Application of vibrational spectroscopy in the quality assessment of Buchu oil obtained from two commercially important Agathosma species (Rutaceae)

Quality assessment of natural raw materials and derived consumer products is often done using conventional analytical techniques such as liquid and gas chromatography which are expensive and time consuming. This paper reports on the use of vibrational spectroscopy techniques as possible alternatives for the rapid and inexpensive assessment of the quality of ‘buchu oil’ obtained from two South African species; Agathosma betulina and Agathosma crenulata belonging to the Rutaceae family. Samples of A. betulina (55) and A. crenulata (16) were collected from different natural localities and cultivation sites in South Africa. The essential oil was obtained by hydrodistillation and scanned on Near infrared (NIR), mid infrared (MIR) and Raman spectrometers. The spectral data obtained was processed using chemometric techniques and orthogonal partial least squares discriminant analysis (OPLS-DA) was used to clearly differentiate between A. betulina and A. crenulata. The OPLS-DA technique also proved to be a useful tool to identify wave regions that contain biomarkers (peaks) that contributed to the separation of the two species. The three spectroscopy techniques were also evaluated for their ability to accurately predict the percentage composition of seven major compounds that occur in A. betulina ‘buchu’ oil. Using GC–MS reference data, calibration models were developed for the MIR, NIR and Raman spectral data to predict/profile the major compounds in ‘buchu oil’. A comparison of the three spectroscopy techniques showed that MIR together with PLS algorithms produced the best model (R²X = 0.96; R²Y = 0.88 and Q²Ycum = 0.85) for the quantification of six of the seven major oil constituents. The MIR model showed high predictive power for pseudo-diosphenol (R² = 0.97), isomenthone (R² = 0.97), menthone (R² = 0.90), limonene (R² = 0.91), pulegone (R² = 0.96) and diosphenol (R² = 0.85). These results illustrate the potential of MIR spectroscopy as a rapid and inexpensive alternative to predict the major compounds in buchu oil.     

Chromosomal rearrangements associated with pelagic larval duration in Labridae (Perciformes)

The family Labridae is one of the largest and most important groups of reef fishes in the Southern Atlantic. There is a remarkable ecologic interest in this family because of their complex interactions in the reef environment. Predictions of genetic variability in fish based on biological patterns have often been contradictory. The present work aimed to increase the cytogenetic data about the family and verify the possible correlation between larval pelagic phase and chromosomal rearrangements based on the putative basal Perciformes karyotype (2n = 48a). Therefore, cytogenetic analyses were performed in the species Halichoeres brasiliensis (2n = 48, 48a, FN = 48); Halichoeres radiatus (2n = 48, 48a, FN = 48) and in three populations of Halichoeres poeyi (2n = 48, 4m + 44st-a, FN = 52) from Brazilian coastline. A conserved diploid number was observed in all species and populations. Single NORs were identified in H. brasiliensis and in two populations of H. poeyi (BA and RJ), while multiple NORs were observed in H. radiatus and in H. poeyi from Rio Grande do Norte. The constitutive heterochromatin is reduced and distributed over centromeric and pericentromeric regions. The ribosomal sites allowed differentiating two groups of H. poeyi along the Brazilian coast; one of them comprising the population from RN, bearing multiple NORs, and another representing the populations from BA and RJ, bearing single NORs. The recently separated species, H. brasiliensis and H. radiatus, although presenting similar diploid numbers and chromosomal formulae, could be distinguished by the number of NOR-bearing chromosomes. The results revealed an evolutionary pattern chiefly derived from pericentric inversions. The correlation between larval pelagic phase and cytogenetic data on Labridae indicates that the degree of karyotypic diversification reported within this family, ranging from a highly conserved to a derived pattern, is probably influenced by the species-specific duration of larval pelagic phase.     

Cytotoxicity from sulfide exposure in a sulfide-tolerant marine invertebrate

It is unknown whether sulfide-tolerant marine invertebrates suffer cytotoxicity from sulfide exposure in vivo at environmentally-relevant concentrations. We tested this with the mudflat polychaete Glycera dibranchiata. Exposure of live animals to sulfide up to 2.4 mmol l^− 1 for 24 h did not affect animal survival, and animal condition (gross appearance and activity) was unaffected at sulfide concentrations up to 0.25 mmol l^− 1. However, animal condition was decreased at higher sulfide concentrations (P < 0.0001, n = 33). Coelomic fluid obtained from the animals showed decreased erythrocyte count (P = 0.0035, n = 14), indicating cell loss, and increased propidium iodide and Hoechst 33324 staining (P < 0.0001 and P = 0.0003, respectively, n = 14), indicating loss of plasma membrane integrity, even at sulfide concentrations that produced no change in animal condition. When G. dibranchiata were allowed a 72 h recovery period following 24 h sulfide exposure, there was no overall improvement in condition (P ≥ 0.12, n = 7-8), and worms that had been exposed to 1 mmol l^−  1 sulfide still had an erythrocyte count that was less than half that of control animals (P = 0.028, n = 7). The inability to completely recover the cell count was at least partially due to impaired production of new erythrocytes, since analysis of BrdU incorporation indicated that erythrocyte proliferation was reduced from 2% per day in control animals to 0.12% per day in animals exposed to 1 mmol l^− 1 sulfide (P = 0.010, n = 21). Together, these findings indicate that at least some sulfide-tolerant marine invertebrates experience significant cellular injury and impaired tissue proliferation when exposed to environmentally relevant sulfide concentrations, even when the appearance and behavior of the animal appear unaffected.     

Cercopithecidae from the Pliocene Chiwondo beds, Malawi-rift

There are a total of 16 fossil cercopithecid specimens, representing at least 10 individuals, from the Chiwondo Beds of northern Malawi. The majority of this material is derived from the Middle Pliocene Unit 3A, but one specimen is from the Early Pliocene Unit 2. This latter specimen is from a papionin of indeterminate genus similar in size to Parapapio ado and Pliopapio alemui. Among the specimens from Unit 3A, two species can be diagnosed: an indeterminate species of Theropithecus, and a species of Parapapio similar in dental size to P. broomi. Neither of the genera from Unit 3A are exclusive to either East Africa or South Africa. Their relative abundances, however, are more similar to Middle Pliocene South African sites where Parapapio is the most common primate genus, and Theropithecus is comparatively rare. This is in contrast to similarly-aged East African sites where Theropithecus is by far the most abundant genus.     

Comparative cytogenetics of opisthorchid species (Trematoda, Opisthorchiidae)

In the present study karyotypes and chromosomes of five species of the family Opisthorchiidae (Opisthorchis felineus (Rivolta, 1884), O. viverrini (Poirier, 1886), Metorchis xanthosomus (Creplin, 1846), M. bilis (Braun, 1893), and Clonorchis sinensis (Cobbold, 1875)) were compared. Karyotypes of O. felineus, M. xanthosomus, M. bilis and C. sinensis consist of two pairs of large meta- and submetacentrics and five pairs of small chromosomes (2n = 14). The karyotype of O. viverrini is 2n = 12, which indicates a fusion of two chromosomes of opisthorchid ancestral karyotype. Analysis of mitotic and meiotic chromosomes was performed by heterologous in situ hybridization of microdissected DNA probes obtained from chromosomes 1 and 2 of O. felineus and chromosomes 1 and 2 of M. xanthosomus. Results of chromosome staining (C- and AgNOR-banding) and FISH of telomeric probes and ribosomal DNA probe on opisthorchid chromosomes were used for chromosome comparison. Data on chromosome number in opisthorchid species were also discussed.     

Annesorhiza asparagoides (Apiaceae), a new species from the Cederberg Mountains, Western Cape Province, South Africa

A highly localised new species from the Cederberg Mountains near Wuppertal in the Western Cape Province is described. Annesorhiza asparagoides B.-E. Van Wyk, collected for the first time in 2009, differs from all other species of Annesorhiza (and the closely related Chamarea) in the unusual leaf structure, with crowded, subsessile, acicular leaf segments, resulting in dense, bottlebrush-like pinnae. The new species has a cluster of 10 or more slender roots, small (< 150 mm long), sparsely hairy leaves and small (± 8 mm long), oblong, conspicuously ribbed, homomericarpic fruits.     

Host associations and evolutionary relationships of avian blood parasites from West Africa

The host specificity of blood parasites recovered from a survey of 527 birds in Cameroon and Gabon was examined at several levels within an evolutionary framework. Unique mitochondrial lineages of Haemoproteus were recovered from an average of 1.3 host species (maximum = 3) and 1.2 host families (maximum = 3) while lineages of Plasmodium were recovered from an average of 2.5 species (maximum = 27) and 1.6 families (maximum = 9). Averaged within genera, lineages of both Plasmodium and Haemoproteus were constrained in their host distribution relative to random expectations. However, while several individual lineages within both genera exhibited significant host constraint, host breadth varied widely among related lineages, particularly within the genus Plasmodium. Several lineages of Plasmodium exhibited extreme generalist host-parasitism strategies while other lineages appeared to have been constrained to certain host families over recent evolutionary history. Sequence data from two nuclear genes recovered from a limited sample of Plasmodium parasites indicated that, at the resolution of this study, inferences regarding host breadth were unlikely to be grossly affected by the use of parasite mitochondrial lineages as a proxy for biological species. The use of divergent host-parasitism strategies among closely related parasite lineages suggests that host range is a relatively labile character. Since host specificity may also influence parasite virulence, these results argue for considering the impact of haematozoa on avian hosts on a lineage-specific basis.     

Glycoprotein 60 diversity in C. hominis and C. parvum causing human cryptosporidiosis in NSW, Australia

Management and control of cryptosporidiosis in human requires knowledge of Cryptosporidium species contributing to human disease. Markers that are able to provide information below the species level have become important tools for source tracking. Using the hypervariable surface antigen, glycoprotein 60 (GP60), C. hominis (n = 37) and C. parvum (n = 32) isolates from cryptosporidiosis cases in New South Wales, Australia, were characterised. Extensive variation was observed within this locus and the isolates could be divided into 8 families and 24 different subtypes. The subtypes identified have global distributions and indicate that anthroponotic and zoonotic transmission routes contribute to sporadic human cryptosporidiosis in NSW.     

Identification of zoonotic Giardia genotypes in fish

Apart from a single record in a shark, there have been no published studies conducted on Giardia genotypes in fish. The present study investigated the prevalence of Giardia in cultured fingerlings (n = 227), wild freshwater (n = 227) and wild marine/estuarine species (n = 255) of fish in Western Australia by PCR amplification at the 18S rRNA, glutamate dehydrogenase (gdh), triose phosphate isomerase (tpi) and beta-giardin (bg) loci. Results revealed a low prevalence of Giardia, 3.8% (27/709), in fish hosts. The zoonotic Giardia species, Giardia duodenalis assemblages A, B as well as G. duodenalis assemblage E and Giardia microti were detected. The identification of zoonotic species of Giardia highlights the public health importance of investigating parasites within fish host species.     

Effect of the environmental conditions on essential oil profile in two Dinaric Salvia species: S. brachyodon Vandas and S. officinalis L.

Two species belonging to the genus Salvia (Salvia brachyodon Vandas and Salvia officinalis L.) from Dalmatian region were studied for their essential oil composition, genome size and base composition. These species showed the same chromosome number (2n = 14), similar genome size (0.95 and 0.97 pg/2C) and base composition (38.52 and 38.55 GC%), respectively. This is the first estimation of DNA content and base composition for both species.     

Genetic diversity of Aegiceras corniculatum (Myrsinaceae) revealed by amplified fragment length polymorphism (AFLP)

Aegiceras corniculatum is a cryptoviviparous mangrove tree distributed in the Indo-West Pacific. The genetic structure of 13 populations of A. corniculatum from South China, Malay Peninsula, Sri Lanka, and North Australia, was assessed by amplified fragment length polymorphism (AFLP) markers. Our results showed a relatively high level of genetic variation at the species level (P = 92%, H_E = 0.294 and H_s = 0.331 ± 0.001). The value of G_ST was 0.698, suggesting significant genetic differentiation among populations. At the population level, however, genetic diversity was low (P = 24%, H_E = 0.086 and H_s = 0.127 ± 0.001). When populations were grouped according to geographic regions, i.e., South China, Malay Peninsula and Sri Lanka, it was inferred from analysis of molecular variance (AMOVA) that about half the total variation (49%) was accounted for differentiation between regions. A UPGMA dendrogram based on genetic distance also revealed five major clades corresponding to geographical regions within the distribution of A. corniculatum, although the precise relationships among the clades were not fully concordant with expected geographical delineations and need further study.     

Larsenia salina gen. nov., sp. nov., a new member of the family Halomonadaceae based on multilocus sequence analysis

Two Gram-staining-negative, moderately halophilic bacteria, strains M1-18^T and L1-16, were isolated from a saltern located in Huelva (Spain). They were motile, strictly aerobic rods, growing in the presence of 3–25% (w/v) NaCl (optimal growth at 7.5–10% [w/v] NaCl), between pH 4.0 and 9.0 (optimal at pH 6.0–7.0) and at temperatures between 15 and 40 °C (optimal at 37 °C). Phylogenetic analysis based on 16S rRNA gene sequence comparison showed that both strains showed the higher similarity values with Chromohalobacter israelensis ATCC 43985^T (95.2–94.8%) and Chromohalobacter salexigens DSM 3043^T (95.0–94.9%), and similarity values lower than 94.6% with other species of the genera Chromohalobacter, Kushneria, Cobetia or Halomonas. Multilocus sequence analysis (MLSA) based on the partial sequences of atpA, rpoD and secA housekeeping genes indicated that the new isolates formed an independent and monophyletic branch that was related to the peripheral genera of the family Halomonadaceae, Halotalea, Carnimonas and Zymobacter, supporting their placement as a new genus of the Halomonadaceae. The DNA–DNA hybridization between both strains was 82%, whereas the values between strain M1-18^T and the most closely related species of Chromohalobacter and Kushneria were equal or lower to 48%. The major cellular fatty acids were C_18:1ω7c/C_18:1ω6c, C_16:0, and C_16:1ω7c/C_16:1ω6c, a profile that differentiate this new taxon from species of the related genera. We propose the placement of both strains as a novel genus and species, within the family Halomonadaceae, with the name Larsenia salina gen. nov., sp. nov. The type strain is M1-18^T (= CCM 8464 = CECT 8192^T = IBRC-M 10767^T = LMG 27461^T).     

Distribution of repetitive DNA sequences in chromosomes of five opisthorchid species (Trematoda, Opisthorchiidae)

Genomes of opisthorchid species are characterized by small size, suggesting a reduced amount of repetitive DNA in their genomes. Distribution of repetitive DNA sequences in the chromosomes of five species of the family Opisthorchiidae (Opisthorchis felineus 2n = 14 (Rivolta, 1884), Opisthorchis viverrini 2n = 12 (Poirier, 1886), Metorchis xanthosomus 2n = 14 (Creplin, 1846), Metorchis bilis 2n = 14 (Braun, 1890), Clonorchis sinensis 2n = 14 (Cobbold, 1875)) was studied with C- and AgNOR-banding, generation of microdissected DNA probes from individual chromosomes and fluorescent in situ hybridization on mitotic and meiotic chromosomes. Small-sized C-bands were discovered in pericentric regions of chromosomes. Ag-NOR staining of opisthorchid chromosomes and FISH with ribosomal DNA probe showed that karyotypes of all studied species were characterized by the only nucleolus organizer region in one of small chromosomes. The generation of DNA probes from chromosomes 1 and 2 of O. felineus and M. xanthosomus was performed with chromosome microdissection followed by DOP-PCR. FISH of obtained microdissected DNA probes on chromosomes of these species revealed chromosome specific DNA repeats in pericentric C-bands. It was also shown that microdissected DNA probes generated from chromosomes could be used as the Whole Chromosome Painting Probes without suppression of repetitive DNA hybridization. Chromosome painting using microdissected chromosome specific DNA probes showed the overall repeat distribution in opisthorchid chromosomes.     

Chromosome numbers of miscellaneous Malagasy Acanthaceae

Meiotic chromosome numbers are reported for 12 species in eight genera of Acanthaceae from Madagascar. Chromosome numbers of 11 species are reported for the first time. Counts inMendoncia (n=19) andNeuracanthus (n=20) are the first for these genera. A new chromosome number (n=30) is reported inJusticia. Systematic implications of the chromosome counts are addressed and basic chromosome numbers for these eight genera of Malagasy Acanthaceae are discussed.     

Resilience and elasticity of intertidal communities in response to harvesting of the alien mussel Mytilus galloprovincialis

Besides the direct impacts of exploitation on target species, indirect effects on non-target species are unavoidable and find expression in changes in community structure. We quantified the effects of experimental harvesting of Mytilus galloprovincialis on intertidal communities on the South African west coast. In the mid- and low-shore, four months of harvesting at intensities greater than F = 0.3 and F = 0.6 respectively, resulted in significant changes in community composition. These changes were driven by progressively greater spatial dominance by the macroalgae Cladophora flagelliformis, Porphyra capensis and Ulva species as harvesting intensity increased. Four months after cessation of harvesting, community structure had not recovered and even areas subjected to as little as F = 0.3 supported significantly altered communities in both zones. The fact that substantial community changes were induced by even low-intensity exploitation is indicative of low resilience to harvesting. The densities and cover of the dominant taxa returned to a pre-harvest state 16 months after the cessation of harvesting, but multivariate analyses indicated that the overall community composition required 32 months for Cover Letter full recovery. Although these communities displayed elasticity within three years, it is recommended that should a M. galloprovincialis fishery be established in the region, harvesting be implemented at a maximum intensity of F = 0.3. This approach would maximise yields and protect associated intertidal communities.     

Combined deletion of DAZ2 and DAZ4 copies of Y chromosome DAZ gene is associated with male infertility in Tunisian men

The relationship between male infertility and AZFc micro-deletions that remove multiple genes of the Y chromosome varies among countries and populations. The purpose of this study was to analyze the prevalence and the characteristics of different Deleted in azoospermia (DAZ) gene copy deletions and their association with spermatogenic failure and male infertility in Tunisian men. 241 infertile men (30.7% azoospermic (n = 74), 31.5% oligozoospermic (n = 76) and 37.7% normozoospermic (n = 91)) and 115 fertile healthy males who fathered at least one child were included in the study. Three DAZ-specific single nucleotide variant loci and six bi-allelic DAZ-SNVs (I–VI) were analyzed using polymerase chain reaction (PCR)–restriction fragment length polymorphism and PCR. Our findings showed high frequencies of infertile men (73.85%) and controls (78.26%) having only three DAZ gene copies (DAZ1/DAZ2/DAZ3 or DAZ1/DAZ3/DAZ4 variants); so deletion of DAZ2 or DAZ4 were frequent both in infertile (36.5% and 37.3%, respectively) and fertile groups (33.9% and 44.3%, respectively) and removing DAZ4 copy was significantly more frequent in oligospermic than in normospermic men (p = 0.04) in infertile group. We also report for the first time that simultaneous deletion of both DAZ2 and DAZ4 copies was significantly more common in infertile men (12.4%) than in fertile men (4.3%) (p = 0.01). However, deletions of DAZ1/DAZ2 and DAZ3/DAZ4 clusters were very rare. Analysis of DAZ gene copies in Tunisian population, suggested that the simultaneous deletion of DAZ2 and DAZ4 gene copies is associated with male infertility, and that oligospermia seems to be promoted by removing DAZ4 copy.     

Body size and body shape in early hominins - implications of the Gona Pelvis

Discovery of the first complete Early Pleistocene hominin pelvis, Gona BSN49/P27, attributed to Homo erectus, raises a number of issues regarding early hominin body size and shape variation. Here, acetabular breadth, femoral head breadth, and body mass calculated from femoral head breadth are compared in 37 early hominin (6.0-0.26 Ma) specimens, including BSN49/P27. Acetabular and estimated femoral head sizes in the Gona specimen fall close to the means for non-Homo specimens (Orrorin tugenesis, Australopithecus africanus, Paranthropus robustus), and well below the ranges of all previously described Early and Middle Pleistocene Homo specimens. The Gona specimen has an estimated body mass of 33.2 kg, close to the mean for the non-Homo sample (34.1 kg, range 24-51.5 kg, n = 19) and far outside the range for any previously known Homo specimen (mean = 70.5 kg; range 52-82 kg, n = 17). Inclusion of the Gona specimen within H. erectus increases inferred sexual dimorphism in body mass in this taxon to a level greater than that observed here for any other hominin taxon, and increases variation in body mass within H. erectus females to a level much greater than that observed for any living primate species. This raises questions regarding the taxonomic attribution of the Gona specimen. When considered within the context of overall variation in body breadth among early hominins, the mediolaterally very wide Gona pelvis fits within the distribution of other lower latitude Early and Middle Pleistocene specimens, and below that of higher latitude specimens. Thus, ecogeographic variation in body breadth was present among earlier hominins as it is in living humans. The increased M-L pelvic breadth in all earlier hominins relative to modern humans is related to an increase in ellipticity of the birth canal, possibly as a result of a non-rotational birth mechanism that was common to both australopithecines and archaic Homo.     

Species-genomic relationships among the tribasic diploid and polyploid Carthamus taxa based on physical mapping of active and inactive 18S–5.8S–26S and 5S ribosomal RNA gene families,and the two tandemly repeated DNA sequences

In the genus Carthamus (2n = 20, 22, 24, 44, 64; x = 10, 11, 12), most of the homologues within and between the chromosome complements are difficult to be identified. In the present work, we used fluorescent in situ hybridisation (FISH) to determine the chromosome distribution of the two rRNA gene families, and the two isolated repeated DNA sequences in the 14 Carthamus taxa. The distinctive variability in the distribution, number and signal intensity of hybridisation sites for 18S–26S and 5S rDNA loci could generally distinguish the 14 Carthamus taxa. Active 18S–26S rDNA sites were generally associated with NOR loci on the nucleolar chromosomes. The two A genome taxa, C. glaucus ssp. anatolicus and C. boissieri with 2n = 20, and the two botanical varieties of B genome C. tinctorius (2n = 24) had diagnostic FISH patterns. The present results support the origin of C. tinctorius from C. palaestinus. FISH patterns of C. arborescens vis-à-vis the other taxa indicate a clear division of Carthamus taxa into two distinct lineages. Comparative distribution and intensity pattern of 18S–26S rDNA sites could distinguish each of the tetraploid and hexaploid taxa. The present results indicate that C. boissieri (2n = 20) is one of the genome donors for C. lanatus and C. lanatus ssp. lanatus (2n = 44), and C. lanatus is one of the progenitors for the hexaploid (2n = 64) taxa. The association of pCtKpnI-2 repeated sequence with rRNA gene cluster (orphon) in 2–10 nucleolar and non-nucleolar chromosomes and the consistent occurrence of pCtKpnI-1 repeated sequence at the subtelomeric region in all the taxa analysed indicate some functional role of these sequences.     

Semicircular canal system in early primates

Mammals with more rapid and agile locomotion have larger semicircular canals relative to body mass than species that move more slowly. Measurements of semicircular canals in extant mammals with known locomotor behaviours can provide a basis for testing hypotheses about locomotion in fossil primates that is independent of postcranial remains, and a means of reconstructing locomotor behaviour in species known only from cranial material. Semicircular canal radii were measured using ultra high resolution X-ray CT data for 9 stem primates (“plesiadapiforms”; n = 11), 7 adapoids (n = 12), 4 omomyoids (n = 5), and the possible omomyoid Rooneyia viejaensis (n = 1). These were compared with a modern sample (210 species including 91 primates) with known locomotor behaviours. The predicted locomotor agilities for extinct primates generally follow expectations based on known postcrania for those taxa. “Plesiadapiforms” and adapids have relatively small semicircular canals, suggesting they practiced less agile locomotion than other fossil primates in the sample, which is consistent with reconstructions of them as less specialized for leaping. The derived notharctid adapoids (excluding Cantius) and all omomyoids sampled have relatively larger semicircular canals, suggesting that they were more agile, with Microchoerus in particular being reconstructed as having had very jerky locomotion with relatively high magnitude accelerations of the head. Rooneyia viejaensis is reconstructed as having been similarly agile to omomyids and derived notharctid adapoids, which suggests that when postcranial material is found for this species it will exhibit features for some leaping behaviour, or for a locomotor mode requiring a similar degree of agility.     

Comparative growth kinetics and virulence of four different isolates of entomopathogenic fungi in the house fly (Muscadomestica L.)

Virulence (speed of kill) of a fungal entomopathogen against a particular host insect depends on biological properties of the specific isolate-host combination, together with factors such as fungal dose. How these intrinsic and extrinsic factors affect the actual pattern and extent of fungal growth invivo is poorly understood. In this study we exposed adult house flies (Muscadomestica L.) to surfaces treated with high and low doses of Beauveriabassiana (isolates BbGHA and Bb5344), Metarhiziumanisopliae (strain MaF52) and M.anisopliae var. acridum (isolate Ma189) and used quantitative real-time PCR with species-specific primers to examine the relationship between fungal growth kinetics and virulence. At the highest dose, all fungal isolates killed flies significantly faster than controls, with BbGHA, Bb5344 and MaF52 roughly equivalent in virulence (median survival time (±SE) = 5.0 ± 0.10, 5.0 ± 0.08 and 5.0 ± 0.12 days, respectively) and Ma189 killing more slowly (MST = 8.0 ± 0.20 days). At the lower dose, effective virulence was reduced and only flies exposed to isolates BbGHA and Bb5344 died significantly faster than controls (MST = 12 ± 1.36, 15 ± 0.64, 18 ± 0.86 and 21.0 ± 0.0 days for BbGHA, Bb5344, MaF52 and Ma189, respectively). Real-time PCR assays revealed that flies exposed to surfaces treated with the high dose of spores had greater spore pickup than flies exposed to the low dose for each isolate. After pickup, a general pattern emerged for all isolates in which there was a significant reduction of recovered fungal DNA 48 h after exposure followed by a brief recovery phase, a stable period of little net change in fungal sequence counts, and then a dramatic increase in sequence counts of up to three orders of magnitude around the time of host death. However, while the patterns of growth were similar, there were quantitative differences such that higher final sequence counts were recovered in insects infected with the most lethal isolates and with the higher dose. These results suggest that variation in virulence between isolates, species and doses is determined more by quantitative rather than qualitative differences in fungal growth kinetics.