Characterization of polymorphic microsatellite loci in the aphid species Macrosiphum euphorbiae (Hemiptera: Aphididae)

Fourteen microsatellite loci were isolated and characterized in the potato aphid, Macrosiphum euphorbiae Thomas, by screening a genomic library with the oligonucleotide probes (GA)₁₀ (GT)₁₀ and (GATA)₄. Allelic diversity was estimated in samples collected from potato fields in Tunisia. Ten loci displayed polymorphism that ranged from two to four alleles per locus and the observed heterozygosity ranged from zero to one. These markers could be used to study the population genetic structure of this polyphagous aphid species.     

Effects of jasmonate-induced defenses in tomato on the potato aphid, Macrosiphum euphorbiae

Jasmonates such as jasmonic acid (JA) are plant‐signaling compounds that trigger induced resistance (IR) to a broad range of arthropod herbivores. JA‐dependent defenses are known to reduce the growth and survivorship of many chewing insects, but their impact on piercing–sucking insects such as aphids has not been extensively investigated. In this study, induced resistance was activated in tomato (Lycopersicon esculentum Mill) (Solanaceae) using a foliar application of synthetic JA, and control plants were treated with carrier solution. The life parameters of individual potato aphids and their progeny (Macrosiphum euphorbiae Thomas) (Hemiptera: Aphididae) were evaluated on the unsprayed leaves of plants in order to access the systemic effects of the foliar treatments. IR significantly reduced the longevity and net reproduction of adult aphids, as well as the percentage of juveniles to survive to maturity. These results indicate that JA application induces systemic defenses in tomato that have a direct negative impact on aphid survivorship. This study also examined aphid honeydew excretion, in order to evaluate the potential influence of induced resistance on aphid feeding behavior. The average honeydew production per aphid was comparable on plants with or without JA treatment, indicating that JA‐dependent defenses did not deter feeding. This suggests that the observed effects of JA on aphid survivorship were due to antibiotic rather than antixenotic factors. In addition to studying the effects of JA treatment on a tomato cultivar that is susceptible to aphids, this study also examined the effects of exogenous application of JA on tomato plants that carry the aphid resistance gene, Mi‐1.2. JA application did not significantly enhance or inhibit aphid control on resistant tomato. These findings expand our understanding of the effects of JA‐dependent defenses on piercing–sucking insects, and of the potential interactions between induced resistance and R‐gene mediated aphid resistance in tomato.     

Calling behaviour of the potato aphid Macrosiphum euphorbiae oviparae under laboratory and field conditions

Abstract.  1. The calling behaviour of virgin oviparae of the potato aphid Macrosiphum euphorbiae (Thomas) (Homoptera: Aphididae) was studied at three different constant temperatures under laboratory conditions. The mean age of calling for the first time decreased with a decrease in temperature from 2.9 days at 20 °C to 2.1 days at 10 °C. At all temperature regimes, the mean onset time of calling advanced from about 5 to 3 h after the onset of the photophase, and the mean time spent calling increased by > 4 h over the 8 days.
2. Cohorts of oviparae were also observed at two different periods in late summer–early autumn in the field, to examine the effects of fluctuating abiotic conditions (temperatures, wind velocity, rain) and age on calling behaviour. As under constant laboratory conditions, the mean age of calling for the first time declined with declining temperature, from 3.7 days in early September to 1.6 days at the end of September. Age-related changes in the mean onset time of calling and the mean time spent calling were much less evident under field conditions, due to the inhibitory effects of low temperatures, high winds, and rain on female calling activity.
3. The results are discussed within the context of reproductive success and address a previously proposed hypothesis suggesting that species-specific calling windows may serve as a reproductive isolating mechanism for sympatric aphid species.     

Immediate alteration of Macrosiphum euphorbiae host plant-selection behaviour after biotic and abiotic damage inflicted to potato plants

The effects of potato [Solanum tuberosum L. (Solanaceae)] plant damage on the host plant-selection behaviour of the potato aphid, Macrosiphum euphorbiae Thomas (Homoptera: Aphididae), were studied. The damage inflicted to the plant was only of short duration and observations on aphid behaviour were made immediately following plant damage. The underlying questions of the study were to know how much time it takes for plant defence mechanisms to be activated and if this activation had noticeable repercussions on aphid behaviour. We considered stresses of various natures: biotic (pre-infestation by conspecifics or by Colorado potato beetles) and abiotic (scissor cuts). Aphid responses to host plant semiochemicals were investigated using a darkened arena bioassay and the probing behaviour was assessed using the electrical penetration graph technique. Aphids were attracted to their host plant (undamaged or damaged). In a preference test (undamaged plant vs. damaged plant), plants previously infested by conspecifics were preferred to undamaged plants, but this preference was not observed for heterospecific and abiotic damage. However, aphid probing behaviour was not modified on plants previously infested by conspecifics, whereas some changes were observed subsequently to heterospecific and abiotic damages. Our data present evidence that plants can respond to biotic and abiotic stresses soon after the damage is inflicted and when the damage is of short duration. The diverse consequences of these various local plant responses on M. euphorbiae behaviour are discussed in the context of plant defence strategies against aphid colonization.     

Biological parameters and thermal requirements of the parasitoid Praon volucre (Hymenoptera: Braconidae) with Macrosiphum euphorbiae (Hemiptera: Aphididae) as host

The effect of temperature on the biology of Praon volucre (Haliday, 1833) (Hymenoptera: Braconidae) in Macrosiphum euphorbiae (Thomas, 1878) (Hemiptera: Aphididae) hosts was studied and the thermal requirements of the parasitoid were determined. Experiments were carried out at 16, 19, 22, 25, and 28 ± 1°C with 70 ± 10% relative humidity and 12 h photophase. The developmental time of P. volucre males and females decreased between 16 and 23°C. The highest percentages of aphid parasitism and adult parasitoid emergence were observed at 16, 19, and 22°C. The sex ratio (expressed as % females) decreased between 16 and 23°C. Male and female longevity was high between 19 and 22°C and decreased strongly at 25°C. The lower temperature threshold for P. volucre was 5.17°C and the thermal constant was 243 degrees-days.     

Comparative analysis of Solanum stoloniferum responses to probing by the green peach aphid Myzus persicae and the potato aphid Macrosiphum euphorbiae

Abstract Plants protect themselves against aphid attacks by species‐specific defense mechanisms. Previously, we have shown that Solanum stoloniferum Schlechtd has resistance factors to Myzus persicae Sulzer (Homoptera: Aphididae) at the epidermal/mesophyll level that are not effective against Macrosiphum euphorbiae Thomas (Homoptera: Aphididae). Here, we compare the nymphal mortality, the pre‐reproductive development time, and the probing behavior of M. persicae and M. euphorbiae on S. stoloniferum and Solanum tuberosum L. Furthermore, we analyze the changes in gene expression in S. stoloniferum 96 hours post infestation by either aphid species. Although the M. euphorbiae probing behavior shows that aphids encounter more probing constrains on phloem activities–longer probing and salivation time– on S. stoloniferum than on S. tuberosum, the aphids succeeded in reaching a sustained ingestion of phloem sap on both plants. Probing by M. persicae on S. stoloniferum plants resulted in limited feeding only. Survival of M. euphorbiae and M. persicae was affected on young leaves, but not on senescent leaves of S. stoloniferum. Infestation by M. euphorbiae changed the expression of more genes than M. persicae did. At the systemic level both aphids elicited a weak response. Infestation of S. stoloniferum plants with a large number of M. persicae induced morphological changes in the leaves, leading to the development of pustules that were caused by disrupted vascular parenchyma and surrounding tissue. In contrast, an infestation by M. euphorbiae had no morphological effects. Both plant species can be regarded as good host for M. euphorbiae, whereas only S. tuberosum is a good host for M. persicae and S. stoloniferum is not. Infestation of S. stoloniferum by M. persicae or M. euphorbiae changed the expression of a set of plant genes specific for each of the aphids as well as a set of common genes.     

The impact of Meu1-mediated resistance in tomato on longevity, fecundity and behavior of the potato aphid, Macrosiphum euphorbiae

The effect of the tomato resistance gene, Meu1, on feeding, longevity, fecundity and developmental rate of the pink biotype of the potato aphid, Macrosiphum euphorbiae (Thomas) (Hemiptera, Aphididae), was determined using nearly isogenic tomato (Lycopersicon esculentum Mill, Solanaceae) lines. Aphid mortality was significantly higher on resistant plants, with 60% of the aphids dying by the 4th day of exposure. By the 10th day, all the aphids on the resistant plants were dead whereas 100% of the aphids on susceptible plants were alive. Meu1-mediated resistance resulted in significantly decreased fecundity with a ten-fold decrease in the net fertility rate (4.5 and 45.7 progeny per aphid on resistant and susceptible tomato, respectively). A qualitative analysis showed that honeydew was produced by aphids on resistant and susceptible plants, suggesting that aphids initiate feeding on both plant types. However, significantly lower quantities of honeydew were present when aphids were caged on resistant plants. There were also significant differences in aphid location on resistant and susceptible leaves. Experiments evaluating behavior in less than 24 h showed that aphids left resistant leaves after relatively short exposure (3–6 h). Aphids transferred from resistant to susceptible tomato at intervals between 3 h and 24 h resumed feeding as evidenced by presence of honeydew. Although the mechanism by which Meu1-mediated resistance operates is not yet known, our data suggest that resistance factors act rapidly after initiation of feeding and that lower fecundity and longevity are related to reduction in aphid feeding.     

Inheritance of partial resistance to the leaf aphids Macrosiphum euphorbiae and Uroleucon sonchi in lettuce

The genetics of partial resistance (PR) of lettuce to the leaf aphid species Macrosiphum euphorbiae and Uroleucon sonchi was studied in F₁ and F₂ generations of six crosses between four cultivars. Partial resistance inherited mainly additively. With M. euphorbiae the estimates of broad-sense F₂ heritability were low and non-significant in crosses between parents of the same resistance level (S x S and PR x PR), and high (0.69-0.76) in crosses between parents of different resistance level (PR x S). The level of resistance in lettuce cultivars ‘Charan’ and ‘Marbello’ led to a marked reduction in percentage of plants with colonies of M. euphorbiae in a field experiment. With U. sonchi the highest estimates of F₂ heritability were found in ‘Charan x ‘Avoncrisp’ (0.51) and ‘Charan’ x ‘Chou de Naples' (0.48). In the cross ‘Charan’ x ‘Marbello’ (PR x PR) no significant segregation for resistance to M. euphorbiae was found, indicating that these cultivars may have the same genetic constitution for resistance to M. euphorbiae.‘Charan’ has additional genes for resistance to U. sonchi, that are not, or only weakly linked to the gene(s) for resistance to M. euphorbiae. The heritability estimates for the partial resistance to M. euphorbiae and U. sonchi indicate that it is possible to effectively select for these traits in plant breeding programmes.     

Properties of copper-dependent o-diphenol oxidase activity in the potato aphid Macrosiphum euphorbiae (thomas)

Potato aphid Macrosiphum euphorbiae (Thomas) was found to contain high amounts of o-diphenol oxidase activity. Enzyme activity was largely distributed into the postmitochondrial supernatant from Brij-35 extracted aphids and occurs in a latent form that was activated up to 45-fold by pretreatment with isopropanol. The aphid enzyme has a broad pH optimum near 6, and utilized L-dopa (K_m = 1.4 mM, V_max = 348 nmol/min-mg protein), dopamine, and 4-methylcatechol the best out of the twelve substrates tested. In addition, this activity is a typical copper-dependent oxidase in that it is potently inhibited by phenylthiourea (50% inhibition at 30nM) and other copper chelators, including salicylhydroxamic acid. The above properties are common to most insect tyrosinases. However, the aphid enzyme lacked the o-hydroxylase and laccase components and the optimal activity at higher temperatures that are typical of cuticular tyrosinases of other insects. The high levels of o-diphenol oxidase in aphids compared to other insects is surprising, since the major function associated with these enzymes, that of melanization and sclerotization of cuticle, is of much less importance to aphids. The possibility that aphids use this enzyme to metabolize dietary phenolics is discussed.     

Biological performance of Myzus persicae and Macrosiphum euphorbiae (Homoptera: Aphididae) on seven wild Solanum species

Resistance level of seven accessions of wild Solanum species (Solanaceae) to Myzus persicae and Macrosiphum euphorbiae (Homoptera: Aphididae) was evaluated by measuring survival and fecundity during sleeve cage experiments and population growth on whole plants in a controlled environment. The survival was lowest on the Solanum circaeifolium spp. capsicibaccatum, Solanum pinnatisectum and Solanum trifidum accessions for M. persicae and on the S. circaeifolium spp. capsicibaccatum, Solanum okadae and S. trifidum, accessions with M. euphorbiae. Plant species significantly influenced the fecundity of both aphid species. Aphid population growth on whole plants was negatively affected by the age of the plant, but generally followed the levels of net reproductive rate on different plant species observed during the sleeve cage experiment. The population of M. persicae varied among the seven wild potato accessions with the lowest growth rates on S. circaeifolium spp. capsicibaccatum, Solanum polyadenium, Solanum tarijense and S. trifidum. The number of M. euphorbiae also varied among accessions but the smallest population was collected from S. polyadenium. The results can be used to identify sources of resistance to aphids within those accessions already known to be resistant to the Colorado potato beetle. This study highlights the difficulties involved in developing a high-throughput screening test for aphid resistance compatible with a potato breeding programme.     

Projection pursuit nonparametric regression applied to field counts of the aphid Macrosiphum euphorbiae (Homoptera: Aphididae) on tomato crops in greenhouses

The number of plants that sampling requires in aphid population studies often exceeds one hundred. Thus, only quick and nondestructive methods can be used to sample this pest in a reasonable time interval. We propose a visual method for estimating the density of the aphid Macrosiphum euphorbiae (Thomas) on tomato plants reared in greenhouses. After approximately 1min of visual observation plants can be assigned to abundance classes, the boundaries of which are roughly the powers of square root 10. Precise counts were collected simultaneously on sets of reference plants from the same greenhouse. Projection pursuit nonparametric regression was then used to provide unbiased estimates of aphid densities from the abundance classes and several easily gathered explanatory variables. The robustness of the method was evaluated by testing the models on the complementary data sets from plants in which the aphid densities were precisely counted. In both single and twin-row cultural conditions, for the reference and complementary data sets, the order of magnitude of the error was less than one class rank per plant. The investigation time was reduced by approximately 10-fold compared with the exact counting method. This easy-to-teach field method could be useful in large-scale population surveys and for optimizing integrated pest management strategies.     

温度对桃蚜和马铃薯长管蚜实验种群生命表参数的影响

【目的】桃蚜Myzus persicae (Sulzer)和马铃薯长管蚜Macrosiphum euphorbiae (Thomas)是2种主要的马铃薯害虫。本研究旨在明确温度对这两种马铃薯蚜虫生长发育、存活、繁殖及种群增长的影响。【方法】在室内测定了5个温度（10, 15, 20, 25和30℃）下桃蚜和马铃薯长管蚜的生长发育、存活和繁殖指标,并组建了4个恒温条件下（10,15,20和25℃）的实验种群生命表。【结果】在10~25℃范围内,2种蚜虫的若蚜期、世代历期、成蚜寿命和产蚜期等均随温度的升高而缩短,而30℃高温抑制了其发育、存活和繁殖。2种蚜虫的平均世代历期（T）随温度的升高而缩短,桃蚜和马铃薯长管蚜分别从10℃的30.08 d和35.35 d缩短至25℃的14.28 d和12.95 d。桃蚜的净增殖率（R₀）在15℃时最高（86.00）,其次为20℃（73.75）,再次为25℃（62.49）,最低为10℃（51.00）;马铃薯长管蚜的R0在15℃最高（58.97）,其次为10℃（51.98）,再次为20℃（48.94）,最低为25℃（12.36）。桃蚜的内禀增长率（r_m）随温度的升高而增大,从最小10℃的0.1307增大到25℃的0.2896;马铃薯长管蚜的r_m在20℃时最大（0.2182）,其次为25℃（0.1942）,再次为15℃（0.1485）,最小为10℃（0.1118）。在相同的温度下,桃蚜的发育速率、净增殖率和内禀增长率均高于马铃薯长管蚜。【结论】温度对2种马铃薯蚜虫的生长发育、存活、繁殖及种群增长有显著的影响,桃蚜在马铃薯上的种群增长能力强于马铃薯长管蚜。这一结果为马铃薯蚜虫预测模型的建立奠定了必要的基础,并解释了桃蚜在马铃薯上发生数量多于马铃薯长管蚜的原因。     

Modification of Macrosiphum euphorbiae colonisation behaviour and reproduction on potato plants treated by mineral oil

Although mineral oil spray is one of the most effective ways to control the transmission of non‐persistent aphid‐borne viruses in the field, its mode of action is poorly understood. In this study, the effects of mineral oil treatment of potato plants on host selection behaviour, growth, and reproduction of potato aphids, Macrosiphum euphorbiae (Thomas) (Hemiptera: Aphididae), were investigated. The effects were assessed 30 min, 1 day, and 7 days after treatment, (1) on aphid orientation behaviour by using a Y‐tube olfactometer, and (2) on aphid feeding behaviour by using the electrical penetration graph (EPG) technique. Olfactory experiments showed that the oil had a repulsive effect only 30 min after spraying. EPG experiments showed a slight modification of the aphid feeding behaviour mainly 7 days after treatment. The number of both salivation and sap ingestion events during the phloem phases were increased 7 days after treatment. In addition, irrespective of the time after treatment, xylem ingestion time was increased. Clip cage experiments were set up to assess potential effects of the oil treatment on aphid survival and population parameters. Nymphal mortality was increased on treated plants, whereas fecundity of surviving insects was enhanced. The antagonistic effects of oil treatment on aphids are discussed in a plant protection context.     

Sucrose-dependent increase in oligosaccharide production and associated glycosidase activities in the potato aphid Macrosiphum euphorbiae (Thomas)

Osmotic stress due to high sucrose concentration was imposed on the potato aphid, Macrosiphum euphorbiae (Thomas), in an artificial diet situation. Thin layer chromatography resolved melezitose and six additional honeydew oligosaccharides. Significantly higher levels of these oligosaccharides were recovered from the honeydew of this species in response to feeding on increased dietary sucrose concentrations, pointing to an osmoregulatory response. In addition, carbohydrase activities from the potato aphid were described. A degree of specificity toward hydrolysis of the sucrose substrate over other α-glucosyl sugars was demonstrated. Also, sucrose was optimal for the formation of the oligosaccharides. A strong α-galactosidase activity was found and transgalactosylation ability was indicated. These major trends in carbohydrase activity were also found in the foxglove aphid, Acyrthosiphon solani (Kaltenbach), and the oleander aphid, Aphis nerii Fonscolombe.     

Different exposure methods to neonicotinoids influenced biochemical characteristics in cotton aphid,Aphis gossypii Glover (Hemiptera:Aphididae)

We measured lipid, carbohydrate and protein content in three strains of cotton aphid, Aphis gossypii (Hemiptera: Aphididae) from very resistant to neonicotinoids up to semi sensitive and sensitive strains in different exposure method of spraying to neonicotinoids. We observed changes in energy source rates at which each substrate was metabolised under starvation, selection and residue stress method spraying that assist in metabolisation of their biochemical parameters. These particular exposure methods influenced some of biochemical parameters in cotton aphid. Results indicated that Sugar, glycogen, total protein and lipid in aphids have significant changes. Studies showed that among three strains called Ag-R, Ag-M and Ag-D which have different susceptibilities to neonicotinoids, strain Ag-R was the most tolerated aphids in counter of imidacloprid and thiametoxam, strain Ag-D was somewhat more tolerated to these insecticides and strain Ag-M was the most sensitive strain which has the resistance factor of nearly 890 in starvation method and was different depending on the method of exposure. Among energy sources, the total lipid in susceptible strain were decreasing more than resistant strain, whereas total proteins were increasing in resistant strain compared with sensitive strain. Total Glycogen was affected significantly in the stress condition which caused an increase and was the most resistant strain.     

Effect of eggplant transformed with oryzacystatin gene on Myzus persicae and Macrosiphum euphorbiae

Abstract:  The effect of a genetically modified eggplant line expressing oryzacystatin on Myzus persicae (Sulzer) and Macrosiphum euphorbiae (Thomas) was examined. The transgenic eggplant reduced the net reproductive rate ( R ₀), the instantaneous rate of population increase ( r ), and the finite rate of population increase ( λ ) of both aphids species compared with a control eggplant line. The mean generation time ( T ) of the aphids was unaffected by the transgenic plants. Age-specific mortality rates of M. persicae and M. euphorbiae were higher on transgenic plants. These results indicate that expression of oryzacystatin in eggplant has a negative impact on population growth and mortality rates of M. persicae and M. euphorbiae and could be a source of plant resistance for pest management of these aphids.     

Demographic parameters of cotton aphid, Aphis gossypii Glover （Homoptera： Aphididae） on five cotton cultivars

Life table parameters of the cotton aphid, Aphis gossypii Glover, were estimated on five cotton cultivars （‘ Sealand＇ ,‘Siokra＇ ,＇ Vararnin＇ ,‘ Bakhtegan＇ and ‘ Sahel＇ ）. Demographic parameters of the cotton aphid were assessed at 27.5 ± 1 ℃, 65% ＋ 10% RH and a photoperiod of 14：10 （L： D）h. The shortest developmental time for the nymphal stages was 5.5 days on ‘Siokra＇ and the longest was 6.1 days on‘ Sealand＇. The highest offspring per female was 29.6 on ‘Sahel＇ and the lowest was 15.3 on ‘Sealand＇. The rm values varied from 0.272 on ‘Sealand＇ to 0.382 （day^-1） on ‘Varamin＇. Jackknife estimates of the A. gossypii parameters on these cultivars indicated the greatest developmental rate and fecundity on ‘ Varamin＇ and the poorest on ‘ Sealand＇ cultivar.     

Fast chromosomal evolution and karyotype instability: recurrent chromosomal rearrangements in the peach potato aphid Myzus persicae (Hemiptera: Aphididae)

The occurrence of karyotype variations with respect to both chromosome number and structure has been frequently reported in aphids. Here, we review recent data attesting to the presence of recurrent chromosomal changes in the karyotype of the peach potato aphid Myzus persicae, where clones presenting metaphases with different chromosome number (from 12 to 17) have been observed, also comparing plates obtained within the same embryo. According to the available data, M. persicae autosomes 3 and 1 are the chromosomes mostly involved in changes compared to other autosomes, suggesting that they could have sites more susceptible to fragmentation. Chromosomal fissions involving the X chromosomes have also been observed, suggesting that they may have fragile sites located at the termini opposite to the nucleolar organizer regions‐bearing telomere. The presence of holocentric chromosomes and reproduction by apomictic parthenogenesis, together with a constitutive expression of telomerase, could explain the inheritance of the observed chromosomal instability in aphids. Considering that chromosomal changes may affect the host choice and could also favour speciation, it would be intriguing to confirm whether the observed karyotype variants have effects over short temporal and spatial scales.     

Role of host‐plant selection in resistance of wild Solanum species to Macrosiphum euphorbiae and Myzus persicae

Damage to potatoes by Macrosiphum euphorbiae (Thomas) and Myzus persicae (Sulzer) (both Hemiptera: Aphididae) can be controlled through plant resistance. We used ethological experiments and electric penetration graph (EPG) analysis to evaluate the role of host selection in the previously assessed resistance levels of Solanum accessions: Solanum circaeifolium Bitter subsp. capsicibaccatum (Cárdenas) (PI210036), S. chomatophilum Bitter (PI243340 and PI310990), S. okadae Hawkes & Hjert. (PI458367), S. oplocense Hawkes (PI473368), S. pinnatisectum Dunal (PI186553), S. polyadenium Greenm. (PI230463), S. tarijense Hawkes (PI414150), and S. trifidum Correll (PI255538), to M. euphorbiae and M. persicae. Through multivariate analysis, we grouped behavioural variables into factors, which we related to host selection behaviours, and then evaluated whether factors varied between each accession and the susceptible S. tuberosum. None of the factors obtained by ethological experiments differed among accessions. Four of six and three of five factors obtained through EPG varied among accessions for M. euphorbiae and M. persicae, respectively, and were used to suggest resistance characteristics. The resistance to M. persicae of both S. chomatophilum accessions was associated with pathway activity disturbance. Solanum tarijense and S. polyadenium resistance to M. persicae resulted from leaf surface characteristics, which may be trichomes. Solanum oplocense and S. trifidum resistance to M. euphorbiae resulted from the wound response system, whereas S. pinnatisectum resistance may stem from nutritionally unbalanced or toxic phloem sap. Solanum polyadenium resistance to M. euphorbiae was phloem‐based. Solanum circaeifolium ssp. capsicibaccatum resistance to M. persicae, and the resistance of PI243340 S. chomatophilum and S. tarijense to M. euphorbiae were not related to host selection and therefore were presumably due to physiologically active compounds.