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Crinum brachynema is a bulbous plant belonging to the family Amaryllidaceae which is restricted to Western India. Due to its high rarity and low distribution range, it has been classified as “critically endangered”. Establishing an efficient and unprecedented somatic embryogenesis protocol is necessary for its conservation and large-scale propagation. In this study, regeneration was achieved through somatic embryogenesis using bulb explants on MS media supplemented with various concentrations of 2,4-D alone and in combination with N6-benzyl-adenine. Different advanced phases with maturation of somatic embryo were obtained on MS medium with different ratios of picloram and thidiazuron (TDZ). The highest number of somatic embryos (50.33 ± 1.52) was obtained after eight weeks in the medium supplemented picloram (2.0 mg L−1) in combination with TDZ (0.5 mg L−1). MS medium with reduced concentration of salts in combination with GA3 (1.0 mg L−1) was used for somatic embryo germination. The maximum embryo germination frequency (82.22) was recorded on half-strength MS medium fortified with 1 mg L−1 GA3. The genetic true-to-typeness of regenerated plants was confirmed by ISSR, SCoT and RAPD primers based molecular analyses. This confirmed their genetic homogeneity compared to the mother plant and it also demonstrated the reliability of our somatic embryogenesis system for C. brachynema. The protocol developed may facilitate efforts in reintroduction, restoration, and ex situ conservation of C. brachynema in its natural habitat and its potential commercial utilization.Key messageWe provided the first report on somatic embryogenesis system in C. brachynema. SEM indicated the morphogenesis and several molecular markers revealed genetic homogeneity of the regenerated plants.

Crinum brachynema (Amaryllidaceae) is an endemic and critically endangered bulbous medicinal plant of the Northern Western Ghats of India. This species contains an important and promising alkaloid called galanthamine (GAL), an anti-Alzheimer drug. This investigation aimed to establish a reproducible protocol for in vitro regeneration of C. brachynema using twin scales as initial explants for the first time. Thus, cultures were established on Murashige and Skoog (MS) medium with cytokinins (6-benzyladenine (BA), and meta-Topolin (mT). The establishment and growth of shoots from the explants was achieved on MS medium supplemented with BA alone and in combination with 1-naphthaleneacetic acid (NAA). In vitro shoots were cultured on MS medium containing 0.5–10 µM of mT, of which 5.0 µM mT gave the highest shoot regeneration frequency (11.10). In general, mT proved superior as cultures produced a maximum number of regenerants compared to the control. Rooted plants with bulblets were successfully acclimatized in the greenhouse where they were phenotypically similar to the mother plants. Antioxidant activity was estimated using oxygen radical scavenging capacity (ORAC) model. Cultures on mT exhibited a significant increase in antioxidant capacity compared to the control. These findings highlight the beneficial effect and validate the rising importance of mT for in vitro regeneration studies. This study will serve as a potential protocol to conserve and restore the medicinally important C. brachynema, while the regenerated C. brachynema may be subsequently manipulated to further increase the accumulation of antioxidant potential.Key MessageAn efficient regeneration strategy was developed for conservation of Crinum brachynema, a critically endangered medicinal plant, using twin-scale explants; meta-Topolin enhanced in vitro shoot proliferation and improved regenerants antioxidant activity.

Surveys for egg-parasitoid wasps were conducted in Rafsanjan, Iran, on two species of Pentatomidae (Hemiptera) found in pistachio orchards, Acrosternum arabicum Wagner and Brachynema signatum Jakovlev. Five species of Scelionidae (Platygastroidea) were recovered, including one that is here described as new: Psix saccharicola (Mani), Trissolcus colemani (Crawford), T. darreh Talamas sp. nov. , T. perepelovi (Kozlov), and T. semistriatus (Nees). In addition to describing a new species, we report new host associations, provide COI barcodes for four of these species, and discuss host-related intraspecific variation in T. darreh and T. perepelovi .

Insect symbionts offer an opportunity to deal with the anticipated elevated demand for novel pest management strategies. One approach is the disruption of essential symbionts required by the pests. In the present study, we examined the effects of symbiont elimination strategies, high temperature and sterilization agents, on the fitness of three stink bugs, Brachynema germari Kolenati, Acrosternum heegeri Fieber, and Acrosternum arabicum Wagner by using demographic approach. In the high-temperature experiments, almost all insects exhibited severe fitness defects, including elevated nymphal mortality and reduced population growth parameters (especially intrinsic rate of increase, r), as well as significant reductions in the gut symbiont titers. In the egg surface sterilization assays, we experimentally assessed the effects of sterilization agents on the bugs and their symbionts and observed similar fitness defects to those observed under the high-temperature condition. According to the results, we concluded that the host's defective phenotypes are attributable not to the heat stress itself but to the suppression of the symbiont titer, which highlights the possibility that global warming and elevated temperature may negatively affect this mutualism.Together, the results suggest the biological importance of the bacterial symbiont for the host that might help us for better management of these important pests in the future.

Pistacia vera L. is one of the most important horticultural crops in Iran. The stink bugs Acrosternum arabicum and Brachynema germari are two of the key pests that cause significant direct and indirect damage on Pistacia vera. Egg parasitoids have been considered as potential biological control agents of pistachio green stink bugs. Among them, Trissolcus semistriatus and Psix saccharicola are the most abundant and efficient parasitoid for A. arabicum in pistachio orchards. In this study, we assessed lethal and sublethal effects of two commonly used insecticide products (fenitrothion and a binary mixture of lambda-cyhalothrin + thiamethoxam) on these two parasitoid wasps under laboratory conditions. The median lethal concentration (LC50) values for fenitrothion and thiamethoxam + lambda-cyhalothrin in P. saccharicola and T. semistriatus were estimated as 1.03, 0.48 and 0.87, 0.98 mg a.i./liter, respectively. In terms of sublethal effects, insecticide treatments altered the type of functional response from type III to type II in T. semistriatus. However, P. saccharicola exhibited a type II functional to density of A. arabicum for all treatments, although attack rates were lower for insecticide-exposed wasps while handling times increased. Our results show that sublethal effects of insecticides further reduce the efficacy of biological control agents. Effective integrated pest management programs should avoid antagonistic interactions between chemical and biological control methods. The results of this study provide useful information to develop comprehensive integrated pest management programs for stink bugs in pistachio orchards.

Females of Trissolcus agriope (Platygastridae) avoid host (Brachynema signatum) eggs parasitized by conspecifics or Ooencyrtus pityocampae (Encyrtidae), but females of the latter species will superparasitize and multiparasitize, although they mostly attack unparasitized eggs. Females of T. agriope were more efficient in the laboratory and parasitized more hosts in 24 and 48 h. In multiparasitized hosts, O. pityocampae was a superior larval competitor and could complete also development as a facultative hyperparasitoid. When females of both species foraged together, T. agriope parasitized significantly more than O. pityocampae, about 50 % in both cases, although O. pityocampae almost doubled its parasitism rate as the exposure period was lengthened from 24 to 48 h. When O. pityocampae followed T. agriope in sequential foraging bouts, the former species successfully parasitized more hosts than the latter. The advisability of co-releases of both species to improve biological control of first generation B. signatum in Iranian pistachio orchards is discussed.

Galanthamine, an isoquinoline alkaloid and the current drug of choice for treatment of mild to moderate Alzheimer disease, is mainly obtained from Amaryllidaceae members. At present, the production of galanthamine from available natural plant sources is not sufficient to meet the demands of pharmaceutical industry. Additionally, overexploitation of the plant material leads to the depletion of available natural wild populations. Therefore, there is a need to explore additional natural sources for the extraction of this drug. Thus the aim of this study is to determine galanthamine content in five Indian Crinum species. The bulbs of each Crinum species were extracted with methanol. Extracts were analysed by high performance liquid chromatography. The chromatographic separation was performed using an isocratic system with a mobile phase of methanol : 5 mM (NH4)2HPO4 (55:45 v/v) applied at a flow rate 0.8 ml/min using a UV detector at 288 nm. Among all the Crinum species studied, the highest galanthamine content was found in the bulbs of C. malabaricum Lekhak & S.R. Yadav (0.308 ± 0.004%), followed by C. viviparum (Lam.) R. Ansari & V.J. Nair (locality Ratnagiri; 0.262 ± 0.042%). However, C. brachynema Herb, and C. pratense Herb, (locality Borbet) showed the lowest and equal galanthamine content (0.029 ± 0.000%) in their bulbs. Galanthamine was not detected in the bulbs of C. latifolium L. This study identifies novel plant sources of galanthamine, which may be helpful for pharmaceutical production of galanthamine. The present study provides a quantitative comparison of galanthamine among Indian Crinum species.

This research was conducted to develop a composite diet and compare its effects on developmental time, fecundity, survival rate, and life table parameters of pistachio green stink bug, Brachynema germari Kolenati (Hemiptera: Pentatomidae) with a natural diet. The biological parameters of this pest were investigated using two-sex, age-stage life table theory on both composite and natural diets (25 ± 1°C, 50 ± 10% relative humidity, and a photoperiod of 16:8 [L:D] h). Based on the preliminary studies, the combination of the green beans (Phaseolus vulgaris L.), sunflower seeds (Helianthus annuus L.), and wild rue capsules (Peganum harmala L.) was introduced into a composite diet. There was no significant difference between the longevity of each stage on diet treatments, but the mean fecundity of female fed on composite diet was about 196.48 ± 12.02 (eggs), which indicated a significant decrease than the fecundity of female fed on the natural diet (265.81 ± 11.67 eggs; P=0.05, t-test). Also, main parameters of life table (r, R0, T, and λ) for composite and natural diets were about 0.0863 ± 0.0031 d–1, 89.03 ± 13.33 eggs, 51.987 ± 0.536 d, and 1.091 ± 0.0034 d–1 and 0.1023 ± 0.003 d–1, 128.75 ± 17.56 eggs, 47.454 ± 0.439 d, and 1.1077 ± 0.0037 d–1, respectively. There was a significant difference between these four parameters on both diets. Despite a significant decrease in fecundity with using composite diets, this diet can satisfy our needs now and the laboratory rearing of pistachio green stink bug did not stop over the year in the absence of host weeds and insect did not diapause.