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A simple method to disperse eggs from lepidopteran scalelike egg masses and to observe embryogenesis
Dispersal and Oviposition Patterns of Lycorma Delicatula (Hemiptera: Fulgoridae) During Oviposition Period in Ailanthus Altissima (Simaroubaceae)
Abstract The spotted lanternfly (SLF), Lycorma delicatula (Hemiptera: Fulgoridae), has the potential to become a global pest and is currently expanding its range in the United States. In this study, we investigated the dispersal patterns of SLF in Ailanthus altissima during its oviposition period in South Korea using fluorescent marking system. Oviposition patterns of SLF were then analyzed by surveying egg masses in A. altissima patches. The recapture rate of fluorescent-marked SLF rapidly decreased < 30% within the first two weeks. During the oviposition period, seven cases of among-patch dispersal of SLF adults were observed with a minimum estimated dispersal distance mainly ranging between 10 - 60 m and a maximum of 1,740 m. Also, the number of A. altissima trees detected with fluorescent-marked SLF increased until late September. Based on the egg mass survey, a total of 159 egg masses were detected from 38 out of 247 A. altissima trees. Furthermore, ca. 80% of egg masses were located < 2.5 m above the ground. Finally, the number of egg masses showed significant positive correlations with the height and diameter at root collar of A. altissima trees; both tree height and DRC were significantly larger from the trees with egg masses.
Cardamine violifohia is an economically-important medicinal plant, and also a valuable plant for strong ability to accumulate selenium (Se) (Ebba et al. 2020). It is not only be used to extract selenium protein and selenium polysaccharide, but also widely used to develop selenium-supplement reagent. In September 2020, root-knot nematodes (RKN; Meloidogyne spp.) infection experiments showed that galls and egg masses were observed on the roots of numerous C. violifolia plants in Enshi (30°32′25.67″ N; 109°48′48.46″ E), Hubei Province, China. Meanwhile, the overground plants of C. violifohia were stunted and leaves were yellow. Almost 5% C. violifohia plants were affected by the disease. The roots with galls were collected, and nematodes were dissected and extracted (Fig. S1). Based on phytopathological clinic, the number of galls on each plant was 91.87 ± 19.01, and egg masses was 15.27 ± 5.36 (n = 15). Nematodes and galls were collected from soil and infected roots (Barker 1985). The morphological diagnostic of the nematode species was measured as follows. Measurements of adult females (n=20), body length = 628.15 ± 73.69 μm, width = 356.77 ± 36.72 μm, stylet length = 15.58 ±0.93 μm. Meanwhile, a high and trapezoidal dorsal arch with thick striations was observed in the perineal region of females. Second-stage juveniles (J2s) (n=20): body length = 377.09 ± 18.19 μm, body width = 15.64 ± 1.24 μm, stylet length = 13.31 ± 1.04 μm, tail length = 42.49 ± 4.64 μm, hyaline tail terminus = 12.35 ± 2.02 μm and presented well developed esophageal glands. Eggs (n=20): length = 80.81 ± 3.47 μm, and width = 37.09 ± 2.98 μm. All the morphological characteristics of the identified species were consistent with the descriptions of Meloidogyne incognita (Kofoid & White, 1919) Chitwood, 1949 (Whitehead, 1968). Molecular identification was carried out by PCR with the M. incognita-specific primers Mi-F/Mi-R (Meng et al. 2004) and 28S rDNA D2/D3 region primers MF/MR (Hu et al. 2011). The target fragments of 955 bp and 478 bp amplified by of the primer pairs Mi-F/Mi-R and MF/MR were observed under a UV light, which confirmed that these nematodes collected from C. violifohia were M. incognita (Fig. S2). Fragments were, sequenced (MZ596342 and MZ566843, respectively) and aligned with available sequences on NCBI, which were 100% identical to the MK410954, MN728679, and MK410953, MF177882 M. incognita sequences, respectively. Pathogenicity testing was conducted to perform Koch’s postulates in a greenhouse by inoculation of 500 J2s from the original population into C. violifohia seedlings (n = 30, 5-6 leaves stage). After 7 weeks, all inoculated plants exhibited the same symptoms that observed in the field initially. Different life stages of M. incognita were observed in dissected galled tissues. The average reproductive factor was 37.30 ± 6.13, which is considered as the pathogenicity of M. incognita to C. violifohia. Therefore, C. violifohia is a suitable host for M. incognita in China. The growers should be informed of the current findings to avoid serious economic losses that might be caused by this pathogenic nematode, and prepare for proper management action. To our knowledge, this is the first report of M. incognita infecting C. violifohia in China.
Chironomids are aquatic insects that undergo a complete metamorphosis of four life stages. Here we studied, for the first time, the microbiota composition of Chironomus circumdatus, a tropical midge species, both from the Mula and Mutha Rivers in Pune, India and as a laboratory-reared culture. We generated a comparative microbial profile of the eggs, larvae and pupae, the three aquatic life stages of C. circumdatus. Non-metric multidimensional scaling analysis (NMDS) demonstrated that the developmental stage had a more prominent effect on the microbiota composition compared to the sampling location. Notably, the microbiota composition of the egg masses from the different sampling points clustered together and differed from laboratory culture larvae. Proteobacteria was the dominant phylum in all the environmental and laboratory-reared egg masses and pupal samples, and in the laboratory-reared larvae, while Fusobacteria was the dominant phylum in the larvae collected from the field environment. The most abundant genera were Cetobacterium, Aeromonas, Dysgonomonas, Vibrio, and Flavobacterium. The ten amplicon sequence variants (ASVs) that most significantly contributed to differences in microbiota composition between the three sampled locations were: Burkholderiaceae (ASVs 04 and 37), C39 (Rhodocyclaceae, ASV 14), Vibrio (ASV 07), Arcobacter (ASV 21), Sphaerotilus (ASV 22), Bacteroidia (ASVs 12 and 28), Flavobacterium (ASV 29), and Gottschalkia (ASV 10). No significant differences were found in the microbial richness (Chao1) or diversity (Shannon H’) of the three sampled locations. In contrast, significant differences were found between the microbial richness of the three life stages. Studying the microbiota of this Chironomus species may contribute to a better understanding of the association of C. circumdatus and its microbial inhabitants.
Tomato (Solanum lycopersicum) is an important vegetable crop in Hainan province, Southern China. In this area, rice and tomato rotation is the most common way for tomato cultivation. During March of 2021, in a field of Yazhou District, Sanya City, Hainan Province, leaves of some tomato plants (cv. Jinsheng) turned yellow, although there were no obvious dwarf plants observed. The tomato plants with yellow leaves exhibiting obvious galls on the roots were collected. Several females and gelatinous egg masses of Meloidogyne spp. were found inside the cortex of the root galls after dissection. The perineal patterns of females (n=12) were dorsal-ventrally oval with low and round dorsal arches, lacking obvious lateral lines. Most of the striae were smooth and sometimes short and irregular striae were observed within them. Morphological measurements of females (n=20) included body length (L) = 569.2 ± 53.6 (457.6 - 662.7) µm, body width (BW) = 342.7 ± 69.8 (245.5 - 457.9) µm, stylet = 11.8 ± 0.7 (10.5 - 13.3) µm, dorsal pharyngeal gland orifice to stylet base (DGO) = 4.0 ± 0.2 (3.7 - 4.6) µm, vulval slit length = 24.1 ± 3.7 (16.7 - 30.7) µm, and vulval slit to anus distance = 16.0 ±1.9 (12.6 - 19.3) µm. The second-stage juveniles (J2s, n=20) had the following morphological characters: L = 440.6 ± 26.7 (395.7 - 488.3) µm, BW = 15.9 ± 1.0 (14.5 - 17.9) µm. stylet = 13.5 ± 0.8 (12.3 - 14.9) µm, tail length = 69.5 ± 3.7 (65.4 - 76.9) µm, hyaline tail terminus = 21.0 ± 2.1 (17.3 - 24.9) µm. These morphological characters matched the original description of Meloidogyne graminicola (Golden and Birchfield, 1968). Ten individual females were transferred to ten different tubes for DNA extraction. The species-specific primers Mg-F3 (5'-TTATCGCATCATTTTATTTG-3') and Mg-R2 (5'-CGCTTTGTTAGAAAATGACCCT-3') were used for the identification of M. graminicola (Htay et al. 2016). For the ten DNA samples, a 369 bp fragment was amplified by this pair of primers, confirming their identities as M. graminicola. The mitochondrial DNA (mtDNA) region between COII and the lRNA gene was amplified using primers C2F3 (5’-GGTCAATGTTCAGAAATTTGTGG-3’) and 1108 (5’-TACCTTTGACCAATCACGCT-3’) (Powers and Harris, 1993). A DNA fragment of 531 bp was obtained and the sequence (GenBank Accession No. MZ576221) was 99.8% identical to the sequences of M. graminicola (GenBank Accession Nos. MH033621, MK616527, and MG356945). Part of the rDNA spanning ITS1, 5.8S gene, and ITS2 was amplified with primers 18S (5’-TTGATTACGTCCCTGCCCTTT-3’) and 26S (5’-TTTCACTCGCCGTTACTAAGG-3’) (Vrain et al. 1992). The sequences from the ITS region were 790 bp (GenBank Accession No. MZ312595) and were all 100% identical to the known sequences of M. graminicola (GenBank Accession Nos. MF320126, HM623442, and KY020414). In glasshouse tests, six 30-day-old tomato plants (cv. Jinsheng) were individually transplanted in pots (V sand :V soil = 3:1) and inoculated with 1500 J2s hatched from the egg masses of collected M. graminicola samples per plant. Two non-inoculated tomato plants served as negative controls. After 50 days, inoculated plants had galled roots similar to those encountered in the field and there were J2s and eggs within the galls. The nematode reproduction factor (RF = final population/initial population) was 5.3. No symptoms were observed on control plants. These results confirmed the nematode’s pathogenicity on tomato. To our knowledge, this is the first time of a natural infection of tomato with M. graminicola in China.
<p>Global change is increasingly impacting coastal marine systems. Organisms inhabiting the intertidal zone may be especially vulnerable to additional anthropogenic influences, which augment the naturally stressful, highly variable conditions to which they are already subjected and may lead to the manifestation of artificially severe carry-over effects (COEs). In marine invertebrates with complex life histories, COEs can occur between life stages, when the conditions experienced by one stage influence the characteristics or performance of the next, as well as trans-generationally, in which case the environment experienced by a parental generation affects offspring. Most of the existing literature surrounding COEs focuses only on those between life stages or generations, seldom both simultaneously, and do so with the implementation of only a single stressor. In nature however, organisms may be affected by both forms of COE, since the presence of one does not preclude the other, and are invariably subjected to multiple co-occurring stressors that can interact in complex ways. Consequently, how trans-generational COEs might impact the propagation of stress through offspring life stages remains unclear, and how these processes operate in a global change context is little understood. It was here aimed to elucidate the role of COEs under ongoing global change by addressing these common literature imitations and taking the novel approach of examining how the effects of multiple, global change-associated stressors carry-over from a parental generation through their offspring’s life stages in order to provide a more realistic representation of the conditions under which COEs manifest in the field. This was done using Siphonaria australis, an intertidal pulmonate limpet that deposits benthic egg masses, from which hatch planktonic veliger larvae. Adult S. australis were subjected to one of four treatments for 4h/day over four weeks to induce trans-generational COEs: a no-stress control, a pollution treatment with added copper (5.0μg/L), a “climate change” treatment with elevated temperature (25°C) and UVR (1.7W/m2), and a full global change treatment incorporating all three stressors. At the end of this period, the egg masses laid under each of these adult treatments were subjected to further experimentation for two weeks by being redistributed among the same four treatments again, so as to produce 16 unique treatment histories of adult-to-egg mass stress. Of these, 11 provided successfully hatching larvae, which were reared and observed for COEs between life stages (from egg to larva) under ambient conditions (ie. no added stressors) for 27 days. In adult S. australis survivor size, the size of egg masses laid and the size of individual eggs varied in complex ways over time and across treatments, while the number of survivors was unaffected by stress. Egg masses were unaffected in terms of hatching time but displayed strong responses to parental and developmental stress exposure through hatching success, and the percentage of viable eggs per egg mass, with the latter clearly declining according to adult treatment severity and both showing trans-generational COEs. Larval characteristics were extremely varied across treatment histories and highly context-dependent as hatching size, size reached by 27 days, growth rate, and size at death all showed evidence of COEs between generations and life stages, as well as interaction between both types of COE, with the number of survivors again being the only unaffected response variable. Overall, trans-generational COEs were slightly more common than those between life stages. These results show that both forms of COE, each triggered by exposure to multiple stressors in progenitors and developmental stages, interact to form highly context-dependent legacies of mostly impaired performance in S. australis larvae. This implies that COEs may become more prominent with worsening stressors in the future and suggests that the role of COEs in the persistence of marine invertebrates under ongoing global change may so far have been underestimated by the existing literature.</p>
<p>Global change is increasingly impacting coastal marine systems. Organisms inhabiting the intertidal zone may be especially vulnerable to additional anthropogenic influences, which augment the naturally stressful, highly variable conditions to which they are already subjected and may lead to the manifestation of artificially severe carry-over effects (COEs). In marine invertebrates with complex life histories, COEs can occur between life stages, when the conditions experienced by one stage influence the characteristics or performance of the next, as well as trans-generationally, in which case the environment experienced by a parental generation affects offspring. Most of the existing literature surrounding COEs focuses only on those between life stages or generations, seldom both simultaneously, and do so with the implementation of only a single stressor. In nature however, organisms may be affected by both forms of COE, since the presence of one does not preclude the other, and are invariably subjected to multiple co-occurring stressors that can interact in complex ways. Consequently, how trans-generational COEs might impact the propagation of stress through offspring life stages remains unclear, and how these processes operate in a global change context is little understood. It was here aimed to elucidate the role of COEs under ongoing global change by addressing these common literature imitations and taking the novel approach of examining how the effects of multiple, global change-associated stressors carry-over from a parental generation through their offspring’s life stages in order to provide a more realistic representation of the conditions under which COEs manifest in the field. This was done using Siphonaria australis, an intertidal pulmonate limpet that deposits benthic egg masses, from which hatch planktonic veliger larvae. Adult S. australis were subjected to one of four treatments for 4h/day over four weeks to induce trans-generational COEs: a no-stress control, a pollution treatment with added copper (5.0μg/L), a “climate change” treatment with elevated temperature (25°C) and UVR (1.7W/m2), and a full global change treatment incorporating all three stressors. At the end of this period, the egg masses laid under each of these adult treatments were subjected to further experimentation for two weeks by being redistributed among the same four treatments again, so as to produce 16 unique treatment histories of adult-to-egg mass stress. Of these, 11 provided successfully hatching larvae, which were reared and observed for COEs between life stages (from egg to larva) under ambient conditions (ie. no added stressors) for 27 days. In adult S. australis survivor size, the size of egg masses laid and the size of individual eggs varied in complex ways over time and across treatments, while the number of survivors was unaffected by stress. Egg masses were unaffected in terms of hatching time but displayed strong responses to parental and developmental stress exposure through hatching success, and the percentage of viable eggs per egg mass, with the latter clearly declining according to adult treatment severity and both showing trans-generational COEs. Larval characteristics were extremely varied across treatment histories and highly context-dependent as hatching size, size reached by 27 days, growth rate, and size at death all showed evidence of COEs between generations and life stages, as well as interaction between both types of COE, with the number of survivors again being the only unaffected response variable. Overall, trans-generational COEs were slightly more common than those between life stages. These results show that both forms of COE, each triggered by exposure to multiple stressors in progenitors and developmental stages, interact to form highly context-dependent legacies of mostly impaired performance in S. australis larvae. This implies that COEs may become more prominent with worsening stressors in the future and suggests that the role of COEs in the persistence of marine invertebrates under ongoing global change may so far have been underestimated by the existing literature.</p>
<p>Salinity, temperature and ultraviolet-B (UV-B) radiation are common environmental stressors in coastal habitats. These stressors are likely to increase in intensity due to the effects of climate change and can have important impacts on population and community dynamics for early development in gastropods that deposit egg masses on rocky shores. The aim of this study was to identify the effects of single and multiple stressors on the development of intertidal and shallow subtidal gastropods with encapsulated embryos. In manipulative experiments I exposed egg masses of the gastropod species Siphonaria australis, Ercolania felina, Pleurobranchaea maculata, Aplysia juliana and Doris wellingtonensis to realistic levels of either salinity, temperature or UV-B radiation, or to a combination of stressors, for different lengths of time. Embryos were then subjected to the most stressful levels of each stressor at either early or late stages of development and at different days of embryonic development. Further, egg masses were exposed to sublethal salinity, temperature and UV-B radiation stress simultaneously, simulating tide pool conditions on a warm sunny summer day. Larvae hatching from stressed and unstressed egg masses were subsequently periodically subjected to increased temperature and UV-B radiation and examined over 10 days to detect possible carry-over effects of exposure to stress in the egg mass. The results revealed that for individual stressors, low salinity (20‰), high temperature (25°C) and high UV-B (1.7 W m ⁻ ² s ⁻ ¹, i.e. a level similar to a sunny NZ summer day) all caused the highest embryonic mortality. The response to stressors was species-specific but overall the intertidal species had lower embryonic mortality than the subtidal species. Generally, chronic exposure had higher impacts on the development of embryos than periodic exposure and early embryonic development stages were most vulnerable to stress. UV-B radiation had particularly damaging effects on embryonic and larval stages for the intertidal pulmonate limpet Siphonaria australis. Further, multiple stressors had synergistic effects and caused high embryonic mortality in the egg mass as well as impacting on the vulnerability of larvae to stressors. This study revealed that stress experienced during embryonic stages can result in sub-lethal damage that increases vulnerability to temperature and decreases vulnerability to UV-B radiation experienced in the larval stage. In total, my results suggest that (1) the effects of different environmental stressors on early development of intertidal and subtidal gastropods are complex and depend on the intensity, duration and time of stress, and are generally species-specific; (2) multiple stressors can act synergistically to affect early development and (3) sublethal exposure to stress in the egg mass can have negative carry-over effects on later larval stages.</p>
<p>Salinity, temperature and ultraviolet-B (UV-B) radiation are common environmental stressors in coastal habitats. These stressors are likely to increase in intensity due to the effects of climate change and can have important impacts on population and community dynamics for early development in gastropods that deposit egg masses on rocky shores. The aim of this study was to identify the effects of single and multiple stressors on the development of intertidal and shallow subtidal gastropods with encapsulated embryos. In manipulative experiments I exposed egg masses of the gastropod species Siphonaria australis, Ercolania felina, Pleurobranchaea maculata, Aplysia juliana and Doris wellingtonensis to realistic levels of either salinity, temperature or UV-B radiation, or to a combination of stressors, for different lengths of time. Embryos were then subjected to the most stressful levels of each stressor at either early or late stages of development and at different days of embryonic development. Further, egg masses were exposed to sublethal salinity, temperature and UV-B radiation stress simultaneously, simulating tide pool conditions on a warm sunny summer day. Larvae hatching from stressed and unstressed egg masses were subsequently periodically subjected to increased temperature and UV-B radiation and examined over 10 days to detect possible carry-over effects of exposure to stress in the egg mass. The results revealed that for individual stressors, low salinity (20‰), high temperature (25°C) and high UV-B (1.7 W m ⁻ ² s ⁻ ¹, i.e. a level similar to a sunny NZ summer day) all caused the highest embryonic mortality. The response to stressors was species-specific but overall the intertidal species had lower embryonic mortality than the subtidal species. Generally, chronic exposure had higher impacts on the development of embryos than periodic exposure and early embryonic development stages were most vulnerable to stress. UV-B radiation had particularly damaging effects on embryonic and larval stages for the intertidal pulmonate limpet Siphonaria australis. Further, multiple stressors had synergistic effects and caused high embryonic mortality in the egg mass as well as impacting on the vulnerability of larvae to stressors. This study revealed that stress experienced during embryonic stages can result in sub-lethal damage that increases vulnerability to temperature and decreases vulnerability to UV-B radiation experienced in the larval stage. In total, my results suggest that (1) the effects of different environmental stressors on early development of intertidal and subtidal gastropods are complex and depend on the intensity, duration and time of stress, and are generally species-specific; (2) multiple stressors can act synergistically to affect early development and (3) sublethal exposure to stress in the egg mass can have negative carry-over effects on later larval stages.</p>
<p>Ozone depletion is a humaninduced global phenomenon that allows increased ultraviolet radiation (UVR) to the Earth's surface. Although UVR is known to be harmful, relatively little is known about how increased UVR impacts natural ecosystems. Ecosystems in New Zealand are particularly at risk, because ozone depletion is much greater here, with levels of biologically harmful UVR up to two times greater than in northern latitudes. In the intertidal environment, potentially negative abiotic stressors are associated with low tide; and organisms inhabiting this environment are particularly vulnerable to UVR. Furthermore, embryos and larvae deposited in this habitat are especially susceptible to these stressors. The aim of this study is to identify the effect of UVR and other environmental stressors on the development of mollusc embryos in New Zealand. Surveys of microhabitats in which egg mass deposition occurs, and what effect this site of deposition has on the survivorship of embryos, revealed that encapsulated embryos of the two common pulmonate limpets Benhamina obliquata and Siphonaria australis are highly vulnerable to the environmental stressors associated with different microhabitats. In particular, egg masses deposited in the sun for both species suffered high mortality. Although, B. obliquata is more susceptible to UVR damage than is S. australis, B. obliquata predominantly deposits egg masses in dry shaded microhabitats while S. australis deposits the majority of its offspring in sunny tidal pools, which surprisingly equated to highest embryonic mortality. Results of manipulative experiments reflected those found in the surveys: egg masses exposed to full spectrum light incurred the greatest embryonic mortality; additionally environmental stressors (e.g. tidal pool conditions and desiccation) synergistically enhanced this mortality. UVR in North America is significantly lower compared to New Zealand; this allowed a unique opportunity to use identical methods to examine the responses of ecologically similar, related species (bubble shell snails in the genus Haminoea), from two regions where UVR naturally differs. Results from surveys and manipulative experiments revealed that the New Zealand species Haminoea zelandiae suffered high embryonic mortality under full spectrum light and this mortality was enhanced by periods of desiccation. The North American species Haminoea vesicula also suffered significant mortality during periods of desiccation, but there were no signs of UVR damage. These results appear to be driven by speciesspecific vulnerability to these stressors and not to ambient UVR intensity in the regions at the time of study. Relative concentrations of the chemical sunscreen compounds, mycosporinelike amino acids (MAAs), varied depending on several factors, but the biggest differences were among species. Analyses revealed that B. obliquata had the highest concentration of MAAs despite suffering high embryonic mortality when exposed to direct sunlight. MAA concentrations in S. australis were intermediate, with H. zelandiae having the lowest concentrations of all three species. MAA concentration for B. obliquata was dependent on stage of development and initial sun exposure at egg mass deposition site, suggesting interactions between MAAs, environmental conditions and embryonic development that need to be further explored. MAA concentrations were higher in S.australis egg masses deposited in spring compared to those deposited in early autumn, which may be driven by a shift in diet or nutrient levels. MAA concentrations did not appear to be correlated with ambient levels of UVR or embryonic survival in S.australis. However, MAA concentrations were related to UV irradiance in both Haminoea species with higher MAA concentrations observed in egg masses initially deposited in the sun compared to those found in the shade. Combined these results suggest: (1) increased UVR due to ozone depletion together with increases in temperatures due to climate change are likely to have strong impacts on the early life stages of these species, unless behavioural and physiological adaptations occur; (2) New Zealand species may be at particularly high risk from UVR damage compared to those from the Northern hemisphere; (3) the role of MAAs as photo-protection in these mollusc species is likely to be species specific, with a variety of abiotic and biotic factors influencing their uptake and sequestration. These experiments in part demonstrate how New Zealand's mollusc species are responding to humaninduced changes in UVR levels.</p>
