Effects of long-term cortisol treatment on growth and osmoregulation of Atlantic salmon and brook trout.

Cortisol is the final product of the hypothalamic-pituitary-interrenal (HPI) axis and acts as a gluco- and mineralo-corticoid in fish. Long-term elevations of cortisol have been linked to reduced growth in fishes, but the mechanism(s) and relative sensitivities of species are still unclear. We carried out experiments to examine the relative effects of cortisol on growth and gill NKA activity in two salmonids: Atlantic salmon (Salmo salar) and brook trout (Salvelinus fontinalis). Treatment with intraperitoneal cortisol implants for 30 days resulted in reduced growth in both species, but with greater sensitivity to cortisol in brook trout. Gill NKA activity was strongly upregulated by cortisol in Atlantic salmon, and weakly upregulated in brook trout but with no statistically significant effect. Cortisol treatment resulted in reduced plasma levels of insulin-like growth factor I and increased plasma growth hormone levels in Atlantic salmon. Our results demonstrate that there are species differences in the sensitivity of growth and osmoregulation to cortisol, even among species in the same family (Salmonidae).

Osmoregulatory role of the intestine in the sea lamprey (Petromyzon marinus).

Lampreys are the most basal vertebrates with an osmoregulatory strategy. Previous research has established that the salinity tolerance of sea lamprey increases dramatically during metamorphosis, but underlying changes in the gut have not been examined. In the present work, we examined changes in intestinal function during metamorphosis and seawater exposure of sea lamprey (Petromyzon marinus). Fully metamorphosed juvenile sea lamprey had 100% survival after direct exposure to 35 parts per thousand seawater (SW) and only slight elevations in plasma chloride (Cl-) levels. Drinking rates of sea lamprey juveniles in seawater were 26-fold higher than juveniles in freshwater (FW). Na+-K+-ATPase (NKA) activity in the anterior and posterior intestine increased 12- and 3-fold, respectively, during metamorphosis, whereas esophageal NKA activity was lower than in the intestine and did not change with development. Acclimation to SW significantly enhanced NKA activity in the posterior intestine but did not significantly change NKA activity in the anterior intestine, which remained higher than that in the posterior intestine. Intestinal Cl- and water uptake, which were observed in ex vivo preparations of anterior and posterior intestine under both symmetric and asymmetric conditions, were higher in juveniles than in larvae and were similar in magnitude of those of teleost fish. Inhibition of NKA by ouabain in ex vivo preparations inhibited intestinal water absorption by 64%. Our results indicate drinking and intestinal ion and water absorption are important to osmoregulation in SW and that preparatory increases in intestinal NKA activity are important to the development of salinity tolerance that occurs during sea lamprey metamorphosis.

Macrophage-derived Wnt signaling increases endothelial permeability during skeletal muscle injury.

OBJECTIVE: The inflammatory response and the presence of macrophages are reported to be necessary for proper muscle regeneration. However, our understanding of the molecular mechanisms governing how macrophages signal to promote muscle regeneration is incomplete. METHODS AND RESULTS: Here we conditionally deleted Wls, which is required for Wnt secretion, from macrophages and examined the impact on endothelial permeability following muscle injury. The expression of Wnt ligands and Wls was increased in the tibialis anterior (TA) of mice 2 days following BaCl2 injury. Loss of macrophage Wls inhibited the loss of endothelial barrier function, as measured by transendothelial resistance and Evans blue dye permeability assays. Interestingly, the blockade in endothelial permeability correlated with reduced VEGF levels and pretreatment of wild type endothelial cells with a VEGFR2 blocking antibody was sufficient to reduce endothelial permeability induced by stimulated macrophage supernatant. We also found that macrophage Wls-null TAs had myocytes with reduced cross-sectional area 7 day post-injury suggesting a delay in muscle regeneration. CONCLUSION: Our results indicate that macrophage-derived Wnt signaling increases endothelial permeability in a VEGF-dependent fashion following muscle injury. Our findings implicate macrophages as a primary source of Wnt ligands following muscle injury and highlight the Wnt pathway as a therapeutic target following injury.

Genetic bases for variation in structure and biological activity of trichothecene toxins produced by diverse fungi.

Trichothecenes are sesquiterpene toxins produced by diverse but relatively few fungal species in at least three classes of Ascomycetes: Dothideomycetes, Eurotiomycetes, and Sordariomycetes. Approximately 200 structurally distinct trichothecene analogs have been described, but a given fungal species typically produces only a small subset of analogs. All trichothecenes share a core structure consisting of a four-ring nucleus known as 12,13-epoxytrichothec-9-ene. This structure can be substituted at various positions with hydroxyl, acyl, or keto groups to give rise to the diversity of trichothecene structures that has been described. Over the last 30 years, the genetic and biochemical pathways required for trichothecene biosynthesis in several species of the fungi Fusarium and Trichoderma have been elucidated. In addition, phylogenetic and functional analyses of trichothecene biosynthetic (TRI) genes from fungi in multiple genera have provided insights into how acquisition, loss, and changes in functions of TRI genes have given rise to the diversity of trichothecene structures. These analyses also suggest both divergence and convergence of TRI gene function during the evolutionary history of trichothecene biosynthesis. What has driven trichothecene structural diversification remains an unanswered question. However, insight into the role of trichothecenes in plant pathogenesis of Fusarium species and into plant glucosyltransferases that detoxify the toxins by glycosylating them point to a possible driver. Because the glucosyltransferases can have substrate specificity, changes in trichothecene structures produced by a fungus could allow it to evade detoxification by the plant enzymes. Thus, it is possible that advantages conferred by evading detoxification have contributed to trichothecene structural diversification. KEY POINTS : • TRI genes have evolved by diverse processes: loss, acquisition and changes in function. • Some TRI genes have acquired the same function by convergent evolution. • Some other TRI genes have evolved divergently to have different functions. • Some TRI genes were acquired or resulted from diversification in function of other genes. • Substrate specificity of plant glucosyltransferases could drive trichothecene diversity.

How repeatable is CTmax within individual brook trout over short- and long-time intervals?

As stream temperatures increase due to factors such as heated runoff from impervious surfaces, deforestation, and climate change, fish species adapted to cold water streams are forced to move to more suitable habitat, acclimate or adapt to increased thermal regimes, or die. To estimate the potential for adaptation, a (within individual) repeatable metric of thermal tolerance is imperative. Critical thermal maximum (CTmax) is a dynamic test that is widely used to measure thermal tolerance across many taxa and has been used in fishes for decades, but its repeatability in most species is unknown. CTmax tests increase water temperature steadily over time until loss of equilibrium (LOE) is achieved. To determine if CTmax is a consistent metric within individual fish, we measured CTmax on the same lab-held individually-marked adult brook trout Salvelinus fontinalis at three different times (August & September 2016, September 2017). We found that CTmax is a repeatable trait (Repeatability ±â€¯S.E.: 0.48 ±â€¯0.14). CTmax of individuals males was consistent over time, but the CTmax of females increased slightly over time. This result indicates that CTmax is a robust, repeatable estimate of thermal tolerance in a cold-water adapted fish.

Proximate composition, lipid utilization and validation of a non-lethal method to determine lipid content in migrating American shad Alosa sapidissima.

Lipid content forms the most important energy reserve in anadromous fish and can limit survival, migration and reproductive success. A fat meter was evaluated and compared with a traditional extractive method of measuring available lipid for migrating American shad Alosa sapidissima in the Connecticut River, U.S.A. The fat meter gives rapid (<10 s) and non-lethal lipid measurements, whereas traditional methods require lethal sampling that is both time consuming and expensive. The fat-meter readings had a strong relationship to traditional lipid extractions for 60 fish, 30 whole body (R2 = 0·72) and 30 fillet only (R2 = 0·81). Additional validation showed that fat-meter readings captured the gradual decrease of lipid in individual fish over time, were not affected by removal of gonads or scales and were stable for fish exposed to water or air for 24 h after death. These experiments indicate that the fat meter can be used as a reliable tool for future A. sapidissima energetic studies, allowing for larger sample sizes and non-lethal sampling.

Trichodiene Production in a Trichoderma harzianum erg1-Silenced Strain Provides Evidence of the Importance of the Sterol Biosynthetic Pathway in Inducing Plant Defense-Related Gene Expression.

Trichoderma species are often used as biocontrol agents against plant-pathogenic fungi. A complex molecular interaction occurs among the biocontrol agent, the antagonistic fungus, and the plant. Terpenes and sterols produced by the biocontrol fungus have been found to affect gene expression in both the antagonistic fungus and the plant. The terpene trichodiene (TD) elicits the expression of genes related to tomato defense and to Botrytis virulence. We show here that TD itself is able to induce the expression of Botrytis genes involved in the synthesis of botrydial (BOT) and also induces terpene gene expression in Trichoderma spp. The terpene ergosterol, in addition to its role as a structural component of the fungal cell membranes, acts as an elicitor of defense response in plants. In the present work, using a transformant of T. harzianum, which is silenced in the erg1 gene and accumulates high levels of squalene, we show that this ergosterol precursor also acts as an important elicitor molecule of tomato defense-related genes and induces Botrytis genes involved in BOT biosynthesis, in both cases, in a concentration-dependent manner. Our data emphasize the importance of a balance of squalene and ergosterol in fungal interactions as well as in the biocontrol activity of Trichoderma spp.

Effects of Trichothecene Production on the Plant Defense Response and Fungal Physiology: Overexpression of the Trichoderma arundinaceum tri4 Gene in T. harzianum.

Trichothecenes are fungal sesquiterpenoid compounds, the majority of which have phytotoxic activity. They contaminate food and feed stocks, resulting in potential harm to animals and human beings. Trichoderma brevicompactum and T. arundinaceum produce trichodermin and harzianum A (HA), respectively, two trichothecenes that show different bioactive properties. Both compounds have remarkable antibiotic and cytotoxic activities, but in addition, trichodermin is highly phytotoxic, while HA lacks this activity when analyzed in vivo. Analysis of Fusarium trichothecene intermediates led to the conclusion that most of them, with the exception of the hydrocarbon precursor trichodiene (TD), have a detectable phytotoxic activity which is not directly related to the structural complexity of the intermediate. In the present work, the HA intermediate 12,13-epoxytrichothec-9-ene (EPT) was produced by expression of the T. arundinaceum tri4 gene in a transgenic T. harzianum strain that already produces TD after transformation with the T. arundinaceum tri5 gene. Purified EPT did not show antifungal or phytotoxic activity, while purified HA showed both antifungal and phytotoxic activities. However, the use of the transgenic T. harzianum tri4 strain induced a downregulation of defense-related genes in tomato plants and also downregulated plant genes involved in fungal root colonization. The production of EPT by the transgenic tri4 strain raised levels of erg1 expression and reduced squalene accumulation while not affecting levels of ergosterol. Together, these results indicate the complex interactions among trichothecene intermediates, fungal antagonists, and host plants.

Evidence for episodic acidification effects on migrating Atlantic salmon Salmo salar smolts.

Field studies were conducted to determine levels of gill aluminium as an index of acidification effects on migrating Atlantic salmon Salmo salar smolts in the north-eastern U.S.A. along mainstem river migration corridors in several major river basins. Smolts emigrating from the Connecticut River, where most (but not all) tributaries were well buffered, had low or undetectable levels of gill aluminium and high gill Na(+) /K(+) -ATPase (NKA) activity. In contrast, smolts emigrating from the upper Merrimack River basin where most tributaries are characterized by low pH and high inorganic aluminium had consistently elevated gill aluminium and lower gill NKA activity, which may explain the low adult return rates of S. salar stocked into the upper Merrimack catchment. In the Sheepscot, Narraguagus and Penobscot Rivers in Maine, river and year-specific effects on gill aluminium were detected that appeared to be driven by underlying geology and high spring discharge. The results indicate that episodic acidification is affecting S. salar smolts in poorly buffered streams in New England and may help explain variation in S. salar survival and abundance among rivers and among years, with implications for the conservation and recovery of S. salar in the north-eastern U.S.A. These results suggest that the physiological condition of outmigrating smolts may serve as a large-scale sentinel of landscape-level recovery of atmospheric pollution in this and other parts of the North Atlantic region.

Temporal dynamics and population genetic structure of Fusarium graminearum in the upper Midwestern United States.

Fusarium graminearum sensu stricto causes Fusarium head blight (FHB) in wheat and barley, and contaminates grains with several trichothecene mycotoxins, causing destructive yield losses and economic impact in the United States. Recently, a F. graminearum strain collected from Minnesota (MN) was determined to produce a novel trichothecene toxin, called NX-2. In order to determine the spatial and temporal dynamics of NX-2 producing strains in MN, North Dakota (ND) and South Dakota (SD), a total of 463 F. graminearum strains were collected from three sampling periods, 1999-2000, 2006-2007 and 2011-2013. A PCR-RFLP based diagnostic test was developed and validated for NX-2 producing strains based on polymorphisms in the Tri1 gene. Trichothecene biosynthesis gene (Tri gene)-based polymerase chain reaction (PCR) assays and ten PCR-restriction fragment length polymorphism (RFLP) markers were used to genotype all strains. NX-2 strains were detected in each sampling period but with a very low overall frequency (2.8%) and were mainly collected near the borders of MN, ND and SD. Strains with the 3ADON chemotype were relatively infrequent in 1999-2000 (4.5%) but increased to 29.4% in 2006-2007 and 17.2% in 2011-2013. The distribution of 3ADON producing strains also expanded from a few border counties between ND and MN in 1999-2000, southward toward the border between SD and MN in 2006-2007 and westward in 2011-2013. Genetic differentiation between 2006-2007 and 2011-2013 populations (3%) was much lower than that between 1999-2000 and 2006-2007 (22%) or 1999-2000 and 2011-2013 (20%) suggesting that most change to population genetic structure of F. graminearum occurred between 1999-2000 and 2006-2007. This change was associated with the emergence of a new population consisting largely of individuals with a 3ADON chemotype. A Bayesian clustering analysis suggested that NX-2 chemotype strains are part of a previously described Upper Midwestern population. However, these analyses also suggest that the NX-2 isolates could represent a distinct population, but that interpretations of population assignment are influenced by the small number of NX-2 strains available for analysis.

Photoperiod control of downstream movements of Atlantic salmon Salmo salar smolts.

This study provides the first direct observations that photoperiod controls the initiation of downstream movement in Atlantic salmon Salmo salar smolts. Under simulated natural day length (LDN) conditions and seasonal increases in temperature, smolts increased their downstream movements five-fold for a period of 1 month in late spring. Under the same conditions, parr did not show changes in downstream movement behaviour. When given a shortened day length (10L:14D) beginning in late winter, smolts did not increase the number of downstream movements. An early increase in day length (16L:8D) in late winter resulted in earlier initiation and termination of downstream movements compared to the LDN group. Physiological status and behaviour were related but not completely coincident: gill Na(+) /K(+) -ATPase activity increased in all treatments and thyroid hormone was elevated prior to movement in 16L:8D treatment. The most parsimonious model describing downstream movement of smolts included synergistic effects of photoperiod treatment and temperature, indicating that peak movements occurred at colder temperatures in the 16L:8D treatment than in LDN, and temperature did not influence movement of smolts in the 10L:14D treatment. The complicated interactions of photoperiod and temperature are not surprising since many organisms have evolved to rely on correlations among environmental cues and windows of opportunity to time behaviours associated with life-history transitions. These complicated interactions, however, have serious implications for phenological adjustments and persistence of S. salar populations in response to climate change.

Migratory patterns of hatchery and stream-reared Atlantic salmon Salmo salar smolts in the Connecticut River, U.S.A.

The timing of downstream migration and detection rates of hatchery-reared Atlantic salmon Salmo salar smolts and stream-reared smolts (stocked 2 years earlier as fry) were examined in the Connecticut River (U.S.A.) using passive integrated transponder (PIT) tags implanted into fish and then detected at a downstream fish bypass collection facility at Turners Falls, MA (river length 192 km). In two successive years, hatchery-reared smolts were released in mid-April and early May at two sites: the West River (river length 241 km) or the Passumpsic (river length 450 km). Hatchery-reared smolts released higher in the catchment arrived 7 to 14 days later and had significantly lower detection rates than smolts stocked lower in the catchment. Hatchery-reared smolts released 3 weeks apart at the same location were detected downstream at similar times, indicating that early-release smolts had a lower average speed after release and longer residence time. The size and gill Na(+) /K(+) -ATPase (NKA) activity of smolts at the time of release were significantly greater for detected fish (those that survived and migrated) than for those that were not detected. Stream-reared pre-smolts (>11·5 cm) from four tributaries (length 261-551 km) were tagged in autumn and detected during smolt migration the following spring. Stream-reared smolts higher in the catchment arrived later and had significantly lower detection rates. The results indicate that both hatchery and stream-reared smolts from the upper catchment will arrive at the mouth of the river later and experience higher overall mortality than fish from lower reaches, and that both size and gill NKA activity are related to survival during downstream migration.

Development of intestinal ion-transporting mechanisms during smoltification and seawater acclimation in Atlantic salmon Salmo salar.

This study investigated the expression of ion transporters involved in intestinal fluid absorption and presents evidence for developmental changes in abundance and tissue distribution of these transporters during smoltification and seawater (SW) acclimation of Atlantic salmon Salmo salar. Emphasis was placed on Na(+) , K(+) -ATPase (NKA) and Na(+) , K(+) , Cl(-) co-transporter (NKCC) isoforms, at both transcriptional and protein levels, together with transcription of chloride channel genes. The nka α1c was the dominant isoform at the transcript level in both proximal and distal intestines; also, it was the most abundant isoform expressed in the basolateral membrane of enterocytes in the proximal intestine. This isoform was also abundantly expressed in the distal intestine in the lower part of the mucosal folds. The protein expression of intestinal Nkaα1c increased during smoltification. Immunostaining was localized to the basal membrane of the enterocytes in freshwater (FW) fish, and re-distributed to a lateral position after SW entry. Two other Nka isoforms, α1a and α1b, were expressed in the intestine but were not regulated to the same extent during smoltification and subsequent SW transfer. Their localization in the intestinal wall indicates a house-keeping function in excitatory tissues. The absorptive form of the NKCC-like isoform (sub-apically located NKCC2 and/or Na(+) , Cl(-) co-transporter) increased during smoltification and further after SW transfer. The cellular distribution changed from a diffuse expression in the sub-apical regions during smoltification to clustering of the transporters closer to the apical membrane after entry to SW. Furthermore, transcript abundance indicates that the mechanisms necessary for exit of chloride ions across the basolateral membrane and into the lateral intercellular space are present in the form of one or more of three different chloride channels: cystic fibrosis transmembrane conductance regulator I and II and chloride channel 3.

Modulating vascular intimal hyperplasia using HSV-1 mutant requires activated MEK.

Outcomes of cardiovascular procedures, such as angioplasty and stent or bypass grafting are limited by failure, predominantly caused by pathological smooth muscle cell (SMC) proliferation, known as intimal hyperplasia. Local delivery of a genetically engineered herpes simplex virus (HSV) is known to block vascular SMC proliferation while allowing for re-endothelialization. However, the mechanism this mutant virus uses to prevent SMC hyperplasia is unknown. The Ras signaling cascade is activated in SMCs undergoing hyperplasia leading to phosphorylation of the mitogen-activated protein kinase (MAPK). In this study we tested the hypothesis that MAPK kinase (MEK) activity is the molecular basis by which SMCs are susceptible to mutant HSV. We show that genetically engineered herpes simplex-1 viruses (HSV-1) can target proliferating SMCs. We demonstrate that the molecular basis of this HSV-1 anti-proliferative effect is MEK activation in SMCs. We demonstrate efficacy and practicality of the MEK-dependent HSV-1 for the treatment of intimal hyperplasia in a clinically relevant in vivo model. Important to this strategy is the ability to modulate the effects by controlling viral dose. These results propel genetically engineered HSV-1 therapy towards clinical evaluation in treatment of intimal hyperplasia.

Involvement of Trichoderma trichothecenes in the biocontrol activity and induction of plant defense-related genes.

Trichoderma species produce trichothecenes, most notably trichodermin and harzianum A (HA), by a biosynthetic pathway in which several of the involved proteins have significant differences in functionality compared to their Fusarium orthologues. In addition, the genes encoding these proteins show a genomic organization differing from that of the Fusarium tri clusters. Here we describe the isolation of Trichoderma arundinaceum IBT 40837 transformants which have a disrupted or silenced tri4 gene, a gene encoding a cytochrome P450 monooxygenase that oxygenates trichodiene to give rise to isotrichodiol, and the effect of tri4 gene disruption and silencing on the expression of other tri genes. Our results indicate that the tri4 gene disruption resulted in a reduced antifungal activity against Botrytis cinerea and Rhizoctonia solani and also in a reduced ability to induce the expression of tomato plant defense-related genes belonging to the salicylic acid (SA) and jasmonate (JA) pathways against B. cinerea, in comparison to the wild-type strain, indicating that HA plays an important function in the sensitization of Trichoderma-pretreated plants against this fungal pathogen. Additionally, the effect of the interaction of T. arundinaceum with B. cinerea or R. solani and with tomato seedlings on the expressions of the tri genes was studied.

Branchial ionocyte organization and ion-transport protein expression in juvenile alewives acclimated to freshwater or seawater.

The alewife (Alosa pseudoharengus) is a clupeid that undergoes larval and juvenile development in freshwater preceding marine habitation. The purpose of this study was to investigate osmoregulatory mechanisms in alewives that permit homeostasis in different salinities. To this end, we measured physiological, branchial biochemical and cellular responses in juvenile alewives acclimated to freshwater (0.5 p.p.t.) or seawater (35.0 p.p.t.). Plasma chloride concentration was higher in seawater-acclimated than freshwater-acclimated individuals (141 mmol l(-1) vs 134 mmol l(-1)), but the hematocrit remained unchanged. In seawater-acclimated individuals, branchial Na(+)/K(+)-ATPase (NKA) activity was higher by 75%. Western blot analysis indicated that the abundance of the NKA α-subunit and a Na(+)/K(+)/2Cl(-) cotransporter (NKCC1) were greater in seawater-acclimated individuals by 40% and 200%, respectively. NKA and NKCC1 were localized on the basolateral surface and tubular network of ionocytes in both acclimation groups. Immunohistochemical labeling for the cystic fibrosis transmembrane conductance regulator (CFTR) was restricted to the apical crypt of ionocytes in seawater-acclimated individuals, whereas sodium/hydrogen exchanger 3 (NHE3) labeling was present on the apical surface of ionocytes in both acclimation groups. Ionocytes were concentrated on the trailing edge of the gill filament, evenly distributed along the proximal 75% of the filamental axis and reduced distally. Ionocyte size and number on the gill filament were not affected by salinity; however, the number of lamellar ionocytes was significantly lower in seawater-acclimated fish. Confocal z-series reconstructions revealed that mature ionocytes in seawater-acclimated alewives occurred in multicellular complexes. These complexes might reduce paracellular Na(+) resistance, hence facilitating Na(+) extrusion in hypo-osmoregulating juvenile alewives after seaward migration.

Greenhouse Studies Reveal Increased Aggressiveness of Emergent Canadian Fusarium graminearum Chemotypes in Wheat.

The role of Fusarium graminearum trichothecene-chemotypes in disease outcomes was evaluated by point inoculation in a series of wheat lines with different levels of resistance to Fusarium head blight (FHB). Four inocula, each consisting of a composite of four strains with either 15-acetyldeoxynivalenol (ADON) chemotypes from "traditional" or emergent populations, a 3-ADON chemotype, or a nivalenol (NIV) chemotype, were compared. The evaluated wheat included Canadian lines with different levels of FHB resistance/susceptibility and double haploid lines developed from crosses of these lines. Highly resistant lines were resistant to infection by all of the F. graminearum chemotypes evaluated. In the moderately susceptible/resistant wheat lines, the 3-ADON producers and the emergent 15-ADON population were, in some instances, more aggressive and resulted in higher Fusarium damaged kernel scores and levels of trichothecene accumulation. The data presented in this study demonstrate the importance of growing highly resistant wheat cultivars in the current climate of an evolving F. graminearum population, and suggest that moderate levels of FHB resistance may not be sufficient to minimize trichothecene contamination of grain from F. graminearum-infected wheat.

Sex Differences in Spotted Hyenas.

The apparent virilization of the female spotted hyena raises questions about sex differences in behavior and morphology. We review these sex differences to find a mosaic of dimorphic traits, some of which conform to mammalian norms. These include space-use, dispersal behavior, sexual behavior, and parental behavior. By contrast, sex differences are reversed from mammalian norms in the hyena's aggressive behavior, social dominance, and territory defense. Androgen exposure early in development appears to enhance aggressiveness in female hyenas. Weapons, hunting behavior, and neonatal body mass do not differ between males and females, but females are slightly larger than males as adults. Sex differences in the hyena's nervous system are relatively subtle. Overall, it appears that the "masculinized" behavioral traits in female spotted hyenas are those, such as aggression, that are essential to ensuring consistent access to food; food critically limits female reproductive success in this species because female spotted hyenas have the highest energetic investment per litter of any mammalian carnivore. Evidently, natural selection has acted to modify traits related to food access, but has left intact those traits that are unrelated to acquiring food, such that they conform to patterns of sexual dimorphism in other mammals.

Identification of loci and functional characterization of trichothecene biosynthesis genes in filamentous fungi of the genus Trichoderma.

Trichothecenes are mycotoxins produced by Trichoderma, Fusarium, and at least four other genera in the fungal order Hypocreales. Fusarium has a trichothecene biosynthetic gene (TRI) cluster that encodes transport and regulatory proteins as well as most enzymes required for the formation of the mycotoxins. However, little is known about trichothecene biosynthesis in the other genera. Here, we identify and characterize TRI gene orthologues (tri) in Trichoderma arundinaceum and Trichoderma brevicompactum. Our results indicate that both Trichoderma species have a tri cluster that consists of orthologues of seven genes present in the Fusarium TRI cluster. Organization of genes in the cluster is the same in the two Trichoderma species but differs from the organization in Fusarium. Sequence and functional analysis revealed that the gene (tri5) responsible for the first committed step in trichothecene biosynthesis is located outside the cluster in both Trichoderma species rather than inside the cluster as it is in Fusarium. Heterologous expression analysis revealed that two T. arundinaceum cluster genes (tri4 and tri11) differ in function from their Fusarium orthologues. The Tatri4-encoded enzyme catalyzes only three of the four oxygenation reactions catalyzed by the orthologous enzyme in Fusarium. The Tatri11-encoded enzyme catalyzes a completely different reaction (trichothecene C-4 hydroxylation) than the Fusarium orthologue (trichothecene C-15 hydroxylation). The results of this study indicate that although some characteristics of the tri/TRI cluster have been conserved during evolution of Trichoderma and Fusarium, the cluster has undergone marked changes, including gene loss and/or gain, gene rearrangement, and divergence of gene function.