Discovery of novel trimethylalkanes in the internal hydrocarbons of developing pupae of Heliothis virescens and Helicoverpa zea

Novel trimethyl-branched alkanes which eluted with the monomethylalkanes were identified in the internal lipids of Helicoverpa zea but were not present in Heliothis virescens. Their structures were unique in that the first methyl branch occurred on carbon 2 and the 2nd and 3rd methyl branch points were separated by a single methylene. Novel trimethylalkanes identified from their chemical ionization and electron impact mass spectra were 2,18,20-trimethyltetratriacontane, 2,18,20-trimethylhexatriacontane, and 2,24,26-trimethyldotetracontane. Previous reports did not find these trimethylalkanes in the cuticular surface lipids of larvae, pupae or adults of either species. The internal pupal hydrocarbons of H. virescens and H. zea amounted to 123 μg and 304 μg per pupa, respectively. They consisted of n-alkanes (8 and 4%, respectively) and methyl-branched alkanes (88 and 94%, respectively). The n-alkanes ranged in chain length from approximately 21 to 35 carbons and the methyl-branched alkanes from approximately 26 to 55 carbons vs. methyl-branched alkanes from 28 to 37 carbons previously reported for hydrocarbons from the pupal cuticular surface. The major n-alkane was heptacosane (3.3 and 1.2%, respectively, in H. virescens and H. zea). The major methyl-branched alkanes in H. virescens were methylhentriacontane (15%), methyltritriacontane (12%) and dimethyltritriacontane (10%), and in H. zea were methylnonacosane (17%), dimethylnonacosane (9%) and methylhentriacontane (20%). Except for the novel trimethylalkanes, the methylalkane branch points were predominantly on odd-numbered carbons as has been reported for these and other species.     

Chemical basis for inter-colonial aggression in the stingless bee Scaptotrigona bipunctata (Hymenoptera: Apidae)

Inter-colonial aggression was tested using three colonies of Scaptotrigona bipunctata in a natural setting when their nests were moved and by artificial contact between individuals. Examination of the cuticular lipids of individuals from two colonies kept under identical conditions showed clear differences in their cuticular hydrocarbon profiles. The cuticular lipids were a mixture of hydrocarbons (saturated and unsaturated alkanes and alkenes) within the range of C23-C29. The use of multivariate analysis (PCA and discriminant analysis) showed that seven of the identified surface compounds are enough to separate workers from colonies A and B from each other.     

The effect of ethanol on the phase behavior of membrane lipids extracted from Clostridium thermocellum strains

The phase behavior of aqueous dispersions of extracted lipids from Clostridium thermocellum wild-type and ethanol-tolerant C919 cells has been examined by DSC. The optimum growth temperature of this anaerobe is 60°C. The wild-type lipids exhibit a broad phase transition centered at 30°C; the C919 mutant lipids show a 10°C lower T_m. The direct addition of growth inhibiting concentrations of ethanol has no significant effect on T_m or headgroup mobility (monitored by ²H-NMR) of either set of lipids. In contrast, wild-type cells adapted to growth in ethanol exhibit a broadened and lower T_m (15–25°C plateau); C919 membrane lipids do not exhibit significantly altered phase behavior when adapted to growth in ethanol. Both wild-type and mutant membranes have fatty acid composition changes upon growth in ethanol, which increases lower-melting components. It is concluded that fatty acid changes which occur upon adaptation of the organism to growth in ethanol are secondary responses and not necessarily direct responses to alter membrane fluidity.     

The effect of the entomopathogenic fungus Conidiobolus coronatus on the composition of cuticular and internal lipids of Blatta orientalis females

The composition of cuticular and internal lipids in females of the cockroach Blatta orientalis L. exposed to the entomopathogenic fungus Conidiobolus coronatus is investigated. The compositions of the fatty acids, n‐alkanes, alcohol, sterols and methyl esters in the lipids are chemically characterized. Although contact with virulent colonies of the fungus does not induce insect mortality, significant changes in the lipid profiles, both cuticular and internal, are found. The cuticular extracts of a control group of B. orientalis females contain 24 compounds varying in carbon chain length from C6 to C22. The main cuticular fatty acids identified are: C16:1, C16:0, C18:1 and C18:0_. The cuticular lipids of B. orientalis females after exposure to C. coronatus contain only 14 free fatty acids from C8 to C20. The highest concentrations identified are C16:0, C18:2 and C18:1. Analysis by gas chromatography‐mass spectrometry identifies the presence of a homologous series of n‐alkanes containing from 25 to 31 carbon atoms. In the case of the insects after fungal exposure, the content of the n‐alkanes in the cuticular lipid is two‐fold higher compared with the controls. Of the cuticular lipids, 11 alcohols are found, ranging from C12:0 to C20:0. There is no presence of alcohols in the internal lipids of the control B. orientalis females and in all of the extracts from the B. orientalis females after fungal exposure. In the samples analyzed, the most common sterol is cholesterol. This is present in the cuticular lipids and the internal lipids of all of the insects sampled. The cuticular and internal lipids of females contain five fatty acid methyl esters, ranging in size from C15 to C19.     

Novel very long-chain methyl-branched alcohols and their esters,and methyl-branched alkanes in pupae of the cabbage looper,Trichoplusia ni (Hubner)

Four homologous series of very long-chain methyl-branched alcohols were found in the internal lipids of cabbage looper, Trichoplusia ni, pupae both as free alcohols and as esters. These alcohols were identified based on mass spectra of their TMS and acetate derivatives, and on the Kovats Indices and mass spectra of the alkanes obatained by reduction of their bromide derivatives with LiAlH₄. The four homologous series (from C36 to C46) consisted of monomethyl-, two dimethyl- and a trimethyl-branched alcohol series. The major alcohols (with the corresponding alkanes in parentheses) were identified as 24-methyltetracontan-1-ol (17-methyltetracontane), 24,28-dimethyltetracontan-1-ol (13,17-dimethyltetracontane), 24,36-dimethyltetracontan-1-ol (5,17-dimethyltetracontane) and 24,28,36-trimethyltetracontan-1-ol (5,13,17-trimethyltetracontane). The minor components had an odd number of carbon atoms (C37, C39, C41 and C43) in the carbon chain and the methylalkanes formed from their reduction had methyl branches on the 18- and 14,18-positions. The methylalkanes found internally in pupae were similar to those previously reported in larval cuticular lipids (de Renobales and Blomquist, Insect Biochem. 13, 493–502, 1983). Additional novel methylalkanes identified were 2,X-dimethylalkanes, 5,15-dimethylhentriacontane, 5,23-dimethyltritriacontane, 5,17,23-trimethyltritriacontane and 5,15,23-trimethylpentatriacontane. The methyl branching positions of the major methyl-branched alcohols were different from those of the major methylalkanes.     

Germination and emergence of Sorghum bicolor. genotypic and environmentally induced variation in the response to temperature and depth of sowing

Abstract The germination of Sorghum bicolor seeds of 9 genotypes was tested at temperatures between 8°C and 48°C on a thermal gradient plate. Samples were tested from three regions of the panicle expected to differ in temperature during grain filling. Seeds of a tenth genotype, SPV 354, produced in controlled-environment glasshouses at different panicle temperatures, were tested similarly. In addition, the emergence of SPV 354 was measured from planting depths of 2 and 5 cm at mean soil temperatures of 15, 20 and 25°C. Four methods of calculating mean germination rate for the nine genotypes were compared. Germination characters like base, optimum and maximum temperature (T_b, T_o, T_m), thermal time (θ)and the germination rate at T_o(R_max showed only small differences between methods. There was a range of genotypic variation in all characters: T_b 8.5–11.9°C; T_o, 33.2–37.5°C; T_m, 46.8–49.2°C; θ, 23.4–38.0°Cd; R_max, 0.69–1.14-d_-1. In contrast, mean germinability (G) was between 90% and 100% over the temperature range 13–40°C. Panicle temperature had no effect on any germination character in SPV 354. However, deeper burial increased θ for emergence and decreased G, irrespective of soil temperature except at 5 cm. Increasing panicle temperature, by reducing seed size, reduced G and increased θ by about 10% only at 15°C and 5 cm depth.     

Calorimetric and molecular mechanics studies of the thermotropic phase behavior of membrane phospholipids

In this review, we summarize the results of recent studies on the main phase transition behavior of phospholipid bilayers using the combined approaches of molecular mechanics simulations and high-resolution differential scanning calorimetry. Following a brief overview of the phase transition phenomenon exhibited by the lipid bilayer, we begin with the review by showing how several structural parameters underlying various phospholipids including phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol are defined and determined. Specifically, these structural parameters are obtained with saturated lipids packed in the gel-state bilayer using computer-based molecular mechanics calculations. Then we proceed to present the calorimetric data obtained with the lipid bilayer composed of saturated phospholipids as it undergoes the gel-to-liquid-crystalline phase transition in excess water. The general equations that can correlate the gel-to-liquid-crystalline phase transition temperature (T_m) of the lipid bilayer with the structural parameters of the lipid molecule constituting the lipid bilayer are subsequently presented. From these equations, two tables of predicated T_m values for well over 400 molecular species of saturated phosphatidylcholine and saturated phosphatidylethanolamine are generated. We further review the structure and chain-melting behavior of a large number of sn-1 saturated/sn-2 unsaturated phospholipids. Two T_m-diagrams are shown, from which the effects of the number and the position of one to five cis carbon–carbon double bonds on T_m can be viewed simultaneously. Finally, in the last part of this review, simple molecular models that have been invoked to interpret the characteristic T_m trends exhibited by lipid bilayers composed of unsaturated lipids with different numbers and positions of cis carbon–carbon double bonds as seen in the T_m-diagram are presented.     

Novel wax esters and hydrocarbons in the cuticular surface lipids of the red harvester ant, Pogonomyrmex barbatus

The cuticular surface lipids of the red harvester ant, Pogonomyrmex barbatus, were found to contain minor amounts of novel wax esters, in addition to the major components, hydrocarbons. The wax esters ranged in carbon number from C19 to C31 and consisted of esters of both odd- and even-numbered alcohols and acids. Each wax ester with a given carbon number eluted at several different retention times indicating possible methyl branching in either the fatty acid or alcohol moiety, or in both moieties. Each eluting peak of wax esters consisted of a mixture of wax esters of the same carbon number in which the fatty acid moiety ranged from C8 to C18, and the alcohol moiety ranged from C8 to C17. Some wax esters were largely found on the head indicating they may be of a glandular origin. The hydrocarbons consisted of: n-alkanes, C23 to C33; odd-numbered n-alkenes, C27 to C35; and the major components, methyl-branched alkanes, C26 to over C49. Notable components of the methyl-branched alkanes were 2-methyltriacontane, and the novel trimethylalkanes with a single methylene between the first and second branch points, 13,15,19-trimethylhentriacontane and 13,15,21-trimethyltritriacontane.     

Epicuticular hydrocarbons of the sugarcane borer Diatraea saccharalis (Lepidoptera: Crambidae)

The epicuticular hydrocarbons of the larval, pupal and adult stages of the sugarcane borer Diatraea saccharalis Fabricius (Lepidoptera: Crambidae) are analysed. Dramatic changes are observed between the stages studied. Adult hydrocarbons are mostly saturated, with a predominance of 1–4 methyl‐branched straight carbon skeletons of 37–47 atoms; the major components are isomeric mixtures of internally branched trimethylderivatives of C39, C37 and C41 carbon backbones. By contrast, very small amounts of methyl‐branched components are detected in the pupae, although straight chain hydrocarbons of 23–35 carbons are the prevailing structures (70.7 ± 3.4%) with n‐C29 and n‐C27 as the major components. Unsaturated hydrocarbons (29.0 ± 3.5%) of similar chain lengths elute by gas chromatography of epicuticular extracts as complex mixtures of mono‐, di‐ and trienes; with the degree of unsaturation increasing with chain length. This is the first report of very long chain unsaturated hydrocarbons in cuticular extracts of a larval lepidopteran (93.3 ± 0.6% of the lipid components), with chain lengths in the range 37–53 carbons and up to four double bonds; the major component being C49:3, which co‐elutes with C49:4 and C49:2.     

Unfolding properties of recombinant human serum albumin products are due to bioprocessing steps

We have used differential scanning calorimetry (DSC) to determine the unfolding properties of commercial products of human serum albumin (HSA) prepared from pooled human blood, transgenic yeast, and transgenic rice. The initial melting temperatures (T_m1) for the unfolding transitions of the HSA products varied from 62°C to 75°C. We characterized the samples for purity, fatty acid content, and molecular weight. The effects of adding fatty acids, heat pasteurization, and a low pH defatting technique on the transition temperatures were measured. Defatted HSA has a structure with the lowest stability (T_m of ～62°C). When fatty acids are bound to HSA, the structure is stabilized (T_m of ～64–72°C), and prolonged heating (pasteurization at 60°C) results in a heat‐stabilized structural form containing fatty acids (T_m of ～75–80°C). This process was shown to be reversible by a low pH defatting step. This study shows that the fatty acid composition and bioprocessing history of the HSA commercial products results in the large differences in the thermal stability. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:62–69, 2015     

The cuticular hydrocarbons of the Triatoma sordida species subcomplex (Hemiptera: Reduviidae)

The cuticular hydrocarbons of the Triatoma sordida subcomplex (Hemiptera: Reduviidae: Triatominae) were ana-lysed by gas chromatography and their structures identified by mass spectrometry. They comprised mostly n-alkanes and methyl-branched alkanes with one-four methyl substitutions. n-alkanes consisted of a homologous series from C21-C33 and represented 33-45% of the hydrocarbon fraction; n-C29 was the major component. Methyl-branched alkanes showed alkyl chains from C24-C43. High molecular weight dimethyl and trimethylalkanes (from C35-C39) represented most of the methyl-branched fraction. A few tetramethylalkanes were also detected, comprising mostly even-numbered chains. Several components such as odd-numbered 3-methylalkanes, dimethylalkanes and trimethylalkanes of C37 and C39 showed patterns of variation that allowed the differentiation of the species and populations studied. Triatoma guasayana and Triatoma patagonica showed the most distinct hydrocarbon patterns within the subcomplex. The T. sordida populations from Brazil and Argentina showed significantly different hydrocarbon profiles that posed concerns regarding the homogeneity of the species. Triatoma garciabesi had a more complex hydrocarbon pattern, but it shared some similarity with T. sordida. The quantitative and qualitative variations in the cuticular hydrocarbons may help to elucidate the relationships between species and populations of this insect group.     

Effect of egg‐phosphatidylcholine on the chilling sensitivity and lipid phase transition of Asian elephant (Elephas maximus) spermatozoa

This study was conducted in an effort to improve our understanding of the response of Asian elephant (Elephas maximus, Em) spermatozoa to chilling. Semen was collected from two elephant bulls by means of the manual rectal stimulation method. Five out of seven semen collections were deemed to be suitable for use based on motility (ranging from 20% to 60%) and membrane integrity. We evaluated the chilling sensitivity by incubating the sperm with a fluorescent dye (5‐carboxyfluorescein diacetate (cFDA)) at 16°C, 12°C, 4°C, and 22°C (control). Cells with an intact membrane retained the dye and were identified as viable. The membrane lipid phase transition (LPT) temperature curve was determined with a Fourier transform infrared (FTIR) spectrometer connected to an FTIR microscope. The LPT center, T_m, was determined by statistical analysis. The LPT and T_m were also assessed in fresh spermatozoa and spermatozoa incubated with egg yolk or egg‐phosphatidylcholine (EPC) liposomes at 16°C, 12°C, 4°C, and 26°C (control). The results show that the membrane integrity of spermatozoa incubated at 16°C, 12°C, and 4°C decreased by 39%, 62%, and 67%, respectively, compared to the control. The LPT temperatures were between room temperature (26°C) and 10°C, with T_m at 14–16°C. The T_m for sperm incubated with liposomes or egg‐yolk extender was below the measured range (2°C). Chilling sensitivity was found at a wide range of temperatures and transition temperatures, suggesting the presence of a wide variety of fatty acids (FAs) in the membrane with a high ratio of saturated‐to‐polyunsaturated FAs. Here we show that the protection afforded by the presence of egg yolk or liposomes in the extender is accomplished by shifting the T_m to below the 4°C point at which chilled semen is maintained for transport, or the point at which fast freezing begins to minimize cellular damage. Zoo Biol 0:1–13, 2005. © 2005 Wiley‐Liss, Inc.     

Diurnal variation in repeated sprint performance cannot be offset when rectal and muscle temperatures are at optimal levels (38.5°C)

The present study investigated whether increasing morning rectal temperatures (T_rec) to evening levels, or increasing morning and evening T_rec to an “optimal” level (38.5°C), resulting in increased muscle temperatures (T_m), would offset diurnal variation in repeated sprint (RS) performance in a causal manner. Twelve trained males underwent five sessions [age (mean ± SD) 21.0 ± 2.3 years, maximal oxygen consumption (V?O₂max) 60.0 ± 4.4 mL.kg^–1 min^–1, height 1.79 ± 0.06 m, body mass 78.2 ± 11.8 kg]. These included control morning (M, 07:30 h) and evening (E, 17:30 h) sessions (5-min warm-up), and three further sessions consisting of a warm-up morning trial (M_E, in 39–40°C water) until T_rec reached evening levels; two “optimal” trials in the morning and evening (M_38.5 and E_38.5, in 39–40°C water) respectively, until T_rec reached 38.5°C. All sessions included 3 × 3-s task-specific warm-up sprints, thereafter 10 × 3-s RS with 30-s recoveries were performed a non-motorised treadmill. T_rec and T_m measurements were taken at the start of the protocol and following the warm-up periods. Values for T_rec and T_m at rest were higher in the evening compared to morning values (0.48°C and 0.69°C, p < 0.0005). RS performance was lower (7.8–8.3%) in the M for distance covered (DC; p = 0.002), average power (AP; p = 0.029) and average velocity (AV; p = 0.002). Increasing T_rec in the morning to evening values or optimal values (38.5°C) did not increase RS performance to evening levels (p = 1.000). However, increasing T_rec in the evening to “optimal” level through a passive warm-up significantly reduced DC (p = 0.008), AP (p < 0.0005) and AV (p = 0.007) to values found in the M condition (6.0–6.9%). Diurnal variation in T_rec and T_m is not wholly accountable for time-of-day oscillations in RS performance on a non-motorised treadmill; the exact mechanism(s) for a causal link between central temperature and human performance are still unclear and require more research.     

Drosophila melanogaster infected with Wolbachia strain wMelCS prefer cooler temperatures

1. Temperature plays a fundamental role in the dynamics of host–pathogen interactions. Wolbachia is an endosymbiotic bacteria that infects about 40% of arthropod species, which can affect host behaviour and reproduction. Yet, the effect of Wolbachia on host thermoregulatory behaviour is largely unknown, despite its use in disease vector control programs in thermally variable environments. 2. Here, a thermal gradient was used to test whether Drosophila melanogaster infected with Wolbachia strain wMelCS exhibited different temperature preferences (T_p) to uninfected flies. 3. It was found that Wolbachia‐infected flies preferred a cooler mean temperature (T_p = 25.06 ± 0.25 °C) than uninfected flies (T_p = 25.78 ± 0.24 °C). 4. This finding suggests that Wolbachia‐infected hosts might seek out cooler microclimates to reduce exposure to, and lessen the consequences of, high temperatures. This finding has generated hypotheses that will be fruitful in areas of research for exploring the mechanisms by which the change in T_p occurs in this complex and significant host–pathogen–environment interaction.     

The volatile Dufour's gland components of the harvester ants Pogonomyrmex rugosus and P. barbatus

The volatile Dufour's gland components of Pogonomyrmex rugosus and P. barbatus have been examined and found to be hydrocarbons. Homologous families of alkanes from this gland consisted of normal hydrocarbons ranging from n-dodecane to n-pentadecane and three types of methyl-branched homologues of the general formula C_nH_2n+1CH(CH₃)C_mH_2m+1, where n is 2, 4, or 5 and the sum of n and m is 10, 11, 12, or 13. The dimethyl-branched hydrocarbons 3,5-dimethyldodecane and 3,4-dimethyltridecane were also observed.     

A comparative study of the action of melittin on sphingomyelin and phosphatidylcholine bilayers

To investigate whether lipid solubilization is of relevance in describing the interaction between melittin and biological membranes, we studied melittin-induced polymorphism using model membranes composed of the biological lipid sphingomyelin (bovine brain). The behavior of the system was monitored by solid state ³¹P-NMR and turbidity measurements and compared to the peptides well-characterized action on the synthetic lipid dipalmitoylphosphatidylcholine. It was found that melittin-induced macroscopic changes of sphingomyelin membranes are qualitatively the same as in the case of dipalmitoylphosphatidylcholine bilayers. The sphingomyelin/melittin system is thus proposed to show a reversible vesicle-to-disc transition (fluid-to-gel phase) through an intermediate fusion or aggregation event centered at the main transition temperature, T_m, as reported in the case of saturated phosphatidylcholine. In the case of spontaneous disc formation at 37 °C, the lipid-to-peptide molar ratio in the discoidal objects was determined to be approximately 20 for dipalmitoylphosphatidylcholine and about 12 in the case of natural sphingomyelin. Melittin partition coefficients between membranes and the aqueous medium at 37 °C were found to be 6.1±0.8 mm ^–1 and 3.7±0.4 mm ^–1 for sphingomyelin and dipalmitoylphosphatidylcholine, respectively. For very high peptide quantities (lipid-to-peptide molar ratio, R_i≤5) mixed micelles are formed over the entire temperature range (20° to 60 °C) for both kinds of lipids.     

Effect of whitefly parasitoids on the cuticular lipid composition of Bemisia argentifolii (Homoptera: aleyrodidae) nymphs

Experiments were conducted to determine the effects of whitefly parasitoids on the cuticular lipid composition of the silverleaf whitefly, Bemisia argentifolii Bellows and Perring [=sweetpotato whitefly, Bemisia tabaci (Gennadius), Biotype B] nymphs. The cuticular lipids of B. argentifolii nymphs that had been attacked by parasitic wasps, either Eretmocerus mundus Mercet or Encarsia pergandiella Howard, were characterized by capillary gas chromatography and CGC-mass spectrometry and the results compared with the cuticular lipids of unparasitized nymphs. Previous studies with B. argentifolii nymphs had shown that wax esters were the major components of the cuticular lipids with lesser amounts of hydrocarbons, long-chain aldehydes, and long-chain alcohols. No appreciable changes in lipid composition were observed for the cuticular lipids of E. pergandiella-parasitized nymphs as compared to unparasitized controls. However, the cuticular lipids from nymphs parasitized by E. mundus contained measurable quantities of two additional components in their hydrocarbon fraction. Analyses and comparisons with an authentic standard indicated that the two hydrocarbons were the even-numbered chain length methyl-branched alkanes, 2-methyltriacontane and 2-methyldotriacontane. The occurrences and possible functions of 2-methylalkanes as cuticular lipid components of insects are discussed and specifically, in regard to host recognition, acceptance, and discrimination by parasitoids. Published 2000 Wiley-Liss, Inc.     

Effect of Conidiobolus coronatus on the Cuticular and Internal Lipid Composition of Tettigonia viridissima Males

Conidiobolus coronatus is an entomopathogenic fungus which has a potential as a biological control agent of insects. The cuticular and internal lipid composition of infected and noninfected Tettigonia viridissima males were analyzed by GC/MS. A total of 49 compounds were identified in the infected and noninfected males, including fatty acids, fatty acid methyl esters (FAMEs), n‐alkanes, alcohols, sterols, and other organic compounds. The most abundant components of the cuticular and internal lipids of the insects were fatty acids. After exposure to C. coronatus, the cuticular lipids of the T. viridissima males contained 17 free fatty acids from C(8) to C(22), while the cuticular lipids of the noninfected insects contained only 15 fatty acids from C(12) to C(24). The cuticular and internal lipids of both the infected and the noninfected males also contained five FAMEs from C(15) to C(19), seven n‐alkanes from C(25) to C(34), five alcohols from C(16) to C(25), five sterols, and the following six other organic compounds: azelaic acid, phenylacetic acid, glutaric acid, benzoic acid, sebacic acid, and glycerol. The compounds which were present only in the cuticular lipids of the infected males could be due to fungal infection.     

Use of principal component analysis in interpretation of deoxyribonucleic acid relatedness

Principal component analysis (PCA) of published DNA-relatedness data showed the usefulness of this method in displaying relationships among closely related bacteria. Very similar ordinations were obtained when relative binding ratios (RBR) at 60°C or 75°C or ΔT _m values were used to form the data matrix. A curvilinear relationship and a (quasi) linear relationship were found, respectively, between 75°C and 60°C RBR and ΔT _m and 60°C RBR. These statistical relationships explain the similarity of PCA results using either measurement (60°C RBR, 75°C RBR, or ΔT _m). Use of PCA is suggested to delineate groups within a complex set of DNA-relatedness data. The level of ΔT _m within groups and between groups should help decide whether these groups are genospecies.