Herbivorous insects frequently harbor bacterial symbionts that affect their ecol- ogy and evolution. Aphids host the obligatory endosymbiont Buchnera, which is requiredfor reproduction, together with facultative symbi...Herbivorous insects frequently harbor bacterial symbionts that affect their ecol- ogy and evolution. Aphids host the obligatory endosymbiont Buchnera, which is requiredfor reproduction, together with facultative symbionts whose frequencies vary across aphid populations. These maternally transmitted secondary symbionts have been particularlystudied in the pea aphid, Acyrthosiphon pisum, which harbors at least 8 distinct bacterial species （not counting Buchnera） having environmentally dependent effects on host fitness.In particular, these symbiont species are associated with pea aphid populations feeding on specific plants. Although they are maternally inherited, these bacteria are occasionallytransferred across insect lineages. One mechanism of such nonmaternal transfer is paternal transmission to the progeny during sexual reproduction. To date, transmission of secondarysymbionts during sexual reproduction of aphids has been investigated in only a handful of aphid lineages and 3 symbiont species. To better characterize this process, we investigatedinheritance patterns of 7 symbiont species during sexual reproduction of pea aphids through a crossing experiment involving 49 clones belonging to 9 host-specialized biotypes, and117 crosses. Symbiont species in the progeny were detected with diagnostic qualitative PCR at the fundatrix stage hatching from eggs and in later parthenogenetic generations.We found no confirmed case of paternal transmission of symbionts to the progeny, and we observed that maternal transmission of a particular symbiont species （Serratia symbiotica）was quite inefficient. We discuss these observations in respect to the ecology of the pea aphid.展开更多
RNA in situ hybridization （ISH）, including chromogenic ISH （CISH） and fluorescent ISH （FISH）, has become a powerful tool for revealing the spatial distribution ofgene transcripts in model organisms. Previously, ...RNA in situ hybridization （ISH）, including chromogenic ISH （CISH） and fluorescent ISH （FISH）, has become a powerful tool for revealing the spatial distribution ofgene transcripts in model organisms. Previously, we developed a robust protocol for wholemount RNA CISH in the pea aphid Acyrthosiphon pisum, an emerging insect genomicmodel. In order to improve the resolving capacity of gene detection, we comprehensively surveyed current protocols of whole-mount RNA-FISH and developed protocols that allow,using confocal microscopy, clearer visualization of target messenger RNAs （mRNAs） - including those subcellularly localized and those with spatially overlapping expression. Wefind that Fast dye-based substrate fluorescence （SF）, tyramide signal amplification （TSA）, and TSA Plus all enable identifying gene expression thanks to multiplex amplificationof fluorescent signals. By contrast, methods of direct fluorescence （DF） do not allow visualizing signals. Detection of a single gene target was achieved with SF and TSA Plusfor most mRNAs, whereas TSA only allowed visualization of abundant transcripts such as Apvasl andAppiwi2 in the germ cells. For detection of multiple gene targets using doubleFISH, we recommend： （i） TSA/TSA, rather than TSA Plus/TSA Plus for colocalized mRNAs abundantly expressed in germ ceils, as proteinase K treatment can be omitted;and （ii） SF/TSA Plus for other gene targets such as Apenl and Apen2 as inactivation of enzyme conjugates is not required. SF/SF is not ideal for double FISH experiments due tosignal blurring. Based on these new conditions for RNA-FISH, we have obtained a better understanding of germline specification and embryonic segmentation in the pea aphid.We anticipate that the RNA-FISH protocols for the pea aphid may also be used for other aphids and possibly other insect species, thus expanding the range of species from whichuseful insights into development and evolution may be obtained.展开更多
文摘Herbivorous insects frequently harbor bacterial symbionts that affect their ecol- ogy and evolution. Aphids host the obligatory endosymbiont Buchnera, which is requiredfor reproduction, together with facultative symbionts whose frequencies vary across aphid populations. These maternally transmitted secondary symbionts have been particularlystudied in the pea aphid, Acyrthosiphon pisum, which harbors at least 8 distinct bacterial species （not counting Buchnera） having environmentally dependent effects on host fitness.In particular, these symbiont species are associated with pea aphid populations feeding on specific plants. Although they are maternally inherited, these bacteria are occasionallytransferred across insect lineages. One mechanism of such nonmaternal transfer is paternal transmission to the progeny during sexual reproduction. To date, transmission of secondarysymbionts during sexual reproduction of aphids has been investigated in only a handful of aphid lineages and 3 symbiont species. To better characterize this process, we investigatedinheritance patterns of 7 symbiont species during sexual reproduction of pea aphids through a crossing experiment involving 49 clones belonging to 9 host-specialized biotypes, and117 crosses. Symbiont species in the progeny were detected with diagnostic qualitative PCR at the fundatrix stage hatching from eggs and in later parthenogenetic generations.We found no confirmed case of paternal transmission of symbionts to the progeny, and we observed that maternal transmission of a particular symbiont species （Serratia symbiotica）was quite inefficient. We discuss these observations in respect to the ecology of the pea aphid.
文摘RNA in situ hybridization （ISH）, including chromogenic ISH （CISH） and fluorescent ISH （FISH）, has become a powerful tool for revealing the spatial distribution ofgene transcripts in model organisms. Previously, we developed a robust protocol for wholemount RNA CISH in the pea aphid Acyrthosiphon pisum, an emerging insect genomicmodel. In order to improve the resolving capacity of gene detection, we comprehensively surveyed current protocols of whole-mount RNA-FISH and developed protocols that allow,using confocal microscopy, clearer visualization of target messenger RNAs （mRNAs） - including those subcellularly localized and those with spatially overlapping expression. Wefind that Fast dye-based substrate fluorescence （SF）, tyramide signal amplification （TSA）, and TSA Plus all enable identifying gene expression thanks to multiplex amplificationof fluorescent signals. By contrast, methods of direct fluorescence （DF） do not allow visualizing signals. Detection of a single gene target was achieved with SF and TSA Plusfor most mRNAs, whereas TSA only allowed visualization of abundant transcripts such as Apvasl andAppiwi2 in the germ cells. For detection of multiple gene targets using doubleFISH, we recommend： （i） TSA/TSA, rather than TSA Plus/TSA Plus for colocalized mRNAs abundantly expressed in germ ceils, as proteinase K treatment can be omitted;and （ii） SF/TSA Plus for other gene targets such as Apenl and Apen2 as inactivation of enzyme conjugates is not required. SF/SF is not ideal for double FISH experiments due tosignal blurring. Based on these new conditions for RNA-FISH, we have obtained a better understanding of germline specification and embryonic segmentation in the pea aphid.We anticipate that the RNA-FISH protocols for the pea aphid may also be used for other aphids and possibly other insect species, thus expanding the range of species from whichuseful insights into development and evolution may be obtained.
