Entomopathogenic fungi-mediated biological control of the red palm weevil Rhynchophorus ferrugineus

The red palm weevil (RPW), Rhynchophorus ferrugineus, is an important pest of palms, and difficult to control by conventional methods. Therefore, microbial control is an alternative strategy for controlling RPW. Herein, a total of 15 entomopathogenic fungi (EPFs) were subjected to primary pathogenicity screening against last stage of RPW larvae. The preliminary data showed that four Beauveria bassiana isolates (JEF-484, 158, 462 and 507) and one Isaria fumosorosea isolate (JEF-014) resulted in 100 % mortality within 5–10 days post inoculation (d.p.i.), respectively. According to the time required for RPW mortality, JEF-484, 158, 462 and 014 were further subjected to bioassays using 10⁷ conidia/ml suspensions by spraying method. Based on the results, JEF-484 showed the highest mortality and shortest LT₅₀ on the last stage of RPW larvae, followed by JEF-158. The two isolates also showed good conidial production and high thermal stability compared to the other isolates. Therefore, JEF-484 and JEF-158 were selected for bioassays against RPW egg and the last larval stage with different concentrations of 10⁵, 10⁶ and 10⁷ conidia/ml conidial suspensions by spraying method. For the bioassay at the egg stage, JEF-158 showed a significantly higher ovicidal effect than JEF-484. In the larval bioassay, both EPF isolates showed a dosage-dependent effect on the RPW larvae. JEF-484 caused higher mortality in RPW larvae than JEF-158. In summary, the combination of the 2 promising EPF isolates might provide an opportunity for the practical microbial control of RPW at different life stages in palm tree fields.     

室内饲养红棕象甲的行为观察

报道红棕象甲Rhynchophorus ferrugineus Fab.成虫交配、产卵行为,描述在交配和产卵过程中发生的多种行为。红棕象甲整日均可见其交配行为,但在暗光周期开始0.5h左右明显有一个交配高峰期。其交配持续时间、交配几率不受虫体大小的影响。在无外界因子干扰的条件下,交配持续时间均可达1min。红棕象甲卵为单产,一处1粒,主要是通过口喙蛀孔辅助完成;观察红棕象甲幼虫、蛹的行为,例如取食、争斗、蜕皮、做茧等行为。且随着低龄幼虫向高龄幼虫的过渡,幼虫的取食和钻蛀行为从棕榈植物中心向外围扩散。通过摇动或震动茧后红棕象甲在其中表现的行为,并结合茧的颜色和质地可准确判断茧中存在的3种虫态。     

上海地区红棕象甲的耐寒性研究

红棕象甲Rhynchophorus ferrugineus (Olivier)是我国危害棕榈科植物的重要入侵害虫, 为探明其越冬抗寒性, 利用过冷却点测定仪测定了红棕象甲上海种群不同虫态的过冷却点, 在低温箱内测定了该虫不同虫态的耐寒性, 然后结合田间越冬模拟试验、气象资料和寄主分布情况, 初步确定了红棕象甲在我国的越冬北界。结果表明： 红棕象甲过冷却点随虫态的发育程度的升高而下降, 卵、1龄幼虫、5龄幼虫、9龄幼虫和成虫的平均过冷却点分别为-5.92, -6.42, -7.19, -7.43和-11.84℃, 过冷却点由高到低的顺序依次为: 卵>幼虫>成虫。在6, 24, 48和72 h 4个时间处理下, 各虫态在低温与存活率之间呈显著或极显著的logistic回归, 半致死温度（Ltemp₅₀）均随处理时间的延长而上升, 不同虫态在处理72 h 后, 卵、1龄幼虫、5龄幼虫、9龄幼虫和成虫之间的Ltemp₅₀分别为1.61, -1.67, -2.39, -2.40和-0.40℃, 各虫态耐寒性由弱到强的顺序依次为: 卵<成虫<幼虫。红棕象甲不同发育阶段的过冷却点与其耐寒性并不完全相关, 幼虫和成虫均可能是该虫的越冬虫态。连续两年的田间模拟越冬试验表明, 在上海地区, 红棕象甲的幼虫和成虫的越冬存活率均在60％以上, 说明红棕象甲在上海地区是可以越冬的。根据这些结果, 结合寄主分布情况, 初步将红棕象甲在我国的越冬北界定于北纬35°附近, 即1月份0℃等温线左右。     

Nano essential oils against the red palm weevil,Rhynchophorus ferrugineus Olivier (Coleoptera: Curculionidae)

The red palm weevil, Rhynchophorus ferrugineus, is the biggest pest of various palm species. The aim of this study was to evaluate the impact of the essential oils of purslane, mustard and castor (bulk and nano) on R. ferrugineus and to evaluate the oviposition deterrent effect to prevent new infestations. Prepared concentrations of essential oils (bulk and nano) were tested for their impact on R. ferrugineus larvae, pupae and adults. The most effective oil was purslane oil (bulk and nano), followed by mustard and the least effective was castor. The percent mortality of larvae was 75.2, 45.3 and 17.9% (bulk phase) and 92.5, 84.4 and 65.5% (nano phase) in purslane, mustard and castor, respectively, when treated with the high concentration. The percent mortality of adults was 67.2, 40.0 and 15.6% (bulk phase) and 83.5, 72.9 and 60.0% (nano phase) in purslane, mustard and castor, respectively, when treated with the high concentration. The number of eggs laid decreased with increasing concentration of the tested oils. Purslane essential oil showed good impact on R. ferrugineus larvae and adults followed by mustard and castor oils as bulk and as nano.     

警惕外来危险害虫红棕象甲的扩散

红棕象甲Rhynchophorus ferrugineus(Olivier)是棕榈科植物外来危险害虫,该虫原产印度,20世纪80年代扩散到中东,目前在我国主要分布在海南、广西、广东、台湾、云南、西藏(墨脱)等地区,存在进一步扩张的趋势。文章对该虫的地理起源及分布、生物学特性、寄主及防治等方面作了描述。     

Potential of certain natural extracts for the control of the red palm weevil,Rhynchophorus ferrugineus (Oliver)

Abstract

Laboratory bioassays were conducted to evaluate the potential of 13 plant extracts for the control of the red palm weevil, Rhynchophorus ferrugineus. Results indicated that the ethanol extracts of sea ambrosia (Ambrosia maritima), French cotton (Calotropis procera) and curcuma (Curcuma longa) have superior toxicity as compared to the extracts of other tested plants, estimated LC 50 were 0.723, 0.978 and 1.092%, respectively. In field experiments, the percentage of recovered date palm trees reached 90.91% within 15 days when A. maritima extract was applied. The injection of C. procera and C. longa extracts into the tunnels of infested trees resulted in 86.36% and 85.00% of tree recovery, respectively.     

Sex ratio and the role of mild relative humidity in mating behaviour of red date palm weevil Rhynchophorus ferrugineus Oliv. (Coleoptera: Curculionidae) gamma‐irradiated adults

The influence of gamma radiation on the mating behaviour of the red date palm weevil (RDPW), Rhynchophorus ferrugineus (Olivier), and the efficacy of sterile insect technique (SIT) under different levels of relative humidity (RH) were studied. No adverse effects of gamma radiation were observed on the mating behaviour parameters of the RDPW, such as mate recognition time, mating duration, mating frequency within a 30‐min period and duration between consecutive matings. However, the weevils were sexually stimulated during aggregation. RH significantly affected egg laying as well as egg hatching. Significantly lower egg laying and hatching were recorded at 25% RH than at higher humidity levels, suggesting that low humidity conditions are better for successful SIT manipulations in the field. Lifespan of irradiated RDPW males was significantly shorter than that of un‐irradiated controls, irrespective of RH level.     

Pseudomonas aeruginosa,a facultative pathogen of red palm weevil,Rhynchophorus ferrugineus

Pseudomonas aeruginosa was identified as a facultative pathogen of red palm weevil. Intra-haemocoelic injection of the pathogen within larvae and pre-pupae was more effective at killing the insects [with a median lethal dose (LD₅₀) of 9×10² to 2×10³ bacteria/insect] than inoculation by force feeding (LD₅₀ of 10⁵ to 4×10⁵ bacteria/insect) or by wading the insects in a suspension of the pathogen (LD₅₀ of 10⁵ to 2×10⁵ bacteria/insect). Injection of 3×10³ bacteria/insect killed 69% of larvae; small larvae were more susceptible (LD₅₀ of 9×10⁵ bacteria/larva) than either larger larvae (LD₅₀ of 10³ bacteria/larva) or pre-pupa. The median time to death of the small larvae following injection of P. aeruginosa was about 6 days but that following force feeding or wading was about 8 days. A secondary invader, Serratia marcescens, had no effect on the pathogenicity of P. aeruginosa but hastened death of larvae by about 3 days.A. Banerjee and T.K. Dangar were with the Central Plantation Crops Research Institute, Regional Station, Kayangulam 690 533, Kerala, India. They are now with the Central Rice Research Institute. Cuttack 753 006, Orissa, IndiaCPCRI research paper no. 870.     

A large‐scale gene discovery for the red palm weevil Rhynchophorus ferrugineus (Coleoptera: Curculionidae)

The red palm weevil (RPW; Rhynchophorus ferrugineus) is a devastating pest of palms, prevalent in the Middle East as well as many other regions of the world. Here, we report a large‐scale de novo complementary DNA (cDNA) sequencing effort that acquired ～5 million reads and assembled them into 26 765 contigs from 12 libraries made from samples of different RPW developmental stages based on the Roche/454 GS FLX platform. We annotated these contigs based on the publically available known insect genes and the Tribolium castaneum genome assembly. We find that over 80% of coding sequences (CDS) from the RPW contigs have high‐identity homologs to known proteins with complete CDS. Gene expression analysis shows that the pupa and larval stages have the highest and lowest expression levels, respectively. In addition, we also identified more than 60 000 single nucleotide polymorphisms and 1 200 simple sequence repeat markers. This study provides the first large‐scale cDNA dataset for RPW, a much‐needed resource for future molecular studies.     

Potential role of microbial pathogens in control of red palm weevil (Rhynchophorus ferrugineus) ‐ A Review

The invasive Red Palm Weevil (RPW), Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae), is one of the most destructive pests of ornamental and economically important palms globally. It has been found in 50 % of date‐growing and 15 % of coconut‐producing countries in the world. Synthetic organic insecticides have been the default method to combat this pest, but they are clearly inefficient due to the secretive nature of the insect and there is concern about non‐target effects from blanket spraying. For this reason, there is increasing interest in biological control methods such as the possible use of microbial entomopathogens, which might be incorporated into IPM approaches. In this review we summarize research work on microbial control agents, their effectiveness against RPW and their integration with other control measures.     

Studies on host preference and its biological effects on the red palm weevil,Rhynchophorus ferrugineus Olivier in Egypt The fruit trees

A comparative bioassay (choice or non-choice) was developed to evaluate the efficacy of different species of fruit trees as alternative host plants on the morphological and biological aspects of the red palm weevil, Rhynchophorus ferrugineus and also the latent effect of feeding the larvae on pupal and adult stages. Highly significant differences exist between averages of larval body weight of R. ferrugineus larvae fed on fruit trees. The lowest average of larval weight occurred in the larvae fed on the pieces of sour orange, mandarin followed by olive and lime trees. A small increase in body weight appeared in larvae fed on mango, fig and guava in comparison with those fed on date palm trees (control). The larval mortality reached the highest percentages (100%) by obligatory feeding of R. ferrugineus insects on olive, lime, sour orange and mandarin followed by mango (97%) and guava (96%) compared with control (0%), respectively. The latent effect of obligatory feeding on the larvae of R. ferrugineus was highly decreased during pupation (4%, 4% and 8%) when the insect fed on mango, guava and fig, compared with 100% pupation in the case of larvae fed on ornamental palm or date palm, respectively. Most pupae resulting from the treated larvae by R. ferrugineus failed in adult emergence especially those insects fed on olive, lime, sour orange and mandarin trees compared to untreated insects (95.6–96.2%), respectively. Data also proved that if an insect is forced to feed on a non-preferred host plant, this results in death of the insect often during the larval stage, a decrease of pupation or failure of adult emergence. These results led to the conclusion that mango, fig, and guava trees are believed to be vulnerable hosts for red weevil attack. The lowest percentage of food consumption appeared in those larvae fed on mandarin, sour orange, lime and olive trees compared with those fed on palm. The present results emphasise the need to test the efficiency of the compounds extracted from these host plants if it is considered as an insecticide or an alternative host protectant for management of the red palm weevil R. ferrugineus.     

Isolation and identification of bacterial strains with insecticidal activities from Rhynchophorus ferrugineus Oliver (Coleoptera: Curculionidae)

Several pathogenic bacteria strains were isolated from dead larvae of the weevil, Rhynchophorus ferrugineus Oliver, collected on palm trees in Taiwan, China. Strains of pure cultures were temporarily named TW‐1, TW‐2, TW‐3, TW‐4, TW‐5, TW‐6, TW‐7, TW‐8, TW‐9, TW‐10, TW‐11, TW‐12, TW‐13 and TW‐14. These strains were identified by synthetically using colony morphological characters, physiological and biochemical tests and molecular biological analysis. Our results showed that these 14 isolated strains belonged to four genera and five species. The expected approximate 1400‐bp bands were obtained through 16S rDNA universal primer amplification. The sequencing results obtained from a commercial sequencing company were initially compared with a corresponding database from NCBI using BLAST, and a phylogenetic tree was constructed using mega 5.1 software. The phylogenetic results indicated that these isolated strains had more than 99% homology with type strains. To further confirm these strains, numerous physiological and biochemical indexes were confirmed. TW‐1, TW‐2, TW‐3, TW‐9, TW‐12, TW‐13 and TW‐14 were identified as Serratia marcescens; TW‐4, TW‐5, TW‐8 and TW‐10 as Staphylococcus sciuri; TW‐6 as Klebsiella pneumonia subsp. pneumonia; TW‐7 as Proteus vulgaris and TW‐11 as Proteus mirabilis. The preliminary single‐dose bioassay on fourth instar larvae showed that S. marcescens had a higher virulence at a density of 1.89 OD₆₀₀ (56.37% mortality) than the other strains. The corrected mortalities of the other strains were all less than 50%. The lowest toxicities were found for P. vulgaris and P. mirabilis where the corrected mortalities were only 28.18% and 25.57%, respectively. LC₅₀ of S. marcescens was 1.2 × 10⁷ CFU/ml inoculums. Our results indicated that S. marcescens from facultative cultivable bacterial flora isolated from R. ferrugineus could potentially be used as a microbial control agent for this widespread pest.     

Evaluation of phylogenetic relationship between three phenotypically different forms of Red date palm weevil Rhynchophorus ferrugineus Oliv. using PCR-based RAPD technique

Abstract

Red date palm weevil Rhynchophorus ferrugineus Oliv. is a widespread major pest of date palm in the Kingdom of Saudi Arabia. Three different forms (black and brown with and without spots on thoracic region) were investigated using PCR-based RAPD technique. Although weevils were collected from the same geographical region of Al-Hassa in Saudi Arabia, the banding profile acquired suggested that black and brown colored morphs are genetically closer compared to the brown with spots. Intra color variation remained minimum in black but brown and brown spotted morphs exhibited more genetic variation. This genetic variation may be either due to the generation of new mutants from the non-spotted or spotted weevil or they may belong to a different race.     

Antioxidant status and ultrastructural defects in the ovaries of red palm weevils (Rhynchophorus ferrugineus) intoxicated with spinosad

Red palm weevil, Rhynchophorus ferrugineus, destroys palms almost all over the world, especially in the Middle East, where dates are a strategic crop in arid places. In view of the urgent need to combat this destructive pest, effective pesticides with high environmental safety should be sought. Spinosad is a pesticide of bacterial origin that is presumed to have a high degree of environmental safety and is effective in combating a wide range of insect pests. In this study, the efficacy of spinosad was evaluated in females of R. ferrugineus. The lethal concentration for 50% of the treated females was calculated at 44.3 ppm. The effects of spinosad concentrations of 10, 50 and 200 ppm on the activity of catalase (CAT), glutathione S‐transferase (GST) and superoxide dismutase (SOD) in the ovaries were assessed. In addition, the pathological effects of these concentrations were documented on the ultrastructure of the follicle cells and ooplasm. The results showed a significant increase in CAT activity only in response to treatment with 200 ppm. Treating the females with different spinosad concentrations resulted in varying intensity changes in cell organelles, where the most pronounced sign of programmed cell death was at the concentration 200 ppm. This study demonstrates the possibility of using spinosad as an insecticide against females of R. ferrugineus.     

Functional compounds from the integument of adult red palm weevil Rhynchophorus ferrugineus

This study deals with the analysis of the integument of the red palm weevil Rhynchophorus ferrugineus of both sexes using the GC–MS technique. The results of the study revealed many promising compounds. These include aspidofractinine-3-methanol (kopsinyl alcohol) which was found in the acetone extract of the sternum of females, and 3-buten-2-ol (32-B) which was found in the extracts of sternum and tergum of males. This compound the aggregation pheromone was secreted by males. Additionally, compounds with methoxy groups were found. These may be responsible for insects’ resistance. This study, through separation and identification of these compounds, aims to open a new possibility for their future medical and therapeutic usage.     

Identification and enzymatic characterisation of digestive glucosidases from gut of red palm weevil,Rhynchophorus ferrugineus (Olivier, 1790) (Coleoptera: Curculionidae)

Red palm weevil, Rhynchophorus ferrugineus (Olivier, 1790) (Coleoptera: Curculionidae) is a serious pest of date worldwide. Thus, damage to palms (almost exclusively to Phoenix canariensis Hort) has been recorded in various places. Thus, the aim of the current study was to investigate two major digestive enzymes of this insect, α-D-glucosidase and β-D-glucosidase. The results showed that α-D-glucosidase and β-D-glucosidase are present in the insect gut mainly in the midgut and hindgut but trace amounts of the both enzymes were found in the foregut. Optimum temperature for α- and β-glucosidases was found to be 50 and 40?°C, respectively, and pH values were 4.0. The activity of glucosidases were increased by NaCl and KCl and inhibited by some compounds such as MgCl₂ and CaCl₂. Kinetic parameters showed that K _m of α and β-D-glucosidases was 3.15 and 4.11?mM, respectively. Therefore, it is concluded that in this insect species, both α-glucosidase and β-glucosidase are active but with different amounts. Understanding of the digestive physiology and glucosidase activity of red palm weevil is important when new management strategies based on interfering in the gut physiology of the insects are devised.     

Lethal and trans-generational effects of Metarhizium anisopliae on red palm weevil,Rhynchophorus ferrugineus,using age-stage,two-sex life table

Outbreaks of red palm weevil, Rhynchophorus ferrugineus Olivier, in Pakistan are considered a very serious threat to the date industry, owing to the sudden death of date palm trees. Current integrated management strategy largely depends on insecticides, with negative impacts on the environment. Integration of bio-control strategies incorporating entomopathogenic fungi (EPF) may provide an ecologically safe alternative. In the current study the effectiveness of Metarhizium anisopliae Sorokin against R. ferrugineus was investigated using the immersion method and the effects of this fungus on biological parameters of R. ferrugineus were estimated by age-stage, two-sex life table software. After treatment with M. anisopliae isolate (Ma-M₂), lethal concentration (LC₅₀) 1.2 × 10⁶ spores/mL was determined against R. ferrugineus 3rd instar grubs. M. anisopliae infected grubs showed reduced net reproductive rate R_o (75.55) and mean generation time T (113.886 days) and intrinsic rate of increase r (0.03797) compared to the untreated group. Also, adult longevity of males and females were significantly reduced in the M. anisopliae-infected group (F₁) as compared to the untreated group. Trans-generational studies revealed that grubs stage duration (69.34 days) and fecundity (181.32 eggs) of the M. anisopliae-infected group (F₁) were decreased compared to the untreated group. The use of lasting and adverse effects on the biological parameters of R. ferrugineus, indicate that treatment with EPF may provide an eco-friendly management strategy for this pest.     

The culturable bacterial community of frass produced by larvae of Rhynchophorus ferrugineus Olivier (Coleoptera: Curculionidae) in the Canary island date palm

Aims: Larvae of the red palm weevil (RPW) Rhynchophorus ferrugineus Olivier (Coleoptera: Curculionidae) feed inside palm stem tissues, making galleries and producing a wet fermenting frass. We characterized the culturable bacteria associated with frass produced by tunnelling larvae inside the Canary island date palms and investigated the role of frass and gut bacteria in plant polymers breakdown. Methods and Results: A culture‐dependent method was used to isolate bacteria from frass and noninfested palm tissues. Bacterial isolates were grouped into operational taxonomic units based on polymorphisms in the ITS‐PCR profiles, and representative isolates were identified by partial sequencing of the 16S rRNA gene. Frass bacteria were dominated by 2,3‐butanediol fermenter Enterobacteriaceae. None of the bacterial isolates was able to degrade cellulose; however, cellulolytic and hemicellulolytic bacteria were isolated from the larval gut enrichment cultures. Conclusions: Frass bacteria are specifically associated with the RPW larvae and might play beneficial roles for RPW, other than nutritional, that deserve further investigations. Breakdown of plant polymers probably occurs inside the larvae digestive system. Significance and Impact of the Study: Frass and gut micro‐organisms of R. ferrugineus should be included in studies of the interactions between RPW, its plant hosts, and its enemies.     

Spatial patterns of the red palm weevil and applied entomopathogenic nematode Heterorhabditis bacteriophora

The distribution and abundance of red palm weevil (Rhynchophorus ferrugineus) within palm tree-infected stems at Ismaelia governorate, Egypt, were investigated. Taylor's power law was fit (P ≤ 0.01) to its larvae, pupae and/or adults. The slope value of this power law indicated the clumped distribution of these insect stages. Sample size optimisation needed to achieve a predetermined level of sampling error for the insect stages was calculated. Also, soil samples were taken after uniform application of entomopathogenic nematode Heterorhabditis bacteriophora strain EG₁ to the soil under citrus tree canopies at the rate of 10⁴ infective juveniles/400 cm² plots, and assayed for the nematode using the Galleria bait method. At the first and second sampling dates, the nematode displayed contagious distribution and attained mean insect mortality of 15.3 and 4, respectively. At the third date of sampling, the nematode showed random distribution according to chi-squared test and caused 1.8 mean insect mortality. Evaluation of H. bacteriophora EG₁ as a biocontrol measure for the weevil was discussed based on their investigated dispersion indices.     

Studies on host preference and its biological effects on the Red Palm weevil,Rhynchophorus ferrugineus Olivier in Egypt B. vegetable crops

The investigation on some Egyptian vegetables preference by the Red Palm weevil (RPW), Rhynchophorus ferrugineus has been carried out under laboratory conditions to explore a suitability of alternative host plants. The choice test of insect larvae showed that Taro, Squash, Eggplant, common potato, yellow carrot, tomato and sweet pepper were accepted food, while the Table beet (Vegetable) was rejected. Non-choice feeding of R. ferrugineus larvae on vegetables affected larval body weight and increased larval mortality. Highest rate of pupation was recorded from larvae fed on C. pepo (92%) and B. vulgaris (97%). Adult emergence reached 80% and 92% on C. pepo and Sugar beet (field crop) (field crop). The external skin of sugar cane was not acceptable food for insect larvae, while injured sites and internal soft fibres were preferred as food. Therefore, vegetable fruits, stem of sugarcane are believed to be the most vulnerable hosts for red weevil attack. The alternative host plant examined can be used to monitor the dynamics of the population of RPW in order to take effective preventive measures and most powerful tool available for use in pest management.