Host Plants Identification for Adult Agrotis ipsilon, a Long-Distance Migratory Insect

In this study, we determined the host relationship of Agrotis ipsilon moths by identifying pollen species adhering them during their long-distance migration. Pollen carried by A. ipsilon moths was collected from 2012 to 2014 on a small island in the center of the Bohai Strait, which is a seasonal migration pathway of this pest species. Genomic DNA of single pollen grains was amplified by using whole genome amplification technology, and a portion of the chloroplast rbcL sequence was then amplified from this material. Pollen species were identified by a combination of DNA barcoding and pollen morphology. We found 28 species of pollen from 18 families on the tested moths, mainly from Angiosperm, Dicotyledoneae. From this, we were able to determine that these moths visit woody plants more than herbaceous plants that they carry more pollen in the early and late stages of the migration season, and that the amounts of pollen transportation were related to moth sex, moth body part, and plant species. In general, 31% of female and 26% of male moths were found to be carrying pollen. Amounts of pollen on the proboscis was higher for female than male moths, while the reverse was true for pollen loads on the antennae. This work provides a new approach to study the interactions between noctuid moth and their host plants. Identification of plant hosts for adult moths furthers understanding of the coevolution processes between moths and their host plants.


Introduction
Plants and their insect herbivores represent more than 50% of all known species on earth [1]. The interaction between these two ecological partners is one of the most common and consequential of ecological associations [2]. Flowers and insects co-evolved [3]. Plants depending upon insects for pollination have evolved pollen that adheres readily to the insects' exterior [4]. The yield of many crops usually increases with the number of pollinators, meanwhile, adults of numerous insect species feed on nectar, pollen and other plant exudates that are frequently associated with flowers [3]. The first step in understanding how these ecological associations function is to determine the range of host plants used by particular herbivorous insects.
Noctuidae is the largest family of Lepidoptera, containing over 40,000 currently recognized species [5]. Noctuids are a prominent feature of terrestrial insect faunas and food webs, including many species of economic importance [6]. Within the noctuids, larvae and adults usually differ considerably in their nutritional requirements and food ecology. Often, larvae feed on the structural tissue of their host plants while adults feed primarily or exclusively on plant-provided food supplements such as nectar and pollen [7]. Many studies focus on larval host records by direct observations of herbivory in

Frequency of Pollen Detection Rates on Body Parts of A. ipsilon
For whole body analyses, there was no significant sex-related difference in frequency of pollen occurrence on A. ipsilon moths, either for year groups (2012-2014) ( Table 2   With respect to the phenology of migration, the frequency of pollen deposits on bodies of A. ipsilon in the early part of the migration period (39.7%) and in the late period (28.5%) were both significantly higher than that in the middle period of the migration season (7.8%) during 2012-2014 (F = 12.61, df1 = 2, df2 = 6, p = 0.007) ( Figure 3).  With respect to the phenology of migration, the frequency of pollen deposits on bodies of A. ipsilon in the early part of the migration period (39.7%) and in the late period (28.5%) were both significantly higher than that in the middle period of the migration season (7.8%) during 2012-2014 (F = 12.61, df1 = 2, df2 = 6, p = 0.007) (Figure 3).

Discussion
We identified host plant used by A. ipsilon moths using DNA bar coding, pollen morphology, and known distributions of plants. Our results indicated that A. ipsilon moths fed on nectar from a very wide of plants, including 28 species from 18 families of mainly Dicotyledoneae in the Angiosperms. Kishimoto-Yamada et al. (2013) inferred host plant families of chrysomelid beetles including herbs, shrubs and vines [27]. However, a preference of A. ipsilon for visiting woody plants more than herbaceous plants was detected in our study. Different host plants can play important roles in the population increase of insects [28]. Both quality and quantity of food influence insect development and fecundity [29], and fitness effects may differ between different sources of nectar. Therefore, further research is needed to explore the preference of A. ipsilon moths for particular host plants.
Agrotis ipsilon is a long-distance migratory pest, often undertaking trans-regional migrations. The identification of pollen species can be used to determine the geographical origin of the insect since some plants grow only in certain ecological zones or geographic locations. For example, Hendrix and Showers (1992) found that A. ipsilon adults captured in Iowa and Missouri contained pollen from plants that only grow in south and southwest Texas [4]. Our results found pollen from Ligustrum lucidum Ait./Ligustrum sempervirens (Franch.) Lingelsh, C. sinensis, M. azedarach, Hoboellia parviflora (Hemsl.) Gagnepl./Hoboellia grandiflora Réaub., O. europaea, A. fruticosa, A. fortunei, R. pseudoacacia, llex cornuta Lindl. Et Paxton/Ilex coralline Franch. and C. echinocarpa on A. ipsilon adults captured in Beihuang from late May to June. Since these species grow in the Yangtze River basin and further south (over 800 km) [29], these adults must have foraged on these flowers there, then migrated northward to Beihuang, providing evidence for the northward spring migration of A. ipsilon [22].
Our use of DNA-based pollen analysis to provide species-level plant identifications validates this novel method, which has great potential for the study of plant-herbivore interactions [15]. The Consortium for the Barcode of Life-plant Working Group (CBOL) has recommended the two-locus combination of rbcL + matK as the best plant barcode due to its universality, sequence quality, and species discrimination [30]. Unfortunately, matK is difficult to amplify universally using currently available primer sets [31]. We chose the rbcL intron mainly because it has the highest level of coverage in GenBank among potential barcoding markers; the universality of PCR primers. A recent study also demonstrated that the rbcL sequences successfully identified the host plants families of rolled-leaf

Discussion
We identified host plant used by A. ipsilon moths using DNA bar coding, pollen morphology, and known distributions of plants. Our results indicated that A. ipsilon moths fed on nectar from a very wide of plants, including 28 species from 18 families of mainly Dicotyledoneae in the Angiosperms. Kishimoto-Yamada et al. (2013) inferred host plant families of chrysomelid beetles including herbs, shrubs and vines [27]. However, a preference of A. ipsilon for visiting woody plants more than herbaceous plants was detected in our study. Different host plants can play important roles in the population increase of insects [28]. Both quality and quantity of food influence insect development and fecundity [29], and fitness effects may differ between different sources of nectar. Therefore, further research is needed to explore the preference of A. ipsilon moths for particular host plants.
Agrotis ipsilon is a long-distance migratory pest, often undertaking trans-regional migrations. The identification of pollen species can be used to determine the geographical origin of the insect since some plants grow only in certain ecological zones or geographic locations. For example, Hendrix and Showers (1992) found that A. ipsilon adults captured in Iowa and Missouri contained pollen from plants that only grow in south and southwest Texas [4]. Our results found pollen from Ligustrum lucidum Ait./Ligustrum sempervirens (Franch.) Lingelsh, C. sinensis, M. azedarach, Hoboellia parviflora (Hemsl.) Gagnepl./Hoboellia grandiflora Réaub., O. europaea, A. fruticosa, A. fortunei, R. pseudoacacia, llex cornuta Lindl. Et Paxton/Ilex coralline Franch. and C. echinocarpa on A. ipsilon adults captured in Beihuang from late May to June. Since these species grow in the Yangtze River basin and further south (over 800 km) [29], these adults must have foraged on these flowers there, then migrated northward to Beihuang, providing evidence for the northward spring migration of A. ipsilon [22].
Our use of DNA-based pollen analysis to provide species-level plant identifications validates this novel method, which has great potential for the study of plant-herbivore interactions [15]. The Consortium for the Barcode of Life-plant Working Group (CBOL) has recommended the two-locus combination of rbcL + matK as the best plant barcode due to its universality, sequence quality, and species discrimination [30]. Unfortunately, matK is difficult to amplify universally using currently available primer sets [31]. We chose the rbcL intron mainly because it has the highest level of coverage in GenBank among potential barcoding markers; the universality of PCR primers. A recent study also demonstrated that the rbcL sequences successfully identified the host plants families of rolled-leaf beetles [16]. rbcl is a good DNA barcoding region for plants at the family and genus levels, but for identification at the species level, a combination of pollen morphology and DNA barcoding is required. The efficiency of traditional pollen identification, which is highly dependent on human expertise and prohibitively time-consuming for large-scale studies, is improved by the use of DNA barcoding.
Our results showed that the frequency of pollen deposits on the proboscis was higher on female than male moths, which may be due to differences in the nutrient requirements of the sexes, with females generally having a higher nutrient demand than males [32]. The reason for higher frequencies of pollen deposits on antennae of male vs. female moths may be due to the different shape of the antennae between the sexes. The more branched antennae of male A. ipsilon moths are more favorable for pollen retention due to their greater surface area.
For the proboscis, when pollen was found, many grains were typically present, suggesting active contact through feeding rather than casual contact through wind-blown contamination. Pollen were rarely found on the antenna (5.4%), so future studies should focus on examining pollen found on A. ipsilon proboscises. Differences in the frequency of pollen deposits on A. ipsilon throughout the migratory period may be caused by differences in the abundance of nectar plants or the nutritional requirements of the moths, a topic that requires further investigation.
Flowers and insects have co-evolved [8], and the relationship between flowering plants and pollinators can be seen as mutually beneficial. Adult herbivores require nutrients or energy supplements from flowers for reproduction or flight, and flower-visiting herbivores are important pollinators [33]. The yield of many crops are dependent on the pollinators that visit their flowers, and yields are usually increased with the help of pollinators [4]. Our results showed A. ipsilon adults to be effective pollinators of C. mollissima, C. sinensis, M. azedarach, F. virosa, O. europaea and others. A. ipsilon is a long-distance disperser, undertaking regular migration across different agricultural areas. Therefore, the gene exchange of plants can be achieved across large regions with the help of A. ipsilon adults [34]. However, A. ipsilon larvae are pests of many crops, such as cotton, maize, potatoes, beans, and cruciferous vegetables [16]. Ultimately, the more knowledge we have about A. ipsilon, the better and more efficient the management practices that can be developed.

Agrotis ipsilon Sampling
Agrotis ipsilon moths were collected using light traps. Moths were collected from sunset to sunrise, except during power outages or periods of heavy rain, using a vertical-pointing searchlight trap (model DK.Z.J1000B/t, 65.2 cm in diameter, 70.6 cm in height, and 30 in spread angle), equipped with a 1000-W metal halide-lamp (model JLZ1000BT; Shanghai Yaming Lighting Co., Ltd., Shanghai, China) on the top of a house (500 m elevation) on the 2.5 km 2 island of Beihuang (BH, 38˝24 1 N; 120˝55 1 E) in the Bohai Gulf 40 km from the mainland to the north and 60 km from land to the south [34][35][36]. Collection were made every night from April to October during 2012-2014. Samples were collected with nylon net bags (60 mesh) which were changed manually every 2 h each night. Twenty moths (or all individuals if the total captured was <20) were removed from collection bags every morning. Each moth was killed by crushing its thorax, and it was placed into a 2 mL crytube and held at´20˝C in a freezer before microscopic examination.

Pollen Examination and Scanning Electron Microscopy (SEM) Preparation
Because Bryant et al. [37] found only 4% of pollen on Helicoverpa zea legs and eyes and suggested focusing examination efforts on the proboscis, in this study we examined the proboscis and antenna. A. ipsilon adult heads were removed from the body and examined at 200ˆmagnification to make a preliminary identification of pollen adhering to their proboscis or antenna. To prevent contamination, a section of paper towel (9ˆ9 cm) was placed on the microscope stage and changed after each sample, and forceps were also cleaned after each sample. Pollen grains found on a proboscis or antenna were mounted on aluminum stubs coated with gold in a sputter coater and immediately photographed under a Hitachi S-4800 cold field emission scanning electron microscope (Hitachi High-Technologies Co., Tokyo, Japan).

DNA Extraction from a Single Pollen Grain, PCR Amplification, Cloning and DNA Sequencing
Single pollen grains were isolated from the aluminum stubs and placed in individual PCR tubes containing 1 µL of lysis solution (NaOH with a concentration of 0.1 M, plus 2% Tween-20). NaOH and Tween-20 were purchased from the Beijing Chemical Reagent Co., Ltd. (Beijing, China). After the pollen grains were deposited, 5 µL of mineral oil was added. The samples were spun briefly (1 min at 1000ˆg) before incubation at 95˝C for 17 min 30 s to lyse the pollen grains. After lysis, equimolar aliquots of 1 µL Tris-EDTA (TE) buffer were added to neutralize the samples, which were then spun briefly [19]. The resulting solution was used as the template for Whole Genome Amplification (WGA) by illustra GenomiPhi V2 DNA Amplification Kit (GE Healthcare UK Ltd., London, UK). The WGA products of single pollen grains were used to amplify plant plastid DNA.
PCR products were purified with a Gel Extraction Kit (Tiangen, Beijing, China) and ligated directly into the pGEM-T Cloning Vector (Promega, Madison, WI, USA). Each DNA-containing plasmid was isolated from cultured E. coli cells by an alkaline miniprep method. The presence of inserts was verified by PCR with the M 13 forward (5 1 -GTTTTCCCAGTCACGAC-3 1 ) and reverse primers (5 1 -CAGGAAACAGCTATGAC-3 1 ) and sequenced using Sanger sequencing by the company Biomed (Beijing, China).

Pollen Identification and the Characteristics of Pollen Source Plants
Identification of rbcL sequences was performed individually through similarity Basic Local Alignment Search Tool (BLAST) searches against GenBank [40]. The unknown sequence was considered a member of the best hit of the query sequences when it was completely consistent with them, and the unknown species sequence was considered to be the same genus as indicated by its top hits if there were differences between the sequences. Then the species were corrected according to their morphologic features and the known plant distributions. The pollen's morphological features were identified using the published scanning electron microscopy (SEM) images in the atlas of pollen flora of China and pollen flora of China woody plants by SEM [41,42]. The pollen grains that could be classified to genus or species level were used to identify the source plants of pollen.

Data Analysis
Statistical analyses were conducted using SPSS 13.0 (SPSS Inc., Chicago, IL, USA) [43]. The frequency of pollen deposits on A. ipsilon during different migration phases was analyzed using one-way ANOVA, and the differences between the mean values were compared using Tukey's HSD (honestly significant difference) test. The differences in the annual mean frequencies of pollen occurrence on female and male body parts (proboscis, antennae, or total (combined proboscis and antennae)) of A. ipsilon moths, and the differences in the annual mean frequency of pollen deposition on the proboscis or antennae of female, male, and total (female and male) A. ipsilon moths were analyzed by Student's t-tests. The differences in frequency of pollen deposition on female and male body parts (proboscis, antennae or total (proboscis and antennae)) of A. ipsilon moths in each year, the differences of in such rates between proboscis and antennae of female, male and total (female and male) A. ipsilon moths in each year, and the characteristics of pollen source plants were all analyzed by a Chi-squared tests. All proportion data were logit transformed before being analyzed.

Conclusions
This study developed an effective approach for examining noctuid moth-host plant interactions. Unlike larvae that mainly feed on herbaceous plants, A. ipsilon moths obtain nutrition primarily from woody plants. We also found evidence that female A. ipsilon moths have a higher nutrient demand than males. Our results are helpful for understanding the coevolution processes between insects and their host plants, and for developing more efficient management practices of A. ipsilon.
Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/17/ 6/851/s1, Figure S1: Frequencies of pollen deposition on female and male proboscises (A), antennae (B) and total (proboscis and antennae) (C) of A. ipsilon moths; Frequencies of pollen deposition on proboscis and antennae of female (D), male (E) and total (female and male) (F) of A. ipsilon moths. Single asterisk (*) or double asterisks (**) indicates there was significant difference at the 5% or 1% level as determined by chi-squared test, and ns indicates there was no significant difference, Text S1: The rbcl sequences of the examined pollen species.