Neoceratitis asiatica (Becker) (Diptera: Tephritidae), known as wolfberry fruit fly, is a harmful pest of Lycium barbarum (Solanaceae). Neoceratitis asiatica female adults insert the ovipositor into the peel of L. barbarum and lay eggs, causing reductions in yield and economic loss. The symbiotic bacteria Wolbachia spp. have attracted considerable attention and interest by entomologists in recent years. Wolbachia infect many genera of tephritid fruit flies, such as Anastrepha, Bactrocera, Rhagoletis, Dacus, Ceratitis, and Carypomya. Wolbachia can induce complete cytoplasmic incompatibility in novel hosts, leading to complete suppression of laboratory populations by single releases of infected males, which potentially makes it a useful method for pest management. In this study, the infection of Wolbachia in N. asiatica from the Ningxia region in China was detected based on the Wolbachia surface protein gene sequence. The neighbor-joining tree showed Wolbachia in wolfberry fruit fly was wRi strain. This research lays the foundation for further study about Wolbachia in Chinese wolfberry fruit fly, and also provides a basis for the prevention and control of other economically important fruit flies using Wolbachia.
Neoceratitis asiatica (Becker) (Diptera: Tephritidae), known as wolfberry fruit fly, became the most harmful pest to Lycium barbarum (Solanaceae) during the 1950s through the 1970s in the Ningxia region of China (Wu et al. 1963), and yield loss was more than 20%, sometimes as high as 55%. From the 1990s to the beginning of the 21st century, it was a consistently damaging pest (Ren & Hu 2004; Ren 2010). Lycium barbarum, which is called Chinese wolfberry in English, is a source of Chinese herbal medicines. Its dried ripe fruit has beneficial effects on the liver, kidneys, and eyes (Zhao et al. 2009; Xu et al. 2014). Berries, leaves, and roots of L. barbarum contain polysaccharides, amino acids, vitamins, and trace elements that are of high medicinal and nutritional value (Xu et al. 2014). Lycium barbarum is an important economic crop in the northwest region of China, especially in Ningxia, Xinjiang, and Gansu. Inner Mongolia, Qinghai, Hebei, Xizang, Shaanxi, Shanxi, Liaoning, Jiangsu, Zhejiang, and Guangdong Provinces also have this species (Wu & Gao 1964; Hu et al. 2009; Xue & Lin 2009; Xu et al. 2014).
The infestation rate of wolfberry trees has attained 70% in severely affected areas (Zheng 2015). Neoceratitis asiatica female adults insert the ovipositor into the peel of L. barbarum and lay eggs. Initially, the exterior surface of damaged L. barbarum shows no difference from healthy fruit, but in the late stage, white curved stripes appear on the peel. Beneath the peel of infested fruit, the flesh is consumed by N. asiatica and the fruit is full of frass. Under these circumstances, the fruit cannot be used as a commodity or medicine, and thus has no economic value (Wu et al. 1963; Guo et al. 2017; Li et al. 2017).
Neoceratitis asiatica occurs during May to Sep, producing 3 generations per yr. It has 3 fairly unique characteristics. First, N. asiatica is monophagous, feeding only on L. barbarum. Second, the adult female generally lays only 1 egg per fruit. If 2 or 3 eggs are laid in a fruit, only 1 larva survives. Third, adults have no phototaxis, and cannot be baited with sweet or sour wine (Wu & Meng 1963). These traits make it difficult to capture and culture N. asiatica. Investigations into N. asiatica began in the 1960s, and in the nearly half century following, research principally focused on the identification, pattern of occurrence, and control of the pest. Methods combining morphological characteristics and DNA barcoding have been used for its identification (Guo et al. 2017). The mitochondrial genome of this fruit fly also has been studied to determine its phylogenetic status (Su et al. 2017).
The symbiotic bacteria Wolbachia spp. (Anaplasmataceae) have attracted a great deal of attention by entomologists in recent years. Wolbachia were first detected in Culex pipiens (L.) (Diptera: Culicidae) (Hertig 1936). They are thought to have potentially important roles in genetic control of pests because of their effects on the reproduction of their hosts. Wolbachia can induce cytoplasmic incompatibility, parthenogenesis, and feminization of their host (Werren 1997). Cytoplasmic incompatibility provides a reproductive advantage to infected females over uninfected females, resulting in the invasion of Wolbachia into a population (Pan et al. 2018). Wolbachia can interfere with pathogen infection and inhibit some human pathogens, such as dengue and Zika viruses, malaria parasites, and filarial worms (Kambris et al. 2009; Moreira et al. 2009; Bian et al. 2010, 2013; Dutra et al. 2016). Wolbachia also infect many genera of tephritid fruit flies, including Anastrepha, Bactrocera, Rhagoletis, Dacus, Ceratitis, and Carypomya (Jamnongluk et al. 2002; Riegler & Stauffer 2002; Arthofer et al. 2009; Coscrato et al. 2009; Schuler et al. 2011, 2013; Augustinos et al. 2013, 2015; Coats et al. 2013; Karimi & Darsouei 2014; Morrow et al. 2015). Zabalou et al. (2004) transinfected cytoplasmic incompatibility-Wolbachia from Rhagoletis cerasi (L.) to Ceratitis capitata (Wiedemann) (both Diptera: Tephritidae), and Wolbachia induced complete cytoplasmic incompatibility in the novel host, leading to complete suppression of laboratory populations by single releases of infected males. Several studies also reported occurrence of Wolbachia in Chinese populations of Bactrocera dorsalis (Hendel) (Diptera: Tephritidae). However, there were no infections of B. dorsalis by Wolbachia in Yunnan, Fujian, and Wuhan populations (Augustinos et al. 2015). Similar to this result, Liu et al. (2016) also found the absence of Wolbachia in 16 collection sites in Thailand, China (Yunnan, Guizhou, Guangxi, Guangdong, Hainan, Fujian, Zhejiang, Shanghai Provinces), and the lab population. In contrast, Wolbachia had been positively detected in the Fujian, Guangdong, Hainan, Yunnan, and Guangxi Provinces, but the infection rates were very low (0.7–3%) (Sun et al. 2007). That may be the reason why the cytoplasmic incompatibility-Wolbachia based method has not been used in B. dorsalis successfully. However, if we can find Wolbachia strains that can cause cytoplasmic incompatibility in other fruit flies, such as Wolbachia in N. asiatica, we can try to transinfect this specific strain to B. dorsalis to reduce its population. In brief, Wolbachia-induced cytoplasmic incompatibility may be used as an environmentally friendly tool for the biological control of fruit flies. However, lack of a suitable strain prevented the application of Wolbachia in fruit fly management. We believe that assessment of Wolbachia in different species of fruit flies can provide more candidate strains for pest management.
In this study, Wolbachia infection in different developmental stages of N. asiatica from Ningxia Province in China was detected based on wsp gene sequence, and then the phylogenetic relationship of the Wolbachia strain in this fruit fly w as analyzed via neighbor-joining tree building. This research will help us in screening more putative strains with good potential for future applications in pest management, and thus provide a basis for the prevention and control of other economically important fruit flies.
Materials and Methods
Larvae, pupae, and adults were collected in wolfberry trees from Zhongning County, Ningxia (1.090166°E, 37.190000°E) on 30 Sep 2017 (Table 1). All samples were preserved in 100% ethanol and stored at −4 °C before DNA extraction.
DNA EXTRACTION, WSP GENE AMPLIFICATION, AND SEQUENCING
Total genomic DNA was isolated from whole individuals of N. asiatica using the commercial TIANamp Genomic DNA Kit (TIANGEN Biotech Co. Ltd., Beijing, China) following the manufacturer's protocol. Polymerase chain reaction (PCR) was completed in a final volume of 25 μL containing 12.5 μL 2 × Taq PCR MasterMix, 9.5 μL sterilized distilled water, 1 μL DNA as a template, 1 μL forward and 1 μL reverse primer, respectively. Amplifications were performed with general primers (Table 2) for wsp gene (Braig et al. 1998) used the following thermal cycling profile: 95 °C for 3 min; followed by 35 cycles of 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min; then 72 °C for 10 min. The reaction was performed on Veriti TM 96-well Thermal Cycler (Applied Biosystems Inc., Waltham, Massachusetts, USA). After amplification, 5 μL of PCR products were separated in 1.5% (w/v) agarose gels (1 × Tris Acetate-EDTA buffer) and stained with GeneGreen Nucleic Acid Dye (TIANGEN Biotech Co. Ltd., Beijing, China) and visualized under UV light. The PCR products were purified and bi-directional sequenced using the same amplification primers used commercially by the Sangon Biotech Co. Ltd., Shanghai, China.
The sequences obtained from N. asiatica were Basic Local Alignment Search Tool (BLAST) in National Center for Biotechnology Information, Bethesda, Maryland, USA. The wsp gene sequences from this study were aligned using MEGA 7.0 (Temple University, Philadelphia, Pennsylvania, USA) (Kumar et al. 2016), and 3 representative sequences were selected. The 3 sequences then were compared with 24 reference wsp sequences from GenBank representing different Wolbachia strains infecting various hosts (Table 3). We used Gblocks Server ( http://molevol.cmima.csic.es/castresana/Gblocks_server.html) to eliminate poorly aligned positions and divergent regions so that it became more suitable for phylogenetic analysis. The neighbor-joining tree was conducted in MEGA version 7.0, distances were calculated using the Kimura 2-parameter (K2P) model (Kimura 1980) in the bootstrap test (1,000 replications) (Felsenstein 1985). All 3 sequences were submitted to GenBank; the accession numbers MG950140, MG940141, MG950142 were wsp genes in larvae, pupae, and adults of N. asiatica, respectively (Table 1).
Sample information of collection site and accession number.
General primers used to amplify wsp gene.
The BLAST result from National Center for Biotechnology Information showed that all the sequences from the total DNA of Neoceratitis asiatica were the wsp gene of Wolbachia. The sequences of the wsp gene of Wolbachia in different stages of N. asiatica showed that they were identical, and were similar to the wRi strain.
Using the universal primers (81F, 691R) for Wolbachia, we screened 50 larvae, 50 pupae, and 32 adults. Thirteen larvae, 26 pupae, and 32 adults were positively detected in agarose gels.
Phylogenetic analysis of partial sequences of the wsp gene identified in N. asiatica, other fruit flies, and several other arthropod species are shown in Figure 1. In this neighbor-joining tree, 3 wsp gene sequences of Wolbachia in this study were in A Group, and together with the wRi strain in Drosophila simulans. However, the infection of Wolbachia in females and males has not been compared, and should to be the focus of future research.
Wolbachia strains used to construct the phylogenetic tree.
Wolbachia are one of the most abundant symbiotic microbes in arthropods (Werren 1997). In 1 species, when a male is infected by 1 or more kinds of Wolbachia, mating with an uninfected female or carrying a different strain, cytoplasmic incompatibility results in embryonic mortality (Bourtzis et al. 2003). The cytoplasmic incompatibility caused by Wolbachia can be used as an environmentally benign tool for the biological control of fruit flies. Most Wolbachia in fruit flies belonged to group A. Sun et al. (2007) found that B. dorsalis in Yunnan and Hainan provinces carried strain Cuc and Mel of Wolbachia, respectively. Rhagoletis cerasi (L.), Anastrepha spp., and C. capitata (all Diptera: Tephritidae) also had the strain Mel; Dacus destillatoria (Bezzi) contained the strain Des (Coscrato et al. 2009). Ceratitis vesuviana Costa showed infection with Wolbachia between Mors and Riv in the phylogenetic tree (Karimi & Darsouei 2014).
This research confirmed that different development stages of wolfberry fruit fly can be infected with the wRi strain of Wolbachia. wRi have a significant influence on cytoplasmic incompatibility in their natural host D. simulans (Hoffmann et al. 1986; Weeks et al. 2007), which may contribute to the pest management of economically important fruit flies by Wolbachia transmission. Bian et al. (2010) used Wolbachia strain wAlbB to control the mosquito Aedes aegypti (L.) (Diptera: Culicidae), the vector of dengue and Zika viruses. They found this strain could inhibit viral replication and dissemination in Ae. aegypti; thus, the spread of dengue and Zika viruses can be blocked through the control of the insect. They also used Wolbachia strain wAlbB to increase resistance in the mosquito Anopheles stephensi Liston (Diptera: Culicidae) to the human malaria parasite Plasmodium falciparum (Bian et al. 2013). Wolbachia can change the symbiotic relationship and can boost the immune system response to enhance the host's resistance to pathogens (Pan et al. 2018). However, there is still an absence of population control of B. dorsalis using Wolbachia. The low infection rate of Wolbachia in B. dorsalis is the biggest obstacle. Transinfecting Wolbachia strains from other fruit flies maybe a good solution to this problem. This research provided a candidate strain, isolated from wolfberry fruit fly, with the potential for controlling B. dorsalis. In further research, we will focus on the suitability of Wolbachia for population control in B. dorsalis. The potential exists to control not only B. dorsalis, but also other economically important fruit flies.
Our study was the first to document the existence of Wolbachia in Chinese wolfberry fruit fly. We suggest that the wRi strain of Neoceratitis asiatica has the potential to assist in the prevention and control of other economically important fruit flies.
Financial support was provided by the National Key Research and Development Project of China (No. 2016YFC1200605), and the Science and Technology Innovation Pilot Fund Project of the Ningxia Academy of Agriculture and Forestry Sciences (NKYZ-16–0502). We are grateful to Jing Wei of China Agricultural University for help with collecting larvae from wolfberry fruit.
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