Atsushi Mochizuki’s research while affiliated with National Institute for Agro-Environmental Sciences in Japan and other places

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Publications (60)


Production of the transgenic tomato plant
Structure of the introduced transgene: the expression vector pEl2Omega with the gene for MLX56 (Kawazu et al., 2012) was used for tomato transformation. The vector has the duplicated CaMV 35S transcriptional enhancer and TMV omega translational enhancer for overproduction of the transgene (A). Detection of mRNA for MLX56 in transgenic tomato leaves: the level of mRNA for MLX56 was determined by qRT-PCR. (B). Ctrl: Luciferase-expressing control line, 69: line 69 (moderate MLX56-expressing line), 73: line 73 (high MLX56-expressing line). nd: not detected.
Partial purification of the chitin-binding protein in a transgenic tomato plant with the gene for MLX56
The chitin-binding protein of the control and MLX56-transformed tomato plant (MLX56-73) were partially purified using chitin beads. The crude extract and each partially purified fraction were separated by SDS-PAGE and visualized by CBB. T: total crude extract; S: unbound supernatant; W: wash fraction by extraction buffer; U: wash fraction by urea; E: eluted fraction after SDS denaturation that tightly bound to chitin.
Effects of transgenic tomato on the numbers of surviving individuals and development of S. litura larvae
Numbers of surviving individuals from inoculation to 9 days (A). Error bars indicate SE (n = 10). Proportions of the larval instars reared on each tomato line after 3, 6 and 9 days (B). Values not followed by the same letters were significantly different among the three lines at the same day by Tukey-Kramer HSD test (day 3: p < 0.005, F = 8.1033; day 6: p < 0.05, F = 4.1923; day 9: p < 0.01, F = 5.8973). Ctrl: Luciferase-expressing control line, 69: line 69 (moderate MLX56-expressing line), 73: line 73 (high MLX56-expressing line).
Effects of transgenic tomato on the numbers of surviving individuals of F. occidentalis (A), and damage to tomato plants (B) and leaves (C) by the thrips after two weeks
Error bars indicate SE (n = 15). Values not followed by the same letters were significantly different (Tukey-Kramer HSD test: p < 0.0001, F = 14.325). Ctrl: Luciferase-expressing control line, 69: line 69 (moderate MLX56-expressing line), 73: line 73 (high MLX56-expressing line).
The effect of MLX56 on H. vigintioctopunctata larvae
Twelve newly hatched larvae (0.14 mg) were fed tomato leaves of either the control, 69, or 73 line for 9 days, and larval weights were then measured. Larval body size (A), and average body weights (B) on day 9. Error bars indicate SD (n = 12). No mortality was observed for any of the three treatments. Values not followed by the same letters were significantly different (Tukey’s test for multiple comparison, p < 0.0001, F = 139.1). Ctrl: Luciferase-expressing control line, 69: line 69 (moderate MLX56-expressing line), 73: line 73 (high MLX56-expressing line).
Expression of a gene for an MLX56 defense protein derived from mulberry latex confers strong resistance against a broad range of insect pests on transgenic tomato lines
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January 2021

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124 Reads

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8 Citations

Mika Murata

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Insect pests cause serious damage in crop production, and various attempts have been made to produce insect-resistant crops, including the expression of genes for proteins with anti-herbivory activity, such as Bt (Bacillus thuringiensis) toxins. However, the number of available genes with sufficient anti-herbivory activity is limited. MLX56 is an anti-herbivory protein isolated from the latex of mulberry plants, and has been shown to have strong growth-suppressing activity against the larvae of a variety of lepidopteran species. As a model of herbivore-resistant plants, we produced transgenic tomato lines expressing the gene for MLX56. The transgenic tomato lines showed strong anti-herbivory activities against the larvae of the common cutworm, Spodoptera litura. Surprisingly, the transgenic tomato lines also exhibited strong activity against the attack of western flower thrips, Frankliniera occidentalis. Further, growth of the hadda beetle, Henosepilachna vigintioctopunctata, fed on leaves of transgenic tomato was significantly retarded. The levels of damage caused by both western flower thrips and hadda beetles were negligible in the high-MLX56-expressing tomato line. These results indicate that introduction of the gene for MLX56 into crops can enhance crop resistance against a wide range of pest insects, and that MLX56 can be utilized in developing genetically modified (GM) pest-resistant crops.

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Expression of a gene for an MLX56 defense protein derived from mulberry latex confers strong resistance against a broad range of insect pests on transgenic tomato lines

September 2020

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41 Reads

Insect pests cause serious damage in crop production, and various attempts have been made to produce insect-resistance crops, including the expression of genes for proteins with anti-herbivory activity, such as BT toxins. However, the number of available genes with sufficient anti-herbivory activity is limited. MLX56 is an anti-herbivory protein isolated from the latex of mulberry plants, and has been shown to have a strong growth-suppressing activity against the larvae of a variety of lepidopteran species. As a model of herbivore-resistant plants, we produced transgenic tomato lines expressing the gene for MLX56. The transgenic tomato lines showed strong anti-herbivory activities against the larvae of the common cutworm, Spodoptera litura . Surprisingly, the transgenic tomato lines also exhibited strong activity against the attack of the western flower thrips, Frankliniera occidentalis . Further, growth of the hadda beetle, Henosepilachna vigintioctopunctata fed on leaves of transgenic tomato was significantly retarded. The levels of damage caused by both western flower thrips and hadda beetles were negligible in the high-MLX56-expressing tomato line. These results indicate that introduction of the gene for MLX56 into crops can enhance crop resistance against a wide range of pest insects, and that MLX56 can be utilized in developing pest-resistance GM crops.



Loliolide, a Carotenoid Metabolite, Is a Potential Endogenous Inducer of Herbivore Resistance

January 2019

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229 Reads

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63 Citations

Plant Physiology

Jasmonic acid (JA) plays an important role in the induction of herbivore resistance in many plants. However, JA-independent herbivore resistance has been suggested. An herbivore-resistance-inducing substance was isolated from Tobacco mosaic virus-infected tobacco (Nicotiana tabacum) leaves in which a hypersensitive response (HR) was induced and identified as loliolide, which has been identified as a β-carotene metabolite. When applied to tomato (Solanum lycopersicum) leaves, loliolide decreased the survival rate of the two-spotted spider mite, Tetranychus urticae, egg deposition by the same pest, and the survival rate of larvae of the common cutworm Spodoptera litura without exhibiting toxicity against these herbivores. Endogenous loliolide levels increased not only with an infestation by Slitura larvae, but also with the exogenous application of their oral secretions in tomato. A microarray analysis identified cell-wall-associated defense genes as loliolide-responsive tomato genes, and exogenous JA application did not induce the expression of these genes. Suppressor of zeaxanthin-less (szl), an Arabidopsis (Arabidopsis thaliana) mutant with a point mutation in a key gene of the β-carotene metabolic pathway, exhibited the decreased accumulation of endogenous loliolide and increased susceptibility to infestation by the western flower thrip (Frankliniella occidentalis). A pretreatment with loliolide decreased susceptibility to thrips in the JA-insensitive Arabidopsis mutant coronatine-insensitive1 Exogenous loliolide did not restore reduced electrolyte leakage in szl in response to a HR-inducing bacterial strain. These results suggest that loliolide functions as an endogenous signal that mediates defense responses to herbivores, possibly independently of JA, at least in tomato and Arabidopsis plants.


Photos of (a) a stand of Chinese silver grass (Miscanthus sinensis), the host plant of the social spider mite Stigmaeopsis miscanthi, and (b) two adult males of S. miscanthi in fighting position, facing each other with front legs spread out, inside a nest
Geographic distribution of the social spider mite, Stigmaeopsis miscanthi and invasion route of the host plant, Miscanthus sinensis, during the last glacial period in the Japanese archipelago and the surrounding area. Open and filled circles show the mite populations of the low aggression (LW) and high aggression (HG) forms (Saito, 1995; Sato, Egas, et al., 2013; Saito & Sahara, 1999). Open and filled squares show the mite populations in which either of aggression or male weapon morph has not been investigated (Saito, 1995; Saito & Sahara, 1999) and the mite populations which exhibit intermediate aggressiveness between the HG and LW forms (Sato, Egas, et al., 2013). Solid and dotted gray arrows show the primary M. sinensis migration route into Japanese Archipelago and the secondary M. sinensis migration route through East China and Korea Peninsula of the host plant (Clark et al., 2014)
Nine demographic scenarios of the three forms in Stigmaeopsis miscanthi tested by DIYABC. Based on male weapon morph and phylogenetic relationship, 42 populations of the mite were pooled into three form groups: LW, ML, and HG groups. Scenarios 1–4 are based on hypothesis 1; ML form in subtropical region is the ancestor group, and the other two forms with low and high aggressiveness emerged while its migration into the north. Scenarios 5–6 are based on hypothesis 2: LW form showing low aggressiveness is the ancestor group; then, the aggressiveness became stronger gradually resulting in ML and HG forms. Scenarios 7–8 are based on hypothesis 3; ML form with intermediate aggressiveness was generated by admixture of LW and HG forms with low and high aggressiveness. Scenario 9 assumes that the three forms were split at the same time. Blue, green, and orange represent LW, ML, and HG forms, and t# represents timescale measured in the number of generations. For the prior distributions of the parameters used in DIYABC, see Supporting Information Table S2. For present geographic relationship of the three forms, see Figure 4c
(a) Dendrogram created by hierarchical cluster analysis using average relative length of male leg I from 42 populations of Stigmaeopsis miscanthi, (b) histogram of relative length of male leg I of 834 individuals from the populations categorized into the three forms by the cluster analysis, and (c) localities of the populations including unknown‐form populations (indicated as black dots) used in phylogenetic analyses. The populations are grouped into seven regions based on current faunal characteristics and geographic features and numbered in order of north latitude in each region. The average relative length of male leg I used in the analysis is shown in Supporting Information Table S3. Orange, blue, and green show populations categorized as HG form, LW form, and ML form, respectively, and shaded colors in the histogram (b) show the overlaps between ML (green) and LW (blue) and between ML (green) and HG (orange). SR3 marked by asterisk was categorized as LW form in the dendrogram (clade 2 in a); however, because of the average value (Supporting Information Table S3) and the phylogenetic relationship (Figure 5), SR3 was deemed as ML form
Phylogenetic networks of 47 populations of Stigmaeopsis miscanthi and two populations of Stigmaeopsis longus based on (a) parasodium channel gene and (b) mtCOI. Blue, green, and orange represent LW, ML, and HG populations categorized by a cluster analysis (Figure 4a), and black represents unknown‐form populations of S. miscanthi and Stigmaeopsis longus populations. For the localities of S. miscanthi populations, see Figure 4c
Phylogeography of lethal male fighting in a social spider mite

January 2019

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326 Reads

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5 Citations

When males fight for access to females, such conflict rarely escalates into lethal fight because the risks and costs involved, that is, severe injury or death, are too high. The social spider mite, Stigmaeopsis miscanthi, does exhibit lethal male fights, and this male–male aggressiveness varies among populations. To understand the evolution of lethal fighting, we investigated aggressiveness in 42 populations and phylogenetic relationships in 47 populations along the Japanese archipelago. By analysis of the male weapon morph, a proxy for aggressiveness, we confirmed the existence of a mildly aggressive (ML) form, besides the low aggression (LW) and high aggression (HG) forms reported earlier. To evaluate demographic history of these three forms, we employed the approximate Bayesian computation approach using mtCOI sequences and taking into consideration the postlast glacial expansion history of the host plant, Miscanthus sinensis. As results, hierarchical split models are more likely to explain the observed genetic pattern than admixture models, and the ML form in the subtropical region was considered the ancestral group. The inferred demographic history was consistent with the one reconstructed for the host plant in a previous study. The LW form was split from the ML form during the last glacial period (20,000–40,000 years BP), and subsequently, the HG form was split from the ML form at the end of or after the last glacial period (5,494–10,988 years BP). The results also suggest that the mite invaded Japan more than once, resulting in the present parapatric distribution of LW and HG forms in eastern Japan.




Figure 2. Oscillographs of duetting interactions. (a) 12 s of a typical duet between a male and a female of Chrysoperla shahrudensis sp. nov., showing the partners politely exchanging single volleyplus-rumble units or shortest repeated units (SRUs). (b) 12 s of a typical duet between a male and a female of Chrysoperla bolti sp. nov., showing the partners politely exchanging single SRUs. (c) 71 s oscillograph showing a female of C. shahrudensis duetting with a conspecific for the first 24 s, with C. bolti for the next 25 s, and with Chrysoperla adamsi (North America) for the last 22 s. In each of those three duets, the female being tested is capable of synchronizing precisely with the prerecorded playback signal. As discussed in the text, C. shahrudensis shows no such duetting response to playbacks of songs of any carnea-group species other than C. bolti and C. adamsi. All duets were recorded at 25 ± 1°C. 
Figure 4. Adult of (a) Chrysoperla. shahrudensis sp. nov., non-diapausing green form; (b) C. shahrudensis, diapausing sandy-brown form, and (c) Chrysoperla bolti sp. nov., all-season green form. 
Figure 5. Dorsal view of third-instar larval head capsule (left half) of Chrysoperla shahrudensis sp. nov. The drawing represents the typical condition seen in 24 individuals from two populations in Iran. The colour insert is a typical mature third-instar larva of the species. 
Figure 6. Dorsal view of third-instar larval head capsule (left half) of Chrysoperla bolti sp. nov. The drawing represents the typical condition seen in 18 individuals from two populations in northern Kyrgyzstan. The colour insert is a typical mature third-instar larva of the species. 
Relative size, expressed as a ratio, of the basal dilation of the metatarsal claw measured in 18 species and subspecies of the global Chrysoperla carnea-group.
Chrysoperla shahrudensis and Ch.bolti 2018

June 2018

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1,214 Reads

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8 Citations

Chrysoperla shahrudensis sp. nov. is discovered in northern Iran, co-occurring with at least five other cryptic species of the Chrysoperla carnea-group. It is distinguished by the volley period and tonality of its courtship duetting song. Another Asian species from alpine meadows of northern Kyrgyzstan, previously C. ‘adamsi-K’ but here named Chrysoperla bolti sp. nov., has a song distinct from but convergent with both C. shahrudensis and North American Chrysoperla adamsi. Coordinated duets can be established in the laboratory between individuals of C. shahrudensis and recorded songs of either C. bolti or C. adamsi. Such functional song equivalence in distinct allopatric species suggests that repeated episodes of parallel speciation can drive the origin of cryptic species diversity in lacewings. Morphology, life history, and ecology of larvae and adults of C. shahrudensis and C. bolti are then formally described. Adding C. shahrudensis to a large mitochondrial DNA data set for ≈ 21 species shows it to be similar to neither C. adamsi nor C. bolti, further supporting independent, convergent evolution of song rather than song similarity due to relationship. Although C. bolti and C. shahrudensis are both from Asia and share some basic temporal song features


Patterns of reproductive isolation in a haplodiploid - strong postmating, prezygotic barriers among three forms of a social spider mite

March 2018

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104 Reads

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18 Citations

Journal of Evolutionary Biology

In speciation research, much attention is paid to the evolution of reproductive barriers, preventing diverging groups from hybridizing back into one gene pool. The prevalent view is that reproductive barriers evolve gradually as a byproduct of genetic changes accumulated by natural selection and genetic drift in groups that are segregated spatially and/or temporally. Reproductive barriers, however, can also be reinforced by natural selection against maladaptive hybridization. These mutually compatible theories are both empirically supported by studies, analyzing relationships between intensity of reproductive isolation and genetic distance in sympatric taxa and allopatric taxa. Here, we present the – to our knowledge – first comparative study in a haplodiploid organism, the social spider mite Stigmaeopsis miscanthi, by measuring premating and postmating pre‐ and postzygotic components of reproductive isolation, using three recently diverged forms of the mite that partly overlap in home range. We carried out cross experiments and measured genetic distances (mtDNA and nDNA) among parapatric and allopatric populations of the three forms. Our results show that the three forms are reproductively isolated, despite the absence of premating barriers, and that the postmating, prezygotic component contributes most to reproductive isolation. As expected, the strength of postmating reproductive barriers positively correlated with genetic distance. We did not find a clear pattern of prezygotic barriers evolving faster in parapatry than in allopatry, although one form did show a trend in line with the ecological and behavioral relationships between the forms. Our study advocates the versatility of haplodiploid animals for investigating the evolution of reproductive barriers. This article is protected by copyright. All rights reserved.


Molecular phylogeny and morphology of Pseudomallada (Neuroptera: Chrysopidae), one of the largest genera within Chrysopidae

July 2017

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247 Reads

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18 Citations

Zoological Journal of the Linnean Society

Pseudomallada, comprising at least 166 described species, is not only one of the most species-rich genera within the green lacewing family Chrysopidae but also one of the largest within the entire order Neuroptera. Using external morphology, it has not been possible to parse Pseudomallada into smaller taxonomic units such as subgenera or species-groups. Here, we examine morphology, life-history traits, and nuclear DNA sequences in 49 described or putative species of Pseudomallada from all over the world, including 2 closely related species currently assigned to Apertochrysa. On the one hand, we find that body and egg coloration, as well as the mode of egg deposition, have evolved independently within different genetic clusters of species in response to environmental conditions favouring cryptic behaviour or sibling cannibalism. However, three lineages supported by molecular phylogenetic analysis are congruent with three clusters based on the morphology of the gonapsis in the male genitalia; these comprise 29 of the taxa under study (59%). Seventeen additional species cluster in a flavifrons-group based on gonapsis shape, but this cluster is not supported by molecular data. Three molecular singletons were identified, two of which can be assigned to one of the four gonapsis groups. It is clear that at least Apertochrysa eurydera, and probably A. edwardsi, should be moved into the genus Pseudomallada, based on molecular data and the shape of the gonapsis.


Citations (53)


... Gene expression due to the insect attack is one of the most specific molecular defense mechanisms in plants including vegetables. Tomato overexpressed with Cry1A genes enabled it to reduce the damage against a diverse range of insects (Murata et al. 2021). Genes that encode the proteinase inhibitor, phytolectin, amylase inhibitor, and chitinase have been reported in plants, which contributed to the natural defense against insects and microbes (Wang et al. , 2021a. ...

Reference:

Host Plant Resistance to Insects in Vegetable Crops
Expression of a gene for an MLX56 defense protein derived from mulberry latex confers strong resistance against a broad range of insect pests on transgenic tomato lines

... Branches are often coloured (Djernaes et al., 2020), state reconstructions have been depicted by both node labeling and branch colouring (St Laurent et al., 2020), and node/branch support information is commonly overlaid, although this can soon become complex and multifaceted even to the point that a key or so-called "Navajo rug" is required (Bucek et al., 2019;Almeida et al., 2023). Taxonomic groups are variously denoted by nested boxes or inserts (Winterton et al., 2019), branch colouring or terminal labels (St Laurent et al., 2020). We would not suggest removing genus names from terminals for use as clade labels (Str€ umpher et al., 2014). ...

Evolution of green lacewings (Neuroptera: Chrysopidae): an anchored phylogenomics approach: Green lacewing phylogenomics
  • Citing Article
  • February 2019

Systematic Entomology

... The increased astaxanthin levels in the treatment fed with alfalfa-supplemented diets may further contribute to enhanced shrimp survival (Ritu et al., 2023;Thanomchaisanit et al., 2024). Alfalfa powder also contains phytoestrogenic compounds such as Loliolide, a carotenoid metabolite of β-carotene, known for its germicidal properties and its ability to increase survival rates (Hong et al., 2011;Murata et al., 2019). ...

Loliolide, a Carotenoid Metabolite, Is a Potential Endogenous Inducer of Herbivore Resistance

Plant Physiology

... In Japan, Stigmaeopsis sabelisi with lower male aggression, S. miscanthi high-aggression form (hereafter, S. miscanthi HG) with higher male aggression, and S. miscanthi mild-aggression form (hereafter, S. miscanthi ML) with intermediate male aggression are distributed (Saito 1995;Saito and Sahara 1999;Sato et al. 2013a;Sato et al. 2019). S. miscanthi ML is distributed in subtropical regions and is geographically isolated from the two other species (Sato et al. 2013a;Sato et al. 2019) (Fig. 1). ...

Phylogeography of lethal male fighting in a social spider mite

... The family Chrysopidae currently includes about 1,400 species and subspecies, divided into three subfamilies and 82 genera (Garzón-Orduña et al. 2019;Oswald 2023). So far, 100 species in 17 genera have been recorded in Mexico (Tauber and De León 2001;Contreras-Ramos and Rosas 2014;Cancino-López and Contreras-Ramos 2019;Oswald 2023). ...

Evolution of green lacewings (Neuroptera: Chrysopidae): a molecular supermatrix approach: Green lacewing phylogeny
  • Citing Article
  • December 2018

Systematic Entomology

... Meanwhile, C. carnea has become one of many cryptic species in the carnea group, and it is unlikely that the species occurs in Pakistan. In the present study, we prefer to refer C. carnea s.l. to avoid further confusion with C. z. sillemi or other song morphs in the carnea group, because several song morphs (new species) have been found in Iran and Western Asia by Henry et al. (2014Henry et al. ( , 2018. However, C. carnea s.l. ...

Chrysoperla shahrudensis and Ch.bolti 2018

... Copulation between kin species is sometimes reported in spider mites (Acari: Tetranychidae) via mating experiments, suggesting incomplete species recognition in reproductive behavior and the importance of the postzygotic reproductive barrier (Takafuji and Fujimoto 1985;Osakabe 1993;Ben-David et al. 2009;Sato et al. 2018). Sperm precedence is generally biased to the first mated male in spider mites of arrhenotokous parthenogenesis in which fertilized diploid eggs and unfertilized haploid eggs are produced and develop into females and males, respectively (Takafuji 1986;Rodrigues et al. 2020). ...

Patterns of reproductive isolation in a haplodiploid - strong postmating, prezygotic barriers among three forms of a social spider mite
  • Citing Article
  • March 2018

Journal of Evolutionary Biology

... Moreover, the DNA barcode gap analysis (Fig. 6) indicated that 16.75% of species of the DNA barcode reference library of Chinese green lacewings lack a DNA barcode gap. However, compared with the less sampled DNA barcode library of green lacewings from Portugal (Oliveira et al., 2021), the rate of correct identification of our library is much higher, probably due to the large proportion of closely related species (55% of all green lacewing samples) in the Apertochrysa "prasina" species group and the Chrysoperla "carnea" species group in the Portuguese DNA barcode reference library of Neuroptera, which have been noted to be hardly distinguished based on COI (Henry et al., 2014;Duelli et al., 2017). ...

Molecular phylogeny and morphology of Pseudomallada (Neuroptera: Chrysopidae), one of the largest genera within Chrysopidae
  • Citing Article
  • July 2017

Zoological Journal of the Linnean Society

... The type species of Kuwayamachrysa was originally described from Japan by Kuwayama (1936) as Chrysopa kichijoi and later transferred to the genus Apertochrysa Tjeder, 1966. Tsukaguchi & Tago (2018) established Kuwayamachrysa based on adult and larval morphological characters and on molecular evidence given by Mochizuki et al. (2017), all of which show distinct differences between A. kichijoi and other typical Apertochrysa species. ...

Apertochrysa (Neuroptera: Chrysopidae): A heterogeneric phantom?

Zootaxa

... This fertilization strategy is usually followed to obtain non-genetic (Squires et al. 2012;Lewis and Pitcher 2017) and genetic benefits (Kekäläinen et al. 2010;Sagebakken et al. 2011). In many species of different taxa, polyandrous females produce eggs being higher in number, smaller in size, greater in viability and larger in yolk volume (Ward 2000;Omkar 2010;Kawazu et al. 2017) that ensure higher fertilization and hatching success (Jennions et al. 2007;Byrne and Whiting 2008). Evidence also shows that polyandrous females produce offspring having comparatively larger body size (Maklakov and Lubin 2006) and higher survival rate (Croshaw et al. 2017) than the monandrous one. ...

Polyandry increases reproductive performance but does not decrease survival in female Brontispa longissima
  • Citing Article
  • August 2016

Bulletin of Entomological Research