Different types of fruit damages of three internal apple feeders diagnosed with mitochondrial molecular markers

Three tortricid pests, Grapholita dimorpha (Komai), G. molesta (Busck), and Carposina sasakii (Matsumura), are known as internal apple feeders in Korea. To identify young larvae, this study developed two types of molecular markers from their mitochondrial DNA (mtDNA) sequences. To this end, six different loci of mtDNA were sequenced in G. dimorpha: cytochrome oxidase subunit I (460 bp), cytochrome oxidase subunit II (446 bp), cytochrome b (308 bp), NADH dehydrogenase 3 (585 bp), NADH dehydrogenase 4 (ND4, 835 bp), and 16S rRNA (1300 bp). These sequences were compared with those of G. molesta and C. sasakii in order to develop PCR–RFLP and diagnostic primers. ND4 locus was selected to be used for developing a PCR–RFLP marker. ND4-Swa I digests showed two bands for G. dimorpha, one band for G. molesta, and three bands for C. sasakii. On the other hand, species-specific diagnostic PCR primers were developed using ND4 locus. These markers were then applied to diagnose larvae infesting apples to determine species-specific fruit damage patterns, in which G. dimorpha, G. molesta, and C. sasakii showed different feeding behaviors in terms of their main feeding sites in apple fruits.     

Diagnostic molecular markers of six lepidopteran insect pests infesting apples in Korea

Two molecular identification techniques for differentiating six lepidopteran pests infesting apples in Korea are presented. These six species include two internal fruit feeders (Grapholita molesta and Carposina sasakii), two leaf rollers (Adoxophyes sp. and Archips breviplicanus) and two leaf miners (Phyllonorycter ringoniella and Lyonetia prunifoliella). All species occur until near harvest and reduce apple production. A 489 bp fragment of mitochondrial cytochrome oxidase subunit I (COI) was sequenced in these six species. The sequence was used to select species-specific restriction enzyme sites and to design diagnostic polymerase chain reaction (PCR) primers, resulting in the development of restriction fragment length polymorphism (RFLP)-PCR and diagnostic PCR. These methods were reliable and rapid in the identification of these six species.     

Cross-amplification of microsatellites from the codling moth Cydia pomonella to three other species of the tribe Grapholitini (Lepidoptera: Tortricidae)

This study examined cross-species amplification of 33 microsatellite markers, previously developed for Cydia pomonella, in three related fruit moth species of the same tribe (Grapholitini), namely Grapholita molesta, Grapholita funebrana and Grapholita lobarzewskii. Eight microsatellite loci yielded polymorphic products for G. molesta, nine for G. funebrana and 11 for G. lobarzewskii. At all these loci, the number of alleles ranged between four and 11 in G. molesta, and between four and nine in G. funebrana and G. lobarzewskii each. The successful cross-amplified loci can be used for research on population genetics and gene flow of the three target species.     

A diagnostic multiplex polymerase chain reaction method to identify Japanese internal apple-feeding Lepidopteran pests: Grapholita molesta, Grapholita dimorpha (Lepidoptera: Tortricidae), and Carposina sasakii (Lepidoptera: Carposinidae)

We present a multiplex polymerase chain reaction method to differentiate between three Japanese internal apple-feeding pests: the oriental fruit moth, Grapholita molesta (Busck), Grapholita dimorpha Komai, and the peach fruit moth, Carposina sasakii Matsumura. A 1,342-bp fragment of mitochondrial cytochrome oxidase subunit I (COI) was sequenced in each species. The three species showed consistent and diagnostic differences in the region of the COI gene, from which three species-specific forward primers were designed. The forward primers along with one universal reverse primer were used to selectively amplify DNA from specimens of diverse geographic origin for each corresponding target species. This method enabled easy, immediate, and accurate identification of internal feeding Lepidoptera in apples and other fruits.     

Effects of different sex pheromone compositions and host plants on the mating behavior of two Grapholita species

The two congener species Grapholita molesta and Grapholita dimorpha share two major sex pheromone components: cis-8-dodecenyl acetate (Z8-12Ac) and trans-8-dodecenyl acetate (E8-12Ac). In fact, commercial sex pheromone lures composed of only these two major components attract the males of both species. In this study, we aimed to determine the reproductive isolation components of these two species by analyzing the effects of the minor sex pheromone components and host plants. First, different ratios of the two major sex pheromone components were greatly favored by either male species. Sex pheromone gland extracts of G. dimorpha contained a lesser proportion of Z8-12Ac than that of G. molesta. In the three (apple, pear, and peach) orchards investigated in this study, a larger number of G. molesta males were attracted to the 95:5 pheromone mixture (Z8-12Ac and E8-12Ac, respectively), while a larger number of G. dimorpha males were attracted to the 85:15 mixture. Second, there was a significant variation in male attractions in different host plants. G. molesta males were more attracted to the sex pheromone lure in the apple orchards than that in the pear and peach orchards. In contrast, G. dimorpha males were more attracted to the lures in the pear and peach orchards than that in the apple orchard. Third, the minor sex pheromone components were important for reproductive isolation. Among the four minor components tested, addition of (Z)-8-dodecenol (Z8-12OH) to the major sex pheromone components significantly suppressed male attraction in G. dimorpha and slightly elevated male attraction in G. molesta. The discriminating effect of Z8-12OH was further validated using male electroantennogram analysis. These results suggest that reproductive isolation between two congeners can be achieved by variations in the minor sex pheromone components and in the host plants, as well as by changes in the ratio of the two major components.     

Molecular identification of glutathione S-transferase genes and their potential roles in insecticides susceptibility of Grapholita molesta

Grapholita molesta is one kind of serious fruit-boring pests in the world, which caused huge economic losses to the fruit industry including pear and apple. The heavy usage of synthetic insecticides resulted in different degrees of resistance of G. molesta to some commonly used insecticides. However, the tolerance mechanisms of G. molesta to insecticides are still unclear. Glutathione S-transferases (GSTs) belong to the superfamily of detoxifying enzymes and mainly play important roles in detoxification metabolism and insecticide susceptibility of insects. In this study, we first evaluated the toxicity of three insecticides including emamectin benzoate, chlorantraniliprole and lambda-cyhalothrin on G. molesta at different developmental stages. Synergism study showed that the susceptibility of G. molesta larvae to these three insecticides obviously increased when the enzyme activity of GST was inhibited by diethyl maleate compared with the controls. Next, we screened out six GmGST genes which are upregulated after insecticides treatment from a total of 21 GmGST genes and analysed their sequence characteristics and expression profiles. RNAi and bioassay results further revealed that the mortality of 5th instar larvae significantly increased after exposure to LC₃₀ of emamectin benzoate, chlorantraniliprole and lambda-cyhalothrin when silencing of GmGSTo2, GmGSTs1 and GmGSTz1 compared with the controls. In conclusion, this study indicated that GmGSTo2, GmGSTs1 and GmGSTz1 play important roles in insecticides susceptibility of G. molesta to three commonly used insecticides, which is significance for its future integrated management.     

Olfactory responses of Grapholita molesta to different developmental stages of immature fruit of peach,plum, and apple

The Oriental fruit moth, Grapholita molesta (Busck) (Lepidoptera: Tortricidae), is known to shift its host use from stone fruits (the primary hosts) to pome fruits (secondary hosts) as the season progresses. Grapholita molesta recognizes potential hosts by semiochemicals from fruits, but few studies have addressed the olfactory responses of G. molesta to host fruits during the seasonal transition of the moth from stone to pome fruits. So, we investigated the olfactory responses of larvae and gravid females of G. molesta to different fruit stages (collected on May 31, June 11, and June 25) of immature peach, plum, and apple fruits, using both multiple and two choice tests. In the multiple choice tests, for fruits collected after June 11, larvae showed a significant preference for peach as their first choice, compared to plum or apple. The final choice rate (when larvae had stayed on the fruit for more than 1 h) of larvae was also highest for peach fruits collected on June 11 and June 25. In two choice tests (Y-tube test), the response rate and overall choice rate of larvae and mated ovipositing adult females were also highest on peaches collected on May 31 (larvae) and June 25 (adults), respectively. In conclusion, we found that both the adults and larvae of G. molesta were highly attracted to immature peach fruits in laboratory choice tests.     

Model of Grapholita molesta spring emergence in pear orchards based on statistical information criteria

The oriental fruit moth, Grapholita molesta, is becoming a large threat to Korean pear production. Timely management of the egg and early larval stages from the spring emergence is critical to reduce the G. molesta population during the pear growing season. A model was developed to precisely predict the spring occurrence of G. molesta adults as a function of accumulated degree-days. The model was validated with male moth caught in sex pheromone-baited traps placed in pear orchards at two major pear production regions (Icheon and Naju) of Korea in 2010. We applied nine distribution models to describe the cumulative proportions of G. molesta males caught relative to accumulated degree-days. The observed phenology of the G. molesta spring population was well described by the nine models. The predicted dates for the cumulative 50% male moth catches were within a 5 day period. Based on statistical information criteria (Akaike's and Bayes–Schwartz information criteria), we recommend the sigmoid function referred by Brown and Mayer, because of its ease of use and meaningfulness; the parameter “b” denotes the degree-day accumulation at 50% moth emergence. The G. molesta spring emergence model could be applied to determine optimal chemical treatment timing for controlling G. molesta in fruit tree orchards and further help to develop a full-cycle phenology model of G. molesta.     

Factors influencing adult male Grapholita molesta dispersal in commercial Malus and Prunus host crops

The oriental fruit moth (OFM), Grapholita molesta (Busck) (Lepidoptera: Tortricidae), has been an economic pest of apples (Malus spp.) and peaches (Prunus spp.; both Rosaceae) in the eastern USA since the 1930s. Successful management of G. molesta with insecticides requires careful timing of these products based on sex‐pheromone trap captures of male moths. In Pennsylvania, apple and peach orchards are often planted adjacently. Factors such as trap distance from release points, host crop, and wind were considered in relation to male moth movement within and between hosts. Our practical objective was to understand male G. molesta dispersal within and between these two hosts to achieve better management. Recapture of moths decreased over distance; the majority of moths were recaptured 50–100 m from the release point regardless of crop type. Male G. molesta released into commercial apple and peach orchards were most likely to remain in the crop of release and to not exhibit host shifts over the season, even after peach fruit were harvested. Our analyses of wind direction data with respect to recapture showed that the likelihood of recapture was similar regardless of the orientation of the traps relative to the mean wind direction at peak flight times. The apple and peach host crops appeared to affect male G. molesta dispersal to the extent that wind in orchards affects their perception of pheromone. Crop effects were most likely due to the traps closest to the release points being in the same crop as the release points.     

Molecular identification of cryptic bumblebee species from degraded samples using PCR–RFLP approach

The worldwide decline and local extinctions of bumblebees have raised a need for fast and accurate tools for species identification. Morphological characters are often not sufficient, and molecular methods have been increasingly used for reliable identification of bumblebee species. Molecular methods often require high‐quality DNA which makes them less suitable for analysis of low‐quality or older samples. We modified the PCR–RFLP protocol for an efficient and cost‐effective identification of four bumblebee species in the subgenus Bombus s. str. (B. lucorum, B. terrestris, B. magnus and B. cryptarum). We used a short partial mitochondrial COI fragment (446 bp) and three diagnostic restriction enzymes (Hinf I, Hinc II and Hae III) to identify species from degraded DNA material. This approach allowed us to efficiently determine the correct species from all degraded DNA samples, while only a subset of samples 64.6% (31 of 48) resulted in successful amplification of a longer COI fragment (1064 bp) using the previously described method. This protocol can be applied for conservation and management of bumblebees within this subgenus and is especially useful for fast species identification from degraded samples.     

DNA diagnostics to identify internal feeders (Lepidoptera: Tortricidae) of pome fruits of quarantine importance

A diagnostic polymerase chain reaction (PCR) method is presented for differentiating among the North American internal apple-feeding pests codling moth, Cydia pomonella (L.); oriental fruit moth, Grapholita molesta (Busck); lesser appleworm, Grapholita prunivora (Walsh); and cherry fruitworm, Grapholita packardi Zeller. An approximately 470-bp fragment of mitochondrial cytochrome oxidase subunit I (COI) was sequenced in three to six specimens of each species. Consistent and diagnostic differences were observed among the species in two regions of COI from which forward and reverse primers were designed to amplify a 112-116-bp segment of the gene. The primer sets were used to selectively amplify DNA from specimens of diverse geographic origin for each corresponding target species. Protocols were adapted for conventional and quantitative PCR, the latter being substantially faster. The method was validated as a decision-making tool for quarantine identifications for Mexico by representatives of their phytosanitary agency (Sanidad Vegetal). The method can facilitate identification of intercepted internal feeding Lepidoptera in apple and pear for many other importing nations.     

Importance of trap liner adhesive selection for male moth catch (Lepidoptera: Tortricidae) with bisexual attractants

Studies compared moth captures of three pests (Lepidoptera: Tortricidae) of apple, Malus domestica Borkhausen, in delta traps using removable liners coated with either a sticky gel (SG) or a hot‐melt pressure sensitive (HMPS) adhesive. Laboratory and field studies with Cydia pomonella (L.), Choristoneura rosaceana (Harris) and Grapholita molesta (Busck) demonstrated that traps with either liner, baited with a pair of virgin females catch males, but at significantly different levels. In the field, male moth captures in traps with the HMPS liner were significantly greater than in traps with the SG liner for C. rosaceana, and G. molesta; but not for C. pomonella. Similar results were observed in laboratory studies using flight tunnels. Additional studies demonstrated that this difference in moth captures between liners was not due to levels of female mortality, but instead was correlated with the occurrence of the female's ventral abdominal surface becoming stuck in the adhesive. Studies showed that a significantly greater proportion of females of all species had their ventral abdomen stuck in the SG than HPMS adhesive on day 1, but only G. molesta and C. rosaceana on day 3. In addition, the tackiness of the two adhesives affected moth movement on the liner with males and females of all species moving farther on liners with SG than HMPS adhesive. A greater proportion of female G. molesta and C. pomonella were either stuck supined or laterally on the SG than HMPS adhesive, and females in this position captured as many males as when prone and unstuck on the liner. Our studies demonstrate that adhesives can secondarily influence male moth captures on trap liners when used with bisexual attractants, and that adhesive type should be considered when developing action thresholds.     

Molecular and functional characterization of three odorant binding proteins from the oriental fruit moth Grapholita molesta (Busck) (Lepidoptera: Tortricide)

Odorant binding proteins (OBPs) act in recognizing odor molecules and their most well‐studied functions are transporting odors across the sensillum lymph to olfactory receptor neurons within the insect antennal sensillum. The adults of Grapholita molesta highly depend on olfactory cues in locating host plants and selecting oviposition sites, in which OBPs play an important role in perceiving and recognizing host plant volatiles. Exploring the physiological function of OBPs could facilitate our understanding of their importance in insects’ chemical communication. In this study, three OBP genes were cloned and named GmolOBP4, GmolOBP5, and GmolOBP10. Quantitative real‐time PCR results indicated that GmolOBP4 and GmolOBP10 were predominantly expressed in adult antennae and GmolOBP5 was expressed in multiple tissues, including head, legs, and wings in addition to antennae. The binding affinities of the three recombinant GmolOBPs (rGmolOBPs) with four sex pheromone components and twenty‐nine host plant volatiles were measured using 1‐N‐Phenyl‐naphthylamine as a fluorescence probe. The three rGmolOBPs exhibited specific binding properties to potential ligands, GmolOBP4 and GmolOBP10 bound to minor sex pheromone components, such as (Z)‐8‐dodecenyl alcohol and dodecanol, respectively. rGmolOBP4 showed intermediate binding ability with hexanal, benzyl alcohol, and pear ester, rGmolOBP5 had a weak affinity for benzaldehyde, pear ester and, methyl jasmonate, and rGmolOBP10 showed strong binding capacity toward hexanol, decanol, and α‐ocimene. We speculate that the GmolOBP4 and GmolOBP10 have dual functions in perception and recognition of host plant volatiles and sex pheromone components, while GmolOBP5 may serve other function(s).     

Predation by generalist predators on the codling moth versus a closely‐related emerging pest the oriental fruit moth: a molecular analysis

• 1 Biological control by conservation of native natural enemies can, at its best, reduce the need for pesticides and prevent detrimental effects upon the environment. The present study investigated the role of ground‐active generalist predators as natural enemies of two tortricid pests in apple orchards.
• 2 Predation rates were compared on the well established codling moth Cydia pomonella and the emerging oriental fruit moth Grapholita molesta, which has recently switched hosts to apples.
• 3 The present study hypothesized that the ground‐active predators consumed the two tortricid pests in significant numbers without preference, and attacked the pests at different developmental stages.
• 4 Using diagnostic polymerase chain reaction on the gut contents of field‐caught ground‐active predators, no difference in predation rates was found on these two pests. Spiders were the most efficient predators of emergent adult moths in spring, whereas the carabid beetles, feeding on diapausing larvae, were important in the autumn.
• 5 The temporal complementarity between spiders and carabid beetles, attacking different stages of the pests at different times of year, highlights the need for diverse predator assemblages to optimize biological control.

     

Reduction of the response to sex pheromone in the oriental fruit moth,Grapholita molesta (Lepidoptera: Tortricidae) following successive pheromonal exposures

The effects of prior pheromonal experience upon the pheromone- mediated upwind flight response was examined in the oriental fruit moth, Grapholita molesta(Busck) (Lepidoptera: Tortricidae). Adult male G. molestawere subjected to a parallel series of staggered and repeated pheromonal exposures in a sustained-flight wind tunnel. Levels of response to pheromone in male G. molestasignificantly decreased in a (a) rectilinear function with increased ages of individuals, (b) logarithmic function of successive trials, and (c) steeper logarithmic function of successive trials with increased dosage of sex pheromone. The baseline levels of responding were not affected by either the (a) dosage of sex pheromone, (b) posteclosion ages of individuals for their initial exposures once the main effect of age itself was estimated, (c) elapsed time in hours between trials, or (d) discrete days of testing as integral intervals, disregarding hours within days.     

Traps and Baits for Luring <Emphasis Type="Italic">Grapholita molesta</Emphasis> (Busck) Adults in Mating Disruption-Treated Apple Orchards

Grapholita molesta (Busck) is one of the main pests in apple crops in Brazil, where it is controlled by mating disruption (MD) with the use of the synthetic sex pheromone. However, sex-pheromone-based monitoring is not effective in MD-treated areas and may result in losses in production. This work has defined a trap model and a bait for luring G. molesta adults in MD apple orchards. The experiments were conducted in commercial apple orchards located in São Joaquim, SC, Brazil. Three trap models—McPhail, Pot, and Ajar—and three baits—grape juice (25%) (GJ), sugarcane molasses (25%) (SM), and a solution containing brown sugar (8.69%) and terpinyl acetate (0.05%) (TAS)—were assessed for luring G. molesta adults in areas subjected to the mating disruption. The assessments were performed weekly by collecting the insects caught in the traps. In addition, time needed to replace traps was also assessed, as well as the selectivity of the trap/bait set. In the laboratory, G. molesta adults were sexed, and the females were dissected to confirm reproductive status. We discuss our results and sugarcane molasses (25%) captured the least number of G. molesta adults regardless of the tested traps. The Ajar/TAS, Pot/TAS, and McPhail/GJ captured the largest number of G. molesta adults. The Ajar/TAS was the most selective and easier to handle. TAS was efficient in catching G. molesta until 14 days after preparation of the solution. Ajar/TAS has potential to be used in the monitoring of G. molesta in apple orchards.     

Presence of Anopheles culicifacies B in Cambodia established by the PCR-RFLP assay developed for the identification of Anopheles minimus species A and C and four related species

Abstract A polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) assay developed for identification of five species of the Anopheles minimus Theobald group and a related mosquito species of the Myzomyia Series (Diptera: Culicidae) was applied to morphologically identified adult female specimens collected in Ratanakiri Province, north‐eastern Cambodia. In addition to finding An. aconitus Dönitz, An. minimus species A and An. pampanai Büttiker & Beales, some specimens showed a new restriction banding pattern. Siblings of specimens that exhibited this new PCR‐RFLP pattern were morphologically identified as An. culicifacies James sensu lato. Based on nucleotide sequences of the ribonuclear DNA internal transcribed spacer 2 region (ITS2) and the mitochondrial cytochrome oxidase I gene (COI), these specimens were recognized as An. culicifacies species B (sensu Green & Miles, 1980 ), the first confirmed record of the An. culicifacies complex from Cambodia. This study shows that the PCR‐RFLP assay can detect species not included in the initial set‐up and is capable of identifying at least seven species of the Myzomyia Series, allowing better definition of those malaria vector and non‐vector anophelines in South‐east Asia.     

Monitoring oriental fruit moth and codling moth (Lepidoptera: Tortricidae) with combinations of pheromones and kairomones

Experiments were conducted in North and South America during 2012–2013 to evaluate the use of lure combinations of sex pheromones (PH), host plant volatiles (HPVs) and food baits in traps to capture the oriental fruit moth, Grapholita molesta (Busck), and codling moth, Cydia pomonella (L.), in pome and stone fruit orchards treated with sex pheromones. The combination of the sex pheromone of both species (PH combo lure) significantly increased G. molesta and marginally decreased C. pomonella captures as compared with captures of each species with either of their sex pheromones alone. The addition of a HPV combination lure [(E,Z)‐2,4‐ethyl decadienoate plus (E)‐β‐ocimene] or acetic acid used alone or together did not significantly increase the catch of either species in traps with the PH combo lure. The Ajar trap baited with terpinyl acetate and brown sugar (TAS bait) caught significantly more G. molesta than the delta trap baited with PH combo plus acetic acid in California during 2012. The addition of a PH combo lure to an Ajar trap significantly increased catches of G. molesta compared to the use of the TAS bait or PH combo lure alone in 2013. Female G. molesta were caught in TAS‐baited Ajar traps at similar levels with or without the use of additional lures. Ajar traps baited with the TAS bait alone or with (E)‐β‐ocimene and/or PH combo lures caught significantly fewer C. pomonella than delta traps with sex pheromone alone. Ajar traps with 6.4‐mm screened flaps caught similar numbers of total and female G. molesta as similarly baited open Ajar traps, and with a significant reduction in the catch of non‐targets. Broader testing of HPV and PH combo lures for G. molesta in either delta or screened or open Ajar traps is warranted.     

Oviposition preferences in Grapholita molesta: the relative importance of visual and olfactory cues

The oriental fruit moth, Grapholita molesta (Busck) (Lepidoptera: Tortricidae), is a major pest of fruit trees worldwide. Females oviposit on the young leaves of peach [Prunus persica (L.) Batsch (Rosaceae)] shoots at twilight, when light intensity changes markedly. Previous researches have shown that the oviposition response of this moth is guided particularly by visual and olfactory cues from host plants. However, the relative importance of visual and olfactory cues in their oviposition preference is largely unknown. As a crepuscular moth, how do choices change as light intensity drops rapidly from day to night? In the present study, through two‐choice behavioural experiments, the oviposition responses of G. molesta to visual and olfactory cues (alone or in combination) from peach shoots were tested at four light intensities ranging from daylight to starlight. Grapholita molesta showed similar oviposition responses to plant cues under 1 000, 100, 1 and 0.01 mW m^?2 illumination. Olfactory cues from peach shoots attracted significantly more oviposition than the negative control, whereas visual cues alone did not. Furthermore, olfactory cues were more attractive than visual cues, and no significant interaction was observed between the responses to the two cues. Our findings indicate that G. molesta females mainly rely on olfactory cues to recognize oviposition sites, regardless of differences in light intensity. These results do not provide evidence for attraction to visual cues, but the possibility that the brightness of leaves might be used to guide oviposition is discussed.