Environmental RNAi-based reverse genetics in the predatory mite Neoseiulus californicus: Towards improved methods of biological control.

The predatory mite Neoseiulus californicus (McGregor) (Mesostigmata: Phytoseiidae) has been commercialized by manufacturers in the pest control industry and is used worldwide as a natural enemy of spider mites. However, because its genome has not been sequenced, reverse genetics techniques that could be used to analyze gene function have not been established. Here we partially sequenced the gene that encodes the vacuolar-type H+-ATPase (V-ATPase), an ATP-dependent proton pump, in N. californicus (NcVATPase) and then conducted a functional analysis using environmental RNA interference (eRNAi) by orally administering sequence-specific exogenous dsRNA (dsRNA-NcVATPase) to larvae and adult females. The larvae treated with dsRNA-NcVATPase took longer to develop and had lower survivorship, fecundity, and offspring viability at the adult stage than those treated with a control dsRNA. Adult females treated with dsRNA-NcVATPase showed significant reductions in survival, fecundity, and prey consumption, and their endogenous gene expression level of NcVATPase was reduced by approximately 65% compared with the control. Our findings suggest that the NcVATPase gene, silencing of which inhibits feeding and reproduction, is an excellent biomarker for investigating the eRNAi mechanism in N. californicus. The highly efficient experimental system of eRNAi established in this study paves the way for applied research using eRNAi to enhance the predatory ability of N. californicus.

A vegetable oil-based biopesticide with ovicidal activity against the two-spotted spider mite, Tetranychus urticae Koch.

A recently developed biopesticide made of safflower and cottonseed oils has excellent ovicidal activity against the hard-to-control spider mite Tetranychus urticae Koch (Acari: Tetranychidae). It has attracted attention as a sustainable treatment for controlling T. urticae because it has low potential for promoting resistance and little effect on the predatory mite Neoseiulus californicus (McGregor) (Acari: Phytoseiidae), which is an important natural enemy of spider mites. Here, we investigated the mechanism of its ovicidal activity against T. urticae. The oil droplets in the oil-in-water emulsion of the biopesticide strongly adhered to T. urticae eggs, seeped through the chorion being cut during hatching, and inhibited the embryonic rotational movement necessary for cutting and hatching. No adverse effect was observed on N. californicus eggs even in undiluted biopesticide. We conclude that this biopesticide and N. californicus can be used simultaneously in the integrated management of T. urticae in oily biopesticide-tolerant plant species.

A Leaf-Mimicking Method for Oral Delivery of Bioactive Substances Into Sucking Arthropod Herbivores.

Spider mites (Acari: Tetranychidae) are pests of a wide range of agricultural crops, vegetables, and ornamental plants. Their ability to rapidly develop resistance to synthetic pesticides has prompted the development of new strategies for their control. Evaluation of synthetic pesticides and bio-pesticides-and more recently the identification of RNA interference (RNAi) target genes-requires an ability to deliver test compounds efficiently. Here we describe a novel method that uses a sheet-like structure mimicking plant leaves and allows for oral delivery of liquid test compounds to a large number of individuals in a limited area simultaneously (~100 mites cm-2). The main component is a fine nylon mesh sheet that holds the liquid within each pore, much like a plant cell, and consequently allows for greater distribution of specific surface area even in small amounts (10 µl cm-2 for 100-µm mesh opening size). The nylon mesh sheet is placed on a solid plane (e.g., the undersurface of a Petri dish), a solution or suspension of test compounds is pipetted into the mesh sheet, and finally a piece of paraffin wax film is gently stretched above the mesh so that the test mites can feed through it. We demonstrate the use of the method for oral delivery of a tracer dye (Brilliant Blue FCF), pesticides (abamectin and bifenazate), dsRNA targeting the Vacuolar-type H+-VATPase gene, or fluorescent nanoparticles to three species of Tetranychus spider mites (Acari: Tetranychidae) and to the cotton aphid, Aphis gossypii Glover (Hemiptera: Aphididae). The method is fast, easy, and highly reproducible and can be adapted to facilitate several aspects of bioassays.

Impact of global warming scenarios on life-history traits of Tetranychus evansi (Acari: Tetranychidae).

BACKGROUND: The tomato red spider mite, Tetranychus evansi Baker & Pritchard (Acari: Tetranychidae), is an agricultural pest of solanaceous crops. Although T. evansi is of South American subtropical origin, it has recently expanded its distribution range to many tropical and temperate areas around the world. Its potential distribution range in response to scenarios of global warming was recently modeled, confirming its current and possible future distributions. Here, we experimentally investigated the biological traits of T. evansi in the context of the current and future global warming (2100) scenarios. Using an environmental simulation system, we tested the life-history traits of T. evansi under current summer temperatures (as of June, July, and August 2016) and under expected temperature increases based on two IPCC scenarios: RCP2.6 (+ 1 °C) and RCP8.5 (+ 3.7 °C). The mites were introduced into each scenario on 1 June and their sequential progeny were used for testing in each following month. RESULTS: The mite could develop and reproduce under all scenarios. There was a decrease in the duration of lifespan and female fecundity at RCP8.5 during June and August, but this may be compensated for by the high intrinsic rate of increase, which implies faster population growth and shorter generation time. CONCLUSION: Our study and other reports reveal the high adaptability of T. evansi to a wide range of summer temperatures; this may explain its current distribution. We anticipate that global warming will favor the spread of T. evansi and may further expand its distribution to a large area of the globe. These findings should be of ecological and practical relevance for designing prevention and control strategies.

Leaf disc-mediated oral delivery of small molecules in the absence of surfactant to the two-spotted spider mite, Tetranychus urticae.

The two-spotted spider mite (TSSM), Tetranychus urticae, is a chelicerate herbivore with a wide host range and strong ability to develop pesticide resistance. Experimental TSSM populations are easy to maintain, and the recent publication of the complete TSSM genome sequence and development of RNA interference-based reverse genetics protocols make this species an ideal chelicerate model for the study of pesticide resistance and plant-herbivore interactions. In such studies, treated leaf discs are often used for oral delivery of test compounds. When preparing these leaf discs, the organosilicone surfactant Silwet L-77 is used to promote wetting of the leaf surface and distribution of the test compound across the entire leaf surface. Here, we examined the toxicity of Silwet L-77 and found it to be toxic to TSSMs. We then developed a novel means of preparing leaf discs in which a polypropylene sheet rather than Silwet L-77 was used to ensure distribution of a tracer dye across the entire leaf surface. These leaf discs were then successfully used to deliver the tracer dye into the midgut of TSSMs. No significant differences were observed in the survival, fecundity, or feeding activity of TSSMs fed on leaf discs treated with water via our novel method compared with those fed on untreated leaf discs. Thus, our novel method of preparing leaf discs eliminates concerns regarding the bioactivity of surfactants in TSSMs, and we anticipate that it will be useful for improving oral delivery-based bioassays that use TSSMs.

Development and Reproduction of Neoseiulus californicus (Acari: Phytoseiidae) and Tetranychus urticae (Acari: Tetranychidae) Under Simulated Natural Temperature.

Although laboratory observations provide basic knowledge of the development and reproduction of predacious and phytophagous mites, little is known of their behavior under natural conditions. Using a closed system designed to simulate natural climate patterns, we investigated the development and reproduction of the predatory mite Neoseiulus californicus (McGregor) (Acari: Phytoseiidae) and the pest mite Tetranychus urticae Koch (Acari: Tetranychidae) at air temperatures typical of June to October at three latitudes (Aomori, Tottori, and Naha) in Japan. The peaks of development in both species showed similar trends at each location. The shortest developmental times for both species were observed during August in Aomori, from July to September in Tottori and during August and September in Naha. Development of T. urticae was not completed during October in Aomori due to the decreased air temperature. High reproduction (number of eggs produced during 5 d from the first oviposition) of N. californicus was attained at the conditions that shortened the developmental times (i.e., high-temperature months). T. urticae showed a reproduction trend similar to that of N. californicus except for the low number of eggs produced during August in Naha due to the high mortality of adult females and during October in Tottori due to diapause incidence. This information is in agreement with field observations and together might be useful for planning biological control programs for phytophagous mites and for successful establishment of predacious mites in new habitats.

The use of the cannibalistic habit and elevated relative humidity to improve the storage and shipment of the predatory mite Neoseiulus californicus (Acari: Phytoseiidae).

This study investigated the feasibility of using the cannibalistic habits of the mite Neoseiulus californicus (McGregor) and controlling the relative humidity (RH) to prolong the survival time during the storage or shipment of this predatory mite. Three-day-old mated and unmated females were individually kept at 25 ± 1 °C in polypropylene vials (1.5 mL), each containing one of the following items or combinations of items: a kidney bean leaf disk (L), N. californicus eggs (E), and both a leaf disk and the eggs (LE). Because the leaf disk increased the RH in the vials, the RH was 95 ± 2 % under the L and LE treatments and 56 ± 6 % under the E treatment. The median lethal time (LT50) exceeded 50 days for the mated and unmated females under the LE treatment. However, it did not exceed 11 or 3 days for all females under the L or E treatments, respectively. Under the LE treatment, the mated and unmated females showed cannibalistic behavior and consumed an average of 5.2 and 4.6 eggs/female/10 days. Some of the females that survived for LT50 under each treatment were transferred and fed normally with a constant supply of Tetranychus urticae Koch. Unmated females were provided with adult males for 24 h for mating. Only females previously kept at LE treatment produced numbers of eggs equivalent to the control females (no treatment is applied). The results suggested that a supply of predator eggs and leaf material might have furnished nutrition and water vapor, respectively, and that this combination prolonged the survival time of N. californicus during storage. Moreover, this approach poses no risk of pest contamination in commercial products.

Effects of temperature on the life-history traits of Sancassania (Caloglyphus) berlesei (Acari: Astigmatina: Acaridae) feeding on root-knot nematodes, Meloidogyne spp. (Nematoda: Meloidogynidae).

Sancassania (Caloglyphus) berlesei (Michael) is a cosmopolitan and free-living mite that inhabits soil as well as laboratory colonies of insects and fungi and may have a role as a biocontrol agent of nematodes. In this study, we investigated the effects of temperature on the development, reproduction, and food consumption of S. berlesei fed egg masses of root-knot nematodes, Meloidogyne spp., an important group of agricultural pests. Mites were reared at 20, 25 or 30 °C in the dark. The mites could feed on the nematode egg masses, and their developmental time decreased at higher temperatures. Time from the egg to adult was similar in females and males reared at the same temperature. Adult females lived longer than males at 25 °C, but not at 20 or 30 °C. Generally, females showed a higher rate of food consumption than males. Females laid the largest number of eggs at 20 and 25 °C (199.7 and 189.8 eggs/female, respectively), but the intrinsic rate of natural increase was highest at 30 °C (r m = 0.29). In comparing our data with previous reports, we noted that S. berlesei that fed on egg masses of root-knot nematodes showed a longer developmental time and a lower reproductive rate than Sancassania mites that fed on other diets. Nonetheless, the relatively high value of r m (e.g., at 25 and 30 °C) suggests that this mite may have certain advantages as a biocontrol agent of root-knot nematodes.

Rapid cold hardening response in the predatory mite Neoseiulus californicus.

We investigated the rapid cold hardening (RCH) response in the predatory mite Neoseiulus californicus (McGregor) (Acari: Phytoseiidae). On direct exposure, ≤2 % of adult females survived -10 °C for 2 h. However, when acclimatized first at 5 °C for 1 h, 75 % of females survived. RCH could also be induced by acclimatization at 30 °C for 2 h or anoxia (oxygen-free nitrogen) for 1-2 h. All immature stages showed enhanced survival when acclimatized at 5 °C for 2 h before exposure to -10 °C. Acclimatization at 30 °C induced RCH only in eggs and deutonymphs, and anoxia was effective for eggs, larvae, and deutonymphs. The variability among immature stages may be attributed to the cost associated with the acclimatization treatments. Our findings suggest that RCH may promote the survival of N. californicus during unexpected changes in temperatures, and can be an important feature particularly when this natural enemy is introduced to non-native environments.

Air temperature optimisation for humidity-controlled cold storage of the predatory mites Neoseiulus californicus and Phytoseiulus persimilis (Acari: Phytoseiidae).

BACKGROUND: Humidity-controlled cold storage, in which the water vapour pressure is saturated, can prolong the survival of the predatory mites Neoseiulus californicus (McGregor) and Phytoseiulus persimilis Athias-Henriot (Acari: Phytoseiidae). However, information on the optimum air temperature for long-term storage by this method is limited. The authors evaluated the survival of mated adult females of N. californicus and P. persimilis at 5.0, 7.5, 10.0 and 12.5 °C under saturated water vapour condition (vapour pressure deficit 0.0 kPa). RESULTS: N. californicus showed a longer survival time than P. persimilis at all the air temperatures. The longest mean survival time of N. californicus was 11 weeks at 7.5 °C, whereas that of P. persimilis was 8 weeks at 5.0 °C. After storage at 7.5 °C for 8 weeks, no negative effect on post-storage oviposition was observed in N. californicus, whereas the oviposition of P. persimilis stored at 5.0 °C for 8 weeks was significantly reduced. CONCLUSION: The interspecific variation in the response of these predators to low air temperature might be attributed to their natural habitat and energy requirements. These results may be useful for the long-term storage of these predators, which is required for cost-effective biological control.

Desiccation tolerance in diapausing spider mites Tetranychus urticae and T. kanzawai (Acari: Tetranychidae).

We investigated the effects of changes in vapor pressure deficit (VPD) on the survival of diapausing (winter form) and non-diapausing (summer form) spider mites Tetranychus urticae Koch and Tetranychus kanzawai Kishida (Acari: Tetranychidae). Adult females of both species were kept without food at VPDs of 0.0, 0.4, 0.7, 1.5, 1.9, or 2.7 kPa for 3, 6, 9, 12, or 15 days at 25 °C. Diapausing females of both species kept at a VPD of ≥0.4 kPa for ≥6 days clearly tolerated desiccation. Under water-saturated conditions (VPD = 0.0 kPa), in which no desiccation occurred, diapausing females showed high starvation tolerance: 90 % survived for up to 15 days. No interspecific differences in tolerance to desiccation or starvation were observed under most conditions. These results indicate that diapause functions increase tolerance to desiccation and starvation. Such multiple tolerances to harsh environments might support winter survival in spider mites.

Effects of air temperature and water vapor pressure deficit on storage of the predatory mite Neoseiulus californicus (Acari: Phytoseiidae).

To determine the optimum air temperature and water vapor pressure deficit (VPD) for the storage of the predatory mite, Neoseiulus californicus, 3-day-old mated females were stored at air temperatures of 0, 5, 10, or 15 °C and VPDs of 0.1, 0.3, or 0.5 kPa for 10, 20, or 30 days. At 10 °C and 0.1 kPa, 83 % of females survived after 30 days of storage; this percentage was the highest among all conditions. VPDs of 0.3 and 0.5 kPa regardless of air temperature, and an air temperature of 0 °C regardless of VPD were detrimental to the survival of the females during storage. Since the highest survival was observed at 10 °C and 0.1 kPa, the effect of the storage duration on the post-storage quality of the stored females and their progeny was investigated at 25 °C to evaluate the effectiveness of the storage condition. The oviposition ability of the stored females, hatchability, and sex ratio of their progeny were not affected even when the storage duration was extended to 30 days. Although a slight decrease in the survival during the immature stages of progeny was observed when the storage duration was ≥20 days, the population growth of N. californicus may not be affected when individuals stored in these conditions are applied to greenhouses and agricultural fields. The results indicate that mated N. californicus females can be stored at 10 °C and 0.1 kPa VPD for at least 30 days.

A high-performance humidity control system for tiny animals: demonstration of its usefulness in testing egg hatchability of the two-spotted spider mite, Tetranychus urticae.

We developed a computer-based system for controlling water vapor conditions (i.e., humidity) using a two-flow method in which streams of humidified and dehumidified air were combined in an acrylic container. The flow rate of each stream was independently controlled to adjust relative humidity (RH). In this system, humidification from 15 to 90 % RH and dehumidification from 90 to 15 % RH at an air temperature (AT) of 25 °C were properly operated with short time constants of 4.3 and 10 min, respectively. Tetranychus urticae egg hatchability was then examined at 20-95 % RH and 25 °C AT. The coefficients of variation of RH were low (0.3-1.5 %). Egg hatchability in a polystyrene Petri dish was lower at 20 % RH than at 70-95 % RH. A delay in hatching was also observed at 70 % RH for eggs tested on a leaf disk placed on water-soaked cotton; this delay was attributed to the AT being 1.4 °C lower on the leaf surface than on the inner surface of the dish. Our system is expected to be useful for further examination of ecological and behavioral responses in pest mites and for developing novel physical control measures using water vapor.

Night-interrupting light inhibits diapause induction in the Kanzawa spider mite, Tetranychus kanzawai Kishida (Acari: Tetranychidae).

The inhibitory effects of the timing, intensity (I(I)) and period (I(T)) of night-interrupting light on diapause induction of the Kanzawa spider mite (Tetranychus kanzawai) were investigated in a series of laboratory experiments. During a light and dark period of 8 and 16 h d(-1), respectively, a single 1-h night-interrupting light was applied at early (E), middle (M), and late (L) parts of the dark period: i.e., at 3, 7.5, and 12h after the start of the dark period, respectively. No interrupting light was applied in the control treatment. The incidence of diapause was significantly lower in the M treatment (63%) compared to the control treatment (100%). In the E and L treatments, more than 90% of females entered diapause, which was comparable to the control treatment. Since the longest consecutive dark period during the E and L treatments was longer than the critical dark period (CDP) of 10.5-11 hd(-1), during which 50% of females entered diapause, the night-interrupting light probably failed to prevent diapause induction. However, in the M treatment, the longest consecutive dark period was shorter than the CDP; therefore, the night-interrupting light inhibited diapause induction. Moreover, the inhibitory effects of night-interrupting light in the M treatment increased as I(I) and I(T) increased. The dose of night-interrupting light (I(I)×I(T)) was significantly negatively related to the incidence of diapause. The median effective dose for 50% disturbance of diapause induction was 2.5 kJ m(-2) at wavelengths between 350 and 1050 nm. Our results suggest that the longest consecutive dark period and the dose of night-interrupting light should both be considered when a lighting-based physical control is applied to inhibit diapause induction and consequent overwintering of T. kanzawai in commercial agricultural fields.

Effect of photoperiod on immature development and diapause induction in the Kanzawa spider mite, Tetranychus kanzawai (Acari: Tetranychidae).

The diapause response of the Kanzawa spider mite (KSM), Tetranychus kanzawai, was examined. KSMs were reared in aluminum bottles at 18°C with different combinations of light and dark periods created by the light-control unit. The developmental periods for all immature stages tended to decrease as the light period increased. The photoperiodic response curve for diapause induction showed that the critical (=50% diapause) light period was around 13 h days(-1). No diapause induction was observed when the light period was longer than 13.5 h days(-1) or under continuous light. At 13-h days(-1) light period, the developmental period for deutonymphal stage as well as for the total immature stages was longer in diapaused females than in non-diapaused females. These results indicate that immature development as well as diapause induction are affected by photoperiod and further suggest that diapause-inducing stimuli prolong the developmental period especially for the deutonymphal stage of KSMs.