although they lay fewer eggs when their mates are raised on the higher (3.0%) nitrogen diet. This suggests that O. nubilalis females may use male‐derived nitrogen not to supplement egg production, but rather for somatic maintenance. Overall, the present study furthers our understanding of how larval diet can affect adult fitness in Lepidoptera.