Ras2 is important for growth and pathogenicity in Fusarium circinatum.

In this study, we investigated to possible role of Ras2 in Fusarium circinatum- a fungus that causes pine pitch canker disease on many different pine species and has a wide geographic distribution. This protein is encoded by the RAS2 gene and has been shown to control growth and pathogenicity in a number of fungi in a mitogen-activated protein kinase- and/or cyclic adenosyl monophosphate pathway-dependent manner. The aim was therefore to characterize the phenotypes of RAS2 gene knockout and complementation mutants of F. circinatum. These mutants were generated by transforming protoplasts of the fungus with suitable split-marker constructs. The mutant strains, together with the wild type strain, were used in growth studies as well as pathogenicity assays on Pinus patula seedlings. Results showed that the knockout mutant strain produced significantly smaller lesions compared to the complementation mutant and wild type strains. Growth studies also showed significantly smaller colonies and delayed conidial germination in the knockout mutant strain compared to the complement mutant and wild type strains. Interestingly, the knockout mutant strain produced more macroconidia than the wild type strain. Collectively, these results showed that Ras2 plays an important role in both growth and pathogenicity of F. circinatum. Future studies will seek to determine the pathway(s) through which Ras2 controls these traits in F. circinatum.

Evolution of lifestyles in Capnodiales.

The Capnodiales, which includes fungi known as the sooty moulds, represents the second largest order in Dothideomycetes, encompassing morphologically and ecologically diverse fungi with different lifestyles and modes of nutrition. They include saprobes, plant and human pathogens, mycoparasites, rock-inhabiting fungi (RIF), lichenised, epi-, ecto- and endophytes. The aim of this study was to elucidate the lifestyles and evolutionary patterns of the Capnodiales as well as to reconsider their phylogeny by including numerous new collections of sooty moulds, and using four nuclear loci, LSU, ITS, TEF-1α and RPB2. Based on the phylogenetic results, combined with morphology and ecology, Capnodiales s. lat. is shown to be polyphyletic, representing seven different orders. The sooty moulds are restricted to Capnodiales s. str., while Mycosphaerellales is resurrected, and five new orders including Cladosporiales, Comminutisporales, Neophaeothecales, Phaeothecales and Racodiales are introduced. Four families, three genera, 21 species and five combinations are introduced as new. Furthermore, ancestral reconstruction analysis revealed that the saprobic lifestyle is a primitive state in Capnodiales s. lat., and that several transitions have occurred to evolve lichenised, plant and human parasitic, ectophytic (sooty blotch and flyspeck) and more recently epiphytic (sooty mould) lifestyles.

Evaluating the effect of seasonal temperature changes on the efficiency of a rhizofiltration system in nitrogen removal from urban runoff.

The study presents an evaluation of nitrogen removal efficiency of a pilot-scale rhizofiltration system in Pretoria, South Africa. The rhizofiltration system was divided into two sections, one side planted with common reeds (Phragmites australis) and the other side was without plants kept as a control. The objective of the study was to evaluate the influence of seasonal temperature on the removal of nitrogen species from the simulated urban runoff using the rhizofiltration system. The final effluent from the filter was collected bimonthly at different sampling points for 10 months after an application time of 5 min and 25 min. Duplicate samples were taken to determine the concentrations of TKN (Total Kjeldahl nitrogen), ammonium, nitrate and chemical oxygen demand (COD) for the raw influent and final effluent from the rhizofiltration system. Temperature and pH were determined on-site. During the monitoring period, there was no significant difference in the inflow concentration of ammonium in colder and warmer months for both planted and control sides. Furthermore, the composition of the feed medium to the rhizofilter was kept the same in both cold and warm season and for both planted and control sides. The removal of ammonium in colder and warmer months was not significant in both systems. At an average temperature increase of 5.2 °C in the warmer months, the ammonium removal efficiency in the planted side increased by 7.5%, while for the control side the removal efficiency increased by 2.4%. The difference in removal was not significant between the averages of effluent ammonium after an application time of 25 min in colder versus warmer months for the planted and control sides of the system. Furthermore, an increased nitrification rate was more evident in the planted than in the control side, which was subsequently denitrified. It was observed that 60.4% of nitrate concentration was potentially removed in the planted side whereas 45.4% was potentially denitrified in the control side. These results suggest positive correlation between nitrate concentration and the potential for denitrification. The nitrate removal efficiency dropped to 32.2% for the planted site and to 26.1% for the control system in colder months. Temperature had an effect on nitrogen removal, since nitrogen removal efficiency decreased in colder months. Complete nitrogen removal could not be achieved under the operating conditions.

Evaluation of the efficiency of selected wastewater treatment processes in removing selected perfluoroalkyl substances (PFASs).

In this study, the efficiencies of selected wastewater treatment plants (WWTPs) to remove selected perfluoroalkyl substances (PFASs) during wastewater treatment processes were evaluated. For this purpose, influent samples from Daspoort, Zeekoegat and Phola WWTPs, were initially screened for the presence of sixteen different PFASs of which only seven were detected. These include: perfluorobutanoic acid (PFBA), perfluoro-n-pentanoic acid (PFPeA), perfluorohexanoic acid (PFHxA), perfluorooctanoic acid (PFOA), perfluorodecanoic acid (PFDA), perfluorohexane sulfonate (L-PFHxS), and perfluorooctane sulfonate (L-PFOS). To determine the concentrations of these PFASs, wastewater samples were subjected to solid-phase extraction followed by liquid chromatography-tandem mass spectrometry. The results showed that L-PFOS was the dominant compound with the highest concentration of 508 ± 258 ng/L at Daspoort WWTP. Overall, the three WWTPs could not achieve the complete influent-to-effluent removal of the PFASs and the best removals were observed at Zeekoegat WWTP. The removal efficiency of the different unit processes varied from one plant to another and also from each type of PFASs. At Daspoort, the removal efficiency of the primary settling tanks was poor and the highest removal reached 39% for PFHxA. The activated sludge (AS) of this WWTP achieved the highest removal of 84% for the L-PFOS. At Zeekoegat, the AS achieved the highest removal of 94% for the L-PFOS. The anaerobic pond at Phola achieved a higher removal of 80% for the L-PFOS. However, no removal was observed downstream of the biological filter for the same compound. Poor removal efficiency was reported downstream of the wetland at Phola except for the PFOA (16%).

Application of hydrolysis probe analysis to identify clade types of the malaria vector mosquito Anopheles funestus sensu stricto from Muheza, northeastern Tanzania.

A hydrolysis probe analysis (TaqMan assay) was used to study clade types in Anopheles funestus sensu stricto Giles, a major malaria vector in sub-Saharan Africa, with specimens collected from Muheza in Tanga, northeastern Tanzania. A total of 186 An. funestus specimens were analysed, revealing that 176 (94.6%) were of clade I and 10 (5.4%) of clade II. These findings extend the distribution of clade type II from southern Mozambique and northern Zambia to northeastern Tanzania. The technique used can also be of great value in assessing the role and contribution of these clade types in malaria transmission and insecticide resistance frequencies for An. funestus s.s.

Potential Treatment Options in a Post-antibiotic Era.

Following the Golden Age of antibiotic discovery in the previous century, the rate of antibiotic discovery has plummeted during the past 50 years while the incidence of antimicrobial resistance is ever-increasing. Presently, humankind is forced to address a major public health threat in the form of multiple drug resistance and urgent action is required to halt the advent of a post-antibiotic era. This chapter aims to draw the attention to the escalating global crisis of antimicrobial resistance fueled by the irresponsible use of antibiotics in healthcare and animal production sectors. The merits of alternative prevention and treatment options, including vaccines, herbal products, bacteriophages, and improved biosecurity measures are also discussed.

The Influence of Insecticide Resistance, Age, Sex, and Blood Feeding Frequency on Thermal Tolerance of Wild and Laboratory Phenotypes of Anopheles funestus (Diptera: Culicidae).

Resistance to insecticides is a global phenomenon and is increasing at an unprecedented rate. How resistant and susceptible strains of malaria vectors might differ in terms of life history and basic biology is often overlooked, despite the potential importance of such information in light of changing climates. Here, we investigated the upper thermal limits (ULT50) of wild and laboratory strains of Anopheles funestus Giles mosquitoes, including resistance status, sex, age, and blood feeding status as potential factors influencing ULT50. No significant differences in ULT50 were observed between strains displaying different resistance patterns, nor was there a significant difference between wild and laboratory strains. In some instances, strains showed a senescence response, displaying decreased ULT50 with an increase in age, and differences between males and females (females displaying higher ULT50 than males). Blood feeding did not seem to influence ULT50 in any way. For An. funestus, it seems evident that there is no cost to resistance despite what is displayed in other anopheline species. This could have significant impacts for vector control, with resistant populations of An. funestus performing just as well, if not better, than susceptible strains, especially under changing environmental conditions such as those expected to occur with climate change.

Saprophytic and pathogenic fungi in the Ceratocystidaceae differ in their ability to metabolize plant-derived sucrose.

BACKGROUND: Proteins in the Glycoside Hydrolase family 32 (GH32) are carbohydrate-active enzymes known as invertases that hydrolyse the glycosidic bonds of complex saccharides. Fungi rely on these enzymes to gain access to and utilize plant-derived sucrose. In fungi, GH32 invertase genes are found in higher copy numbers in the genomes of pathogens when compared to closely related saprophytes, suggesting an association between invertases and ecological strategy. The aim of this study was to investigate the distribution and evolution of GH32 invertases in the Ceratocystidaceae using a comparative genomics approach. This fungal family provides an interesting model to study the evolution of these genes, because it includes economically important pathogenic species such as Ceratocystis fimbriata, C. manginecans and C. albifundus, as well as saprophytic species such as Huntiella moniliformis, H. omanensis and H. savannae. RESULTS: The publicly available Ceratocystidaceae genome sequences, as well as the H. savannae genome sequenced here, allowed for the identification of novel GH32-like sequences. The de novo assembly of the H. savannae draft genome consisted of 28.54 megabases that coded for 7 687 putative genes of which one represented a GH32 family member. The number of GH32 gene family members appeared to be related to the ecological adaptations of these fungi. The pathogenic Ceratocystis species all contained two GH32 family genes (a putative cell wall and a putative vacuolar invertase), while the saprophytic Huntiella species had only one of these genes (a putative cell wall invertase). Further analysis showed that the evolution of the GH32 gene family in the Ceratocystidaceae involved transposable element-based retro-transposition and translocation. As an example, the activity of a Fot5-like element likely facilitated the assembly of the genomic regions harbouring the GH32 family genes in Ceratocystis. CONCLUSIONS: This study provides insight into the evolutionary history of the GH32 gene family in Ceratocystidaceae. Our findings suggest that transposable elements shaped the evolution of the GH32 gene family, which in turn determines the sucrolytic activities and related ecological strategies of the Ceratocystidaceae species that harbour them. The study also provides insights into the role of carbohydrate-active enzymes in plant-fungal interactions and adds to our understanding of the evolution of these enzymes and their role in the life style of these fungi.

Huntiella decorticans sp. nov. (Ceratocystidaceae) associated with dying Nothofagus in Patagonia.

During a survey of ophiostomatoid fungi in native forests of southern Argentina, several isolates of Huntiella species were obtained from Nothofagus trees. Sequences of multiple gene regions were used to identify these fungi, and their pathogenicity was tested on N. pumilio and N. dombeyi. Phylogenetic analyses revealed a novel taxon described here as H. decorticans sp. nov. Inoculations on N. dombeyi and N. pumilio in the forest showed that H. decorticans is able to produce localized lesions on healthy Nothofagus trees.

Population Growth and Its Impact on the Design Capacity and Performance of the Wastewater Treatment Plants in Sedibeng and Soshanguve, South Africa.

This study investigated the effects of population growth on the performance of the targeted wastewater treatment plants in Sedibeng District and Soshanguve peri-urban area, South Africa. The impact of population growth was assessed in terms of plant design, operational capacity (flow rate) and other treatment process constraints. Between 2001 and 2007, the number of households connected to the public sewerage service increased by 15.5, 17.2 and 37.8% in Emfuleni, Lesedi and Midvaal Local Municipalities, respectively. Soshanguve revealed a 50% increment in the number of households connected to the sewerage system between 1996 and 2001. Except for Sandspruit (-393.8%), the rate of influent flows received by Meyerton increased by 6.8 ML/day (67.8%) and 4.7 ML/day (46.8%) during the dry and wet seasons, respectively. The flow rate appeared to increase during the wet season by 6.8 ML/day (19.1%) in Leeuwkuil and during the dry season by 0.8 ML/day (3.9%) in Rietgat. Underperformance of the existing wastewater treatment plants suggests that the rapid population growth in urban and peri-urban areas (hydraulic overloading of the wastewater treatment plants) and operational constraints (overflow rate, retention time, oxygen supply capacity of the plants and chlorine contact time) resulted in the production of poor quality effluents in both selected areas. This investigation showed that the inefficiency of Meyerton Wastewater Treatment Plant was attributed to the population growth (higher volumes of wastewater generated) and operational constraints, while the cause of underperformance in the other three treatment plants was clearly technical (operational).

Seasonal variation of nutrient loads in treated wastewater effluents and receiving water bodies in Sedibeng and Soshanguve, South Africa.

The discharge of inadequately treated wastewater effluent presents a major threat to the aquatic environment and public health worldwide. As a water-scarce country, South Africa is facing an alarming situation since most of its wastewater discharges are not meeting the permissible limit. The aim of this study was to assess the physicochemical quality of treated wastewater effluents and their impact on receiving water bodies. During the study period, pH, temperature, free chlorine residue (Cl(-)), dissolved oxygen (DO), nitrate (NO3 (-1)), orthophosphate (PO4 (-3)) and chemical oxygen demand (COD) were measured in order to ascertain whether the selected wastewater systems in Sedibeng and Soshanguve complied with the South African and World Health Organization standards during wet and dry seasons. These parameters were analysed for samples collected from raw wastewater influent, treated wastewater effluent and receiving water bodies. The study was carried out between August 2011 and May 2012, and samples were collected on a weekly basis during both seasons. The physicochemical quality of effluents did not comply with the regulatory limits set by South Africa in terms of pH in Meyerton, Rietgat and Sandspruit (pH 7.6 to 8.1); free chlorine in Sandspruit (0.27 ± 0.05 mg/L); nitrate in Leeuwkuil and Rietgat (2.1 and 3.8 mg/L, respectively) during the wet season; orthophosphate in Meyerton during the wet season and in Sandspruit during the dry season (1.3 mg PO4 (-3) as P/L and 1.1 mg PO4 (-3) as P/L, respectively); and chemical oxygen demand in Rietgat during the dry season and in Sandspruit during the wet season (75.5 and 35 mg/L, respectively). Furthermore, the quality of the receiving water bodies did not comply with the South African standards recommended for pH, chemical oxygen demand and orthophosphate and DO (5 mg/L) in Rietgat during the wet season. The geometric mean of the water quality index values ranged between 32.4 and 36.9 for the effluent samples and between 38.1 and 65.7 for the receiving water bodies. These findings revealed that the receiving water bodies were classified as having "poor" quality status, except Leeuwkuil receiving water body (Vaal River) and Sandspruit upstream (Sandspruit stream). The dry season showed a relatively lower water quality index. This situation might be attributed to the higher amount of organic matter and lower microbial activities in the receiving water bodies. This study suggests that wastewater effluents and receiving water systems should be monitored regularly to ensure best practices with regard to nutrient treatment and discharge of wastewater.

Larval salinity tolerance of two members of the Anopheles funestus group.

The Anopheles funestus group (Diptera: Culicidae) is one of the main species groups involved in malaria transmission in the Afrotropical regions. Basic research into this group has been limited because its members are eurygamic (they tend not to mate in confined spaces), which makes laboratory colonization difficult. Currently, only a few An. funestus Giles colonies are available and no colonies of other members of the group have been established. As information on the larval biology of members of the An. funestus group is limited, the present study aims to determine the effects of different salt concentrations on survival rates of the aquatic stages of two members of the An. funestus group, Anopheles funestus and Anopheles rivulorum Leeson. There were statistically significant negative trends in hatch rate and larval survival rate in An. funestus with increasing salt concentrations, with no larvae surviving to pupae at concentrations that included >15% seawater. Anopheles rivulorum, by contrast, showed no significant trends in hatch rate or larval survival with increasing salt concentrations. This is the first report on salinity tolerance in An. rivulorum. A basic understanding of these variations in salinity tolerance provides vital information on the biology, ecology and colony rearing of members of the An. funestus group.

Fungal Planet description sheets: 281-319.

Novel species of fungi described in the present study include the following from South Africa: Alanphillipsia aloeicola from Aloe sp., Arxiella dolichandrae from Dolichandra unguiscati, Ganoderma austroafricanum from Jacaranda mimosifolia, Phacidiella podocarpi and Phaeosphaeria podocarpi from Podocarpus latifolius, Phyllosticta mimusopisicola from Mimusops zeyheri and Sphaerulina pelargonii from Pelargonium sp. Furthermore, Barssia maroccana is described from Cedrus atlantica (Morocco), Codinaea pini from Pinus patula (Uganda), Crucellisporiopsis marquesiae from Marquesia acuminata (Zambia), Dinemasporium ipomoeae from Ipomoea pes-caprae (Vietnam), Diaporthe phragmitis from Phragmites australis (China), Marasmius vladimirii from leaf litter (India), Melanconium hedericola from Hedera helix (Spain), Pluteus albotomentosus and Pluteus extremiorientalis from a mixed forest (Russia), Rachicladosporium eucalypti from Eucalyptus globulus (Ethiopia), Sistotrema epiphyllum from dead leaves of Fagus sylvatica in a forest (The Netherlands), Stagonospora chrysopyla from Scirpus microcarpus (USA) and Trichomerium dioscoreae from Dioscorea sp. (Japan). Novel species from Australia include: Corynespora endiandrae from Endiandra introrsa, Gonatophragmium triuniae from Triunia youngiana, Penicillium coccotrypicola from Archontophoenix cunninghamiana and Phytophthora moyootj from soil. Novelties from Iran include Neocamarosporium chichastianum from soil and Seimatosporium pistaciae from Pistacia vera. Xenosonderhenia eucalypti and Zasmidium eucalyptigenum are newly described from Eucalyptus urophylla in Indonesia. Diaporthe acaciarum and Roussoella acacia are newly described from Acacia tortilis in Tanzania. New species from Italy include Comoclathris spartii from Spartium junceum and Phoma tamaricicola from Tamarix gallica. Novel genera include (Ascomycetes): Acremoniopsis from forest soil and Collarina from water sediments (Spain), Phellinocrescentia from a Phellinus sp. (French Guiana), Neobambusicola from Strelitzia nicolai (South Africa), Neocladophialophora from Quercus robur (Germany), Neophysalospora from Corymbia henryi (Mozambique) and Xenophaeosphaeria from Grewia sp. (Tanzania). Morphological and culture characteristics along with ITS DNA barcodes are provided for all taxa.

Botryosphaeriaceae partially overlap on asymptomatic and symptomatic tissues of Anacardiaceae in agroecosystems and conservation areas in northern South Africa.

Members of the Botryosphaeriaceae are well-known endophytes and stress-related pathogens. We recently characterised the diversity of Botryosphaeriaceae in healthy tissues of three tree species in the Anacardiaceae, namely Sclerocarya birrea, Mangifera indica and Lannea schweinfurthii. Here we ask how that diversity compares with the Botryosphaeriaceae diversity associated with dieback on those tree species. Samples were collected from agroecosystems (Tshikundamalema and Tshipise in Limpopo) and conservation areas (Nwanedi and the Mapungubwe National Park in Limpopo and the Kruger National Park in Mpumalanga) ecosystems. Species were characterised using multigene sequence data and morphological data. Diplodia allocellula, Dothiorella brevicollis, Do. viticola, Lasiodiplodia crassispora, L. mahajangana and Neofusicoccum parvum occurred on both asymptomatic and symptomatic samples. Dothiorella dulcispinea, L. gonubiensis and L. exigua, as well as a previously unknown species described here as Oblongocollomyces ednahkunjekuae sp. nov, only occurred in asymptomatic branches. An interesting aspect of the biology of O. ednahkunjekuaeae is that it appears to be adapted to higher temperatures, with an optimum growth at 30 °C, and faster growth at 35 °C than at 25 °C. Lasiodiplodia pseudotheobromae only occurred in symptomatic branches. Neofusicoccum parvum was notably absent from conservation areas, and in agroecosystem it was most common on M. indica. Only L. crassispora and L. mahajangana overlapped on all three tree species and were the dominant species associated with dieback. These results show that not all Botryosphaeriaceae occurring asymptomatically in an area contribute equally to disease development on a related group of hosts, and that environmental disturbance plays a significant role in the distribution of N. parvum. Citation: Slippers B, Ramabulana E, Coetzee MPA (2024). Botryosphaeriaceae partially overlap on asymptomatic and symptomatic tissues of Anacardiaceae in agroecosystems and conservation areas in northern South Africa. Fungal Systematics and Evolution 13: 131-142. doi: 10.3114/fuse.2024.13.07.

The accuracy of the Thompson score in predicting early outcome in neonates with hypoxic ischaemic encephalopathy treated with therapeutic cooling in a tertiary hospital.

BACKGROUND: Hypoxic ischaemic encephalopathy (HIE) is one of the major contributors to neonatal mortality and morbidity in developing countries. Scarcity of resources limits clinicians in optimally caring for these patients. Optimal utilisation of clinical tools such as the Thompson score (TS) can assist in improving care by classifying the severity of HIE followed by appropriate treatment. OBJECTIVES: The primary objective was to study the correlation of the TS and early neonatal outcomes in infants with HIE who received therapeutic hypothermia (TH). Secondary objectives were to investigate the correlation of blood gas values with the TS, need for resuscitation with TS, target organ damage (TOD) with TS and the most common risk factors associated with HIE in Tembisa Provincial Tertiary Hospital (TPTH). METHODS: This was a retrospective record review of infants admitted with HIE from January 2018 to August 2019 at the TPTH neonatal unit. Infants had to have successfully completed TH. RESULTS: Ninety-three infants met the inclusion criteria, with 32, 48 and 13 being classified into the mild, moderate and severe categories by TS, respectively. The median length of stay (LOS) was noted to rise with a rising TS, recorded to be 7, 8 and 9 days in the mild, moderate and severe groups, respectively. The mortality rate in the study was calculated to be 2.1%, and there was no significant difference across the groups (p=0.231). A need for antiseizure medication (ASM) on discharge was significantly associated with severe HIE (p=0.028). Hypertension was a frequent chronic illness, noted in 11.3% of the mothers. The most frequent perinatal risk factor was meconium aspiration (50.5%), followed by prolonged second stage of labour (PSSL) (17.2%). A higher TS (severe group) was associated with prolonged resuscitation for >10 minutes (p=0.001) and a need for adrenaline (p=0.008). The frequency of cardiac impairment, liver impairment and clinical seizures increased with a higher TS category (p=0.23, p=0.35 and p=0.51, respectively). On blood gas analysis, a low pH and a high base deficit were associated with severe HIE (p=0.027, p=0.061 respectively). CONCLUSION: The TS is still a useful clinical tool in the era of TH as it is able to predict some early neonatal outcomes such as LOS and a need for ASM at discharge. It is also able to demonstrate increased frequency of duration of resuscitation and a need for adrenaline in severely encephalopathic infants compared with mild. A high TS is also associated with severe metabolic acidosis and increased frequency of TOD. Maternal hypertension, meconium-stained liquor and PSSL are the common risk factors for HIE at TPTH.

Post-TB health and wellbeing.

TB affects around 10.6 million people each year and there are now around 155 million TB survivors. TB and its treatments can lead to permanently impaired health and wellbeing. In 2019, representatives of TB affected communities attending the '1st International Post-Tuberculosis Symposium´ called for the development of clinical guidance on these issues. This clinical statement on post-TB health and wellbeing responds to this call and builds on the work of the symposium, which brought together TB survivors, healthcare professionals and researchers. Our document offers expert opinion and, where possible, evidence-based guidance to aid clinicians in the diagnosis and management of post-TB conditions and research in this field. It covers all aspects of post-TB, including economic, social and psychological wellbeing, post TB lung disease (PTLD), cardiovascular and pericardial disease, neurological disability, effects in adolescents and children, and future research needs.

Age-related pyrethroid resistance is not a function of P450 gene expression in the major African malaria vector, Anopheles funestus (Diptera: Culicidae).

Anopheles funestus is a major vector of malaria in most of the African region. Resistance to pyrethroid and carbamate insecticides has been recorded in populations of this species in South Africa and Mozambique. The P450 gene, CYP6P9, has been shown to be highly transcribed in a permethrin (pyrethroid)-resistant laboratory strain, FUMOZ-R, originating from southern Mozambique. We examined the relationship between pyrethroid resistance and gene transcription levels of two closely related genes, CYP6P9 and CYP6P13, in FUMOZ-R. Levels of resistance to 0.75% permethrin were determined based on standard WHO insecticide susceptibility assays using females and males of different ages, ranging from 3 to 30 days old. The transcription levels of the two genes were quantified using qPCR for each age cohort. In the WHO insecticide susceptibility assays, survival of both males and females significantly decreased as age increased. Quantitative analysis of the two genes CYP6P9 and CYP6P13 showed the highest levels of expression at 10 days of age. There was no correlation between expression of these two genes and pyrethroid survival by age. We conclude that the resistance of this mosquito strain to permethrin is not directly related to age-mediated differences in CYP6P9 and CYP6P13 expression.

Deciphering the effect of FUB1 disruption on fusaric acid production and pathogenicity in Fusarium circinatum.

Fusarium circinatum is an important pathogen of pine trees. However, little is known regarding the molecular processes underlying its pathogenesis. We explored the potential role of the phytotoxin fusaric acid (FA) in the pathogenicity of the fungus. FA is produced by products of the FUB biosynthesis gene cluster, containing FUB1-12. Of these, FUB1 encodes the core polyketide synthase, which we disrupted. We used the resulting mutant strain to investigate whether FUB1 and FA production play a role in the virulence of F. circinatum on pine. Our results showed that FA production was abolished both in vitro and in planta. However, bikaverin production was increased in the knockout mutant. FUB1 disruption also corresponded with downregulation of a F. circinatum homologue of LaeA, a master transcriptional regulator of secondary metabolism. Lesion lengths produced by the FUB1 knockout mutant on inoculated Pinus patula seedlings were significantly smaller than those produced by the wild type strain. Collectively, these results show that FUB1 plays a role in FA production in F. circinatum, and that this gene contributes to the aggressiveness of F. circinatum on P. patula. This study will contribute to the limited knowledge we have about the molecular basis of pathogenicity in this fungus.

Evaluation of the pyrrole insecticide chlorfenapyr against pyrethroid resistant and susceptible Anopheles funestus (Diptera: Culicidae).

OBJECTIVE: To evaluate the pyrrole insecticide chlorfenapyr, which has a novel non-neurotoxic mode of action and is a promising alternative to conventional adulticides, against Anopheles funestus. METHOD: The toxicity of a range of concentrations of chlorfenapyr against pyrethroid resistant and susceptible laboratory reared southern African An. funestus was assessed using standard WHO protocols and analysed using probit analysis. RESULTS: The pyrethroid resistant strain showed consistently higher LD50 and LD95 values compared to the susceptible strain, but these differences were not statistically significant and the magnitude was twofold at most. The LD50 values recorded for An. funestus are approximately three-fold higher than those reported elsewhere for other species of anopheline. CONCLUSIONS: Monooxygenase based pyrethroid resistance in An. funestus does not influence the toxic effect of chlorfenapyr. It is unlikely that such a small decrease in susceptibility of An. funestus to chlorfenapyr relative to other anophelines would have any operational implications. Chlorfenapyr is an important addition to insecticides available for malaria vector control, and could be used as a resistance management tool to either circumvent or slow the development of resistance.

Sequence characterization of cytochrome P450 CYP6P9 in pyrethroid resistant and susceptible Anopheles funestus (Diptera: Culicidae).

Anopheles funestus, one of the main African malaria vectors, caused a major malaria outbreak in South Africa during 1999/2000, even though South Africa had an effective vector control program in place. The outbreak was due to pyrethroid resistant An. funestus invading KwaZulu/Natal. Increased activity of cytochrome P450 (monooxygenase) was responsible for the pyrethroid resistance in this species. A monooxygenase gene, CYP6P9, was highly overexpressed in the pyrethroid-resistant strain compared with a susceptible strain. Characterization of this gene as well as the redox partners involved in the catalytic cycle of P450s was investigated. The full length of the CYP6P9 sequence was isolated, sequenced and compared between the pyrethroid-resistant and -susceptible strains. Sequence identity between the two strains was 99.3%; the sequence differences were mainly outside of the conserved regions. The functional significance is still unknown, but it is feasible that these variations are associated with differences in insecticide metabolism. A second CYP6 gene (CYP6P13) was also isolated; it shared close similarities with CYP6P9. The putative redox partners, cytochrome b(5) (cyt b(5)) and NADPH-cytochrome P450 reductase (CPR), were isolated from An. funestus (resistant strain) and showed high levels of sequence identity to other insect cyt b(5) and CPRs. Isolation of the coding sequences CYP6P9 and its cognate redox partners enables expression of functional recombinant protein for biochemical and structural analysis.