Heat stress survival and thermal tolerance of Australian stingless bees.

Stingless bees (Meliponini) are important pollinators throughout the world's tropical and subtropical regions. Understanding their thermal tolerance is key to predicting their resilience to changing climates and increasingly frequent extreme heat events. We examined critical thermal maxima (CTmax), survival during 1-8 h heat periods, chill coma recovery and thermal preference for Australian meliponine species that occupy different climates across their ranges: Tetragonula carbonaria (tropical to temperate regions), T. hockingsi (tropical and subtropical regions only) and Austroplebeia australis (widely distributed including arid regions). We found interspecific differences in thermal tolerance consistent with differences in the climate variability observed in each species' range. Foragers of A. australis had a faster chill coma recovery (288 s) than foragers of T. hockingsi (1059 s) and T. carbonaria (872 s). Austroplebeia australis also had the highest CTmax of 44.5 °C, while the CTmax of the two Tetragonula species was ∼43.1 °C. After a 1-h heat exposure, T. carbonaria foragers experienced 95% mortality at 42 °C, and 100% at 45 °C. Surprisingly, larvae and pupae of both Tetragonula species were more resistant to heat exposure than foragers. Within an enclosed temperature gradient apparatus (17-38 °C), no clear preference was found for foragers; however, they were most frequently observed at ∼18 °C. Results indicate that in some regions of Australia, meliponines already experience periodic heat events exceeding their thermal maxima. Employing effective management strategies (such as nest site insulation and habitat preservation) may be crucial to colony survival under continued climate change.

Bee pollination services and the burden of biogeography.

Native bees augment pollination services in the Northern Hemisphere, especially cultivated apple crops, yet Southern Hemisphere contexts are poorly known. We observed the foraging behaviour of 69 354 invertebrate flower visitors in Australian orchards (two regions, 3 years) to assess the efficacy of pollination service (Peff). Native stingless bees and introduced honey bees were the most abundant visitors and most efficacious pollinators (Tetragonula Peff = 6.16; Apis Peff = 13.02), with Tetragonula becoming important service providers above 22°C. However, visits by tree-nesting stingless bees decreased with distance from native forest (less than 200 m) and their tropical/subtropical distribution precludes pollination service in other major Australian apple-producing regions. More broadly distributed native allodapine and halictine bees transferred the most pollen per-visit, but their low abundances reduce efficacies (Exoneura Peff = 0.03; Lasioglossum Peff = 0.06), resulting in a general dependence on honey bees. This reliance is a burden of biogeography, since key Northern Hemisphere pollinators of apple (Andrena, Apis, Bombus, Osmia) do not naturally occur in Australasia-where there is only 15% generic overlap with Central Asian bees sympatric with wild apple distributions (cf. Palaearctic 66% and Nearctic 46% generic overlaps). The historical biogeography of bees therefore drives an extreme dependence on one introduced species for apple pollination in Australia.

Microbial diversity in stingless bee gut is linked to host wing size and influenced by the environment.

Stingless bees are important social corbiculate bees, fulfilling critical pollination roles in many ecosystems. However, their gut microbiota, particularly the fungal communities associated with them, remains inadequately characterised. This knowledge gap hinders our understanding of bee gut microbiomes and their impacts on the host fitness. We collected 121 samples from two species, Tetragonula carbonaria and Austroplebeia australis across 1200 km of eastern Australia. We characterised their gut microbiomes and investigated potential correlations between bee gut microbiomes and various geographical and morphological factors. We found their core microbiomes consisted of the abundant bacterial taxa Snodgrassella, Lactobacillus and Acetobacteraceae, and the fungal taxa Didymellaceae, Monocilium mucidum and Aureobasidium pullulans, but variances of their abundances among samples were large. Furthermore, gut bacterial richness of T. carbonaria was positively correlated to host forewing length, an established correlate to body size and fitness indicator in insects relating to flight capacity. This result indicates that larger body size/longer foraging distance of bees could associate with greater microbial diversity in gut. Additionally, both host species identity and management approach significantly influenced gut microbial diversity and composition, and similarity between colonies for both species decreased as the geographic distance between them increased. We also quantified the total bacterial and fungal abundance of the samples using qPCR analyses and found that bacterial abundance was higher in T. carbonaria compared to A. australis, and fungi were either lowly abundant or below the threshold of detection for both species. Overall, our study provides novel understanding of stingless bee gut microbiomes over a large geographic span and reveals that gut fungal communities likely not play an important role in host functions due to their low abundances.

Endosymbionts moderate constrained sex allocation in a haplodiploid thrips species in a temperature-sensitive way.

Maternally inherited bacterial endosymbionts that affect host fitness are common in nature. Some endosymbionts colonise host populations by reproductive manipulations (such as cytoplasmic incompatibility; CI) that increase the reproductive fitness of infected over uninfected females. Theory predicts that CI-inducing endosymbionts in haplodiploid hosts may also influence sex allocation, including in compatible crosses, however, empirical evidence for this is scarce. We examined the role of two common CI-inducing endosymbionts, Cardinium and Wolbachia, in the sex allocation of Pezothrips kellyanus, a haplodiploid thrips species with a split sex ratio. In this species, irrespective of infection status, some mated females are constrained to produce extremely male-biased broods, whereas other females produce extremely female-biased broods. We analysed brood sex ratio of females mated with males of the same infection status at two temperatures. We found that at 20 °C the frequency of constrained sex allocation in coinfected pairs was reduced by 27% when compared to uninfected pairs. However, at 25 °C the constrained sex allocation frequency increased and became similar between coinfected and uninfected pairs, resulting in more male-biased population sex ratios at the higher temperature. This temperature-dependent pattern occurred without changes in endosymbiont densities and compatibility. Our findings indicate that endosymbionts affect sex ratios of haplodiploid hosts beyond the commonly recognised reproductive manipulations by causing female-biased sex allocation in a temperature-dependent fashion. This may contribute to a higher transmission efficiency of CI-inducing endosymbionts and is consistent with previous models that predict that CI by itself is less efficient in driving endosymbiont invasions in haplodiploid hosts.

Triangulation of methods using insect cell lines to investigate insecticidal mode-of-action.

BACKGROUND: This study investigated three in vitro models to assist in elucidating possible mode-of-action, which could be adopted to evaluate insecticidal activity of complex, unknown, or multi-constituent formulations. We used a combination of absorbance spectrometry, confocal scanning laser microscopy and microelectrode ion flux estimation (MIFE) to provide insight into potential target sites for insecticides. This study used two insect cell lines and evaluated three pyrethroid insecticides. RESULTS: We observed that the two cell lines produced distinctly different responses. Drosophila melanogaster D.mel-S2 cell line was a useful model to monitor ion flux changes, resulting from insecticides with neural toxicity; however, it was less useful to determine some metabolic pathway indicators of toxic stress. Conversely, the Spodoptera frugiperda Sf9 cell line produced acute reactive oxygen species (ROS) in response to insecticide treatments, but was not highly responsive in electrophysiological experiments. We also showed that the natural, multi-constituent botanical extract of pyrethrum elicited different Na+ , Cl- and Ca2+ ion fluxes than its synthetic, single constituent analogues, α-cypermethrin and esfenvalerate. These two methods used in combination with absorbance spectrometry measuring cell growth inhibition plus cell mortality assays shed some light on cytotoxic responses in differing model cell lines. CONCLUSION: This research highlights the importance of using multiple cell types and interdisciplinary methods to provide a better insight into mode of insecticidal action. This is especially pertinent to novel biopesticide discovery, as the underlying mechanisms for toxicity in initial screening processes are likely to be unknown.

Temporal changes in the microbiome of stingless bee foragers following colony relocation.

Maintaining beneficial interactions with microbial symbionts is vital for animal health. Yet, for social insects, the stability of microbial associations within and between cohorts is largely unknown. We investigated temporal changes in the microbiomes of nine stingless bee (Tetragonula carbonaria) colonies at seven timepoints across a 10-month period when moved between two climatically and florally different sites. Bacterial 16S rRNA gene and fungal ITS amplicon sequencing confirmed that microbiomes varied considerably between colonies initially at site one. However, following relocation, considerable changes occurred in bacterial community composition within each colony, and the microbiome composition became more similar across colonies. Notably, Snodgrassella disappeared and Zymobacter appeared as relatively abundant taxa. Remarkably, bacterial communities within colonies continued to shift over time but remained similar across colonies, becoming dominated by Acinetobacter six months after returning to the original site. Our results indicate that the stingless bee microbiome can undergo major changes in response to the environment, and that these changes can be long-lasting. Such legacy effects have not been reported for corbiculate bees. Further understanding the microbial ecology of stingless bees will aid future management of colonies used in agricultural production.

Chemical Characterization and Biological Activities of Essential Oil Obtained from Mint Timija Cultivated under Mineral and Biological Fertilizers.

Cultivation of mint timija (Mentha suaveolens subsp. timija (Briq.) Harley) constitutes a promising solution to the conservation and sustainable utilization of this Moroccan endemic and threatened species. Optimized agronomic practices require mineral and/or biological fertilizer applications. The aim of this study was to determine the effects of application of a complete (N, P, and K) mineral fertilizer and vesicular arbuscular mycorrhizae (VAM) inoculation on the composition, antioxidant, and insecticidal properties of mint timija essential oils (EOs). The GC-MS analyses identified 27 components representing more than 99.9% of the total oils. Menthone (40.7-49.3%), pulegone (31.3-36.5%), and isomenthone (2.5-4.4%) were found to be the main constituents. Cultivation of mint timija with mineral fertilizer and VAM inoculation induced an increase in menthone content and a parallel decrease of pulegone. Both treatments enhanced the antioxidant activity of the investigated EOs in all assays (IC50 ranged from 2.34 ± 0.03 mg/mL to 6.82 ± 0.25 mg/mL), while no significant difference in the toxicities of these oils against Tribolium confusum du Val. has been observed. Overall, we conclude that cultivation using complete mineral fertilizer and VAM inoculation could be useful in modulating the chemical composition and enhancing the antioxidant activity of the EO of this endemic Moroccan species.

Independent cytoplasmic incompatibility induced by Cardinium and Wolbachia maintains endosymbiont coinfections in haplodiploid thrips populations.

Cardinium and Wolbachia are common maternally inherited reproductive parasites that can coinfect arthropods, yet interactions between both bacterial endosymbionts are rarely studied. For the first time, we report their independent expression of complete cytoplasmic incompatibility (CI) in a coinfected host, and CI in a species of the haplodiploid insect order Thysanoptera. In Pezothrips kellyanus, Cardinium-induced CI resulted in a combination of male development (MD) and embryonic female mortality (FM) of fertilized eggs. In contrast, Wolbachia-induced CI resulted in FM together with postembryonic mortality not previously reported as a CI outcome. Both endosymbionts appeared to not influence fecundity but virgins produced more offspring than mated females. In coinfected individuals, Wolbachia density was higher than Cardinium. Wolbachia removal did not impact Cardinium density, suggesting a lack of competition within hosts. Maternal transmission was complete for Wolbachia and high for Cardinium. Our data support theoretical predictions and empirical detection of high endosymbiont prevalence in field populations of the native range of this pest thrips. However, previous findings of more frequent loss of Wolbachia than Cardinium, particularly in field populations of the host's invasive range, suggest that genetic diversity or varying environmental factors between field populations also play a role in shaping host-endosymbiont dynamics.

First confirmed report of a bacterial brood disease in stingless bees.

Susceptibility to brood pathogens in eusocial stingless bees (Meliponini), alternative pollinators to honey bees, is unknown. Brood losses in managed colonies of the Australian stingless bee, Tetragonula carbonaria, were studied over 20months. We isolated a disease-causing bacterium, Lysinibacillus sphaericus (Firmicutes, Bacillaceae), from worker and queen larvae, brood cell provisions and honey stores. Pathogenicity experiments confirmed this bacterium as the causal organism. It took 22days from infection to first appearance of brood disease symptoms. This is the first confirmed record of a brood pathogen in stingless bees.

Primary parasitoids of red scale (Aonidiella aurantii) in Australia and a review of their introductions from Asia.

We used morphological and molecular differences to confirm the identities of red scale (Aonidiella aurantii) and yellow scale (A. citrina), and their primary parasitoids, in Australia. An extension to the distribution of yellow scale was confirmed. Six primary parasitoids of red scale were identified: Aphytis chrysomphali, A. lingnanensis, A. melinus, Comperiella bifasciata, Encarsia citrina, and E. perniciosi. With the exception of A. lingnanensis, these parasitoids, and a species of Aphelinus, were detected in association with red scale during studies in citrus orchards in coastal New South Wales between 2009 and 2012. Two races of A. melinus were recorded: one from the Indian Subcontinent, the other previously only recorded in China. The studies, and reviews of historical records, led us to conclude that 4 parasitoids, A. lingnanensis, C. bifasciata, and both species of Encarsia, were present in Australia before successful or unsuccessful formal introductions between 1902 and 1970. The A. melinus race previously recorded in China may also have been present before the Indian Subcontinent race was formally introduced in 1961. We suggest the possibility that the natural distribution of some of the parasitoids may include East and Southeast Asia, and parts of Australasia. We found no reports of native armored scales being recorded on species and hybrids of Citrus introduced to Australia, and no reports of introduced armored scales being recorded on native Rutaceae, including 6 species of Citrus. However, we subsequently recorded yellow scale on Geijera parviflora, a native rutaceous tree.

Age and aggregation trigger mating behaviour in the small hive beetle, Aethina tumida (Nitidulidae).

This study aimed to investigate the poorly documented reproductive behaviour of the small hive beetle, Aethina tumida (Nitidulidae), a honey bee (Apis mellifera) parasite. We described the mating behaviour in detail and tested the hypothesis that beetle aggregation plays a vital role in mating in this species. Gender preference was examined in the context of age-dependency and possible chemical communication. Beetles started mating at a high frequency 18 days after emergence from the soil but only if they were aggregated (p < 0.001); mating was infrequent when beetles were paired. Males in aggregation also tried to copulate with males and only copulated more frequently with females at 18 days after emergence from soil (p < 0.001) in contrast to newly emerged, 7-day-old and 60-day-old beetles. Males and females spent more time in social contact with the opposite sex (p < 0.01) when they were 18 days old in contrast to 7-day-old beetles. Filter papers which had been in contact with 21-day-old beetles were highly attractive to similar-aged beetles of the opposite sex (p < 0.01). This suggests that chemical substances produced by the beetles themselves play a role in mating. Mating behaviour was characterised by a short pre-copulation courtship and female aggression towards other females and copulating couples. Both behaviours may be indicative of cryptic female choice. Delayed onset of reproductive behaviour is typical of many polygamous species, whilst the indispensability of aggregation for onset of sexual behaviour seems to be a feature unique to A. tumida. Both strategies support mass reproduction in this parasitic species, enabling A. tumida to overcome its honey bee host colony, and are probably triggered by chemotactic cues..

Chemical characterization and insecticidal properties of essential oils from different wild populations of Mentha suaveolens subsp. timija (Briq.) Harley from Morocco.

The present study is the first investigation of the volatile-oil variability and insecticidal properties of the endemic Moroccan mint Mentha suaveolens subsp. timija (mint timija). The yield of essential oils (EOs) obtained from different wild mint timija populations ranged from 0.20±0.02 to 1.17±0.25% (v/w). GC/MS Analysis revealed the presence of 44 oil constituents, comprising 97.3-99.9% of the total oil compositions. The main constituents were found to be menthone (1.2-62.6%), pulegone (0.8-26.6%), cis-piperitone epoxide (2.9-25.5%), piperitone (0.3-35.5%), trans-piperitone epoxide (8.1-15.7%), piperitenone (0.2-9.6%), piperitenone oxide (0.5-28.6%), (E)-caryophyllene (1.5-11.0%), germacrene D (1.0-15.7%), isomenthone (0.3-7.7%), and borneol (0.2-7.3%). Hierarchical-cluster analysis allowed the classification of the EOs of the different mint timija populations into four main groups according to the contents of their major components. This variability within the species showed to be linked to the altitude variation of the mint timija growing sites. The results of the insecticidal tests showed that all samples exhibited interesting activity against adults of Tribolium castaneum, but with different degrees. The highest toxicity was observed for the EOs belonging to Group IV, which were rich in menthone and pulegone, with LC50 and LC90 values of 19.0-23.4 and 54.9-58.0 µl/l air in the fumigation assay and LC50 and LC90 values of 0.17-0.18 and 0.40-0.52 µl/cm(2) in the contact assay.

Polyploidy versus endosymbionts in obligately thelytokous thrips.

BACKGROUND: Thelytoky, the parthenogenetic development of females, has independently evolved in several insect orders yet the study of its mechanisms has so far mostly focussed on haplodiploid Hymenoptera, while alternative mechanisms of thelytoky such as polyploidy are far less understood. In haplodiploid insects, thelytoky can be encoded in their genomes, or induced by maternally inherited bacteria such as Wolbachia or Cardinium. Microbially facilitated thelytoky usually results in complete homozygosity due to gamete duplication and can be reverted into arrhenotoky, the parthenogenetic development of males, through treatment with antibiotics. In contrast, genetically encoded thelytoky cannot be removed and may result in conservation of heterozygosity due to gamete fusion. We have probed the obligate thelytoky of the greenhouse thrips, Heliothrips haemorrhoidalis (Bouché), a significant cosmopolitan pest and a model species of thelytoky in the haplodiploid insect order Thysanoptera. Earlier studies suggested terminal fusion as a mechanism for thelytoky in this species, while another study reported presence of Wolbachia; later it was speculated that Wolbachia plays a role in this thrips' thelytokous reproduction. RESULTS: By using PCR and sequence analysis, we demonstrated that global population samples of H. haemorrhoidalis were not infected with Wolbachia, Cardinium or any other known bacterial reproductive manipulators. Antibiotic treatment of this thrips did also not result in male production. Some individuals carried two different alleles in two nuclear loci, histone 3 and elongation factor 1 alpha, suggesting heterozygosity. However, the majority of individuals had three different alleles suggesting that they were polyploid. Genetic diversity across both nuclear loci was low in all populations, and absent from mitochondrial cytochrome oxidase I, indicating that this species had experienced genetic bottlenecks, perhaps due to its invasion biology or a switch to thelytoky. CONCLUSIONS: Geographically broad sampling and experimental manipulation revealed low genetic diversity, absence of Wolbachia but presence of three different alleles of nuclear loci in most analysed individuals of obligately thelytokous H. haemorrhoidalis. This suggests that polyploidy may be involved in the thelytokous reproduction of this thrips species, and polyploidy may be a contributing factor in the reproduction of Thysanoptera and other haplodiploid insect orders.

Genetic variation amongst biotypes of Dactylopius tomentosus.

The tomentose cochineal scale insect, Dactylopius tomentosus (Lamarck) (Hemiptera: Dactylopiidae), is an important biological control agent against invasive species of Cylindropuntia (Caryophyllales: Cactaceae). Recent studies have demonstrated that this scale is composed of host-affiliated biotypes with differential host specificity and fitness on particular host species. We investigated genetic variation and phylogenetic relationships among D. tomentosus biotypes and provenances to examine the possibility that genetic diversity may be related to their host-use pattern, and whether their phylogenetic relationships would give insights into taxonomic relatedness of their host plants. Nucleotide sequence comparison was accomplished using sequences of the mitochondrial cytochrome c oxidase I (COI) gene. Sequences of individuals from the same host plant within a region were identical and characterized by a unique haplotype. Individuals belonging to the same biotype but from different regions had similar haplotypes. However, haplotypes were not shared between different biotypes. Phylogenetic analysis grouped the monophyletic D. tomentosus into 3 well-resolved clades of biotypes. The phylogenetic relationships and clustering of biotypes corresponded with known taxonomic relatedness of their hosts. Two biotypes, Fulgida and Mamillata, tested positive for Wolbachia (α-Proteobacteria), a common endosymbiont of insects. The Wolbachia sequences were serendipitously detected by using insect-specific COI DNA barcoding primers and are most similar to Wolbachia Supergroup F strains. This study is the first molecular characterization of cochineal biotypes that, together with Wolbachia sequences, contribute to the better identification of the biotypes of cochineal insects and to the biological control of cacti using host-specific biotypes of the scale.

Insecticidal properties and chemical composition of essential oils of some aromatic herbs from Morocco.

The essential oils (EOs) of Artemisia herba alba, Rutachalepensis and Satureja calamintha aerial parts were analysed by GC/MS and the EOs were tested for their toxicity against two flour beetles, Tribolium castaneum and Tribolium confusum. EO composition showed that the major compounds were pulegone, menthone and menthol for S. calamintha, camphor and ß-thujone for A. herba alba and 2-undecanone for R. chalepensis. Contact bioassay showed that all EOs were toxic to adults of T. castaneum and T. confusum. Higher toxicity was found with the EOs from S. calamintha and R. chalepensis (LD50 of 0.09-0.13 µL/cm(2) and LD90 of 0.17-0.29 µL/cm(2)). In fumigant toxicity test, A. herba alba and S. calamintha EOs produced insecticidal activity with S. calamintha EO being more toxic with LD50 and LD90 values of 10.5 and 19.1 µL/L air, respectively, for T. confusum against 7.8 and 17.4 µL/L air, respectively, for T. castaneum.

The alternative Pharaoh approach: stingless bees mummify beetle parasites alive.

Workers from social insect colonies use different defence strategies to combat invaders. Nevertheless, some parasitic species are able to bypass colony defences. In particular, some beetle nest invaders cannot be killed or removed by workers of social bees, thus creating the need for alternative social defence strategies to ensure colony survival. Here we show, using diagnostic radioentomology, that stingless bee workers (Trigona carbonaria) immediately mummify invading adult small hive beetles (Aethina tumida) alive by coating them with a mixture of resin, wax and mud, thereby preventing severe damage to the colony. In sharp contrast to the responses of honeybee and bumblebee colonies, the rapid live mummification strategy of T. carbonaria effectively prevents beetle advancements and removes their ability to reproduce. The convergent evolution of mummification in stingless bees and encapsulation in honeybees is another striking example of co-evolution between insect societies and their parasites.

The influence of sublethal deposits of agricultural mineral oil on the functional and numerical responses of Phytoseiulus persimilis (Acari: Phytoseiidae) to its prey, Tetranychus urticae (Acari: Tetranychidae).

Occasional pesticide application in integrated pest management to at least part of a crop requires that any biological control agents must re-invade previously sprayed areas in order that resurgent pests can be constrained. The ability of the phytoseiid predatory mite Phytoseiulus persimilis to feed on adult two-spotted spider mite (TSSM) Tetranychus urticae on excised leaf discs in both control conditions and in a treatment with a sub lethal residue of agricultural mineral oil (AMO) was assessed. The predator exhibited a Type II functional response with the asymptote significantly higher in the AMO conditions due to the fact that the prey grew slower and reached a smaller size in this treatment. In terms of prey volume eaten, the satiation level of the predator was unchanged by the AMO deposits. The numbers of eggs produced by adult P. persimilis females at densities of 4, 8 and 16 TSSM adult females/disc in the control were significantly higher than those in the AMO treatment, but were similar for the higher density levels, 32 and 64 prey per disc. Thus the functional response in terms of volume of prey eaten explained the numerical response in terms of predator eggs produced. The presence of AMO deposits when the prey were at high density had no effect on predator efficiency (volume eaten) but resulted in a lower intake than that in control conditions when there was a greater distance between prey.

Cytotoxic clerodane diterpenes from Glossocarya calcicola.

Three clerodane diterpenes were isolated and identified from leaf extract of Glossocarya calcicola. Compound has been characterised as (rel)-10betaH-trans-12xi-(2-methylbut-2(E)-enoyl)-1beta-(isobutanoyl)-6alpha,13xi-dihydroxyclerodan-4(20),8(18)-dien-7,15-dione-15,16-oxide, to which we have assigned the trivial name calcicolin-A. The other two compounds had the same skeletal structure and C-12 substituent but in compound, the C-1 esterifying group becomes 2-methylbut-2(E)-enoic acid and in it becomes 2-methylbutanoic acid. Although anti-insect activity was not observed for G. calcicola, cytotoxicity against insect and human carcinoma cell lines was detected.

Acaricidal and cytotoxic activities of extracts from selected genera of Australian Lamiaceae.

Crude foliar extracts of 67 species from six subfamilies of Australian Lamiaceae were screened by whole organism contact toxicity on the polyphagous mite Tetranychus urticae Koch (Acari: Tetranychidae) by using a Potter precision spray tower. Cytotoxicity assessments against insect cell lines from Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) and Drosophila melanogaster (Meigen) (Diptera: Drosophilidae) also were made. The Spodoptera cell line was more susceptible to extracts than the Drosophila cellline. No direct correlation was observed between the two screening methods, but several interesting relationships were identified. Extracts from subfamilies Ajugoideae, Scutellarioideae, Chloanthoideae, Viticoideae and Nepetoideae showed acaricidal activity, whereas only those from Ajugoideae and Nepetoideae displayed potent cytotoxic effects. A range of activities was observed for the 25 species of Plectranthus, 14 of which showed moderate-to-high contact toxicity against T. urticae. Overall, the lowest toxicity was observed for extracts from the plant subfamily Prostantheroideae, which showed little contact toxicity or cytotoxicity for the 18 extracts studied.