The function and genetic specification of a neglected secretory organ in insects and springtails

Lead Research Organisation: University of Cambridge
Department Name: Zoology


Work over the last century has defined many aspects of insect physiology. This work has served both to exemplify and expand our understanding of general physiological principles, and to document aspects of biology unique to the insects that have then been exploited to control their reproduction and behavior. This proposal focuses on an aspect of basic insect physiology that has largely escaped attention.

Most insects develop, in the embryo, appendages that differentiate uniquely as organs known as pleuropods, with the appearance of secretory or excretory structures. Old experiments from more than 40 years ago suggest that these organs are essential for the insects to hatch, but it is not clear whether they secrete a hatching enzyme, a hormone that controls the secretion of a hatching enzyme from other tissues, or have some other more general function concerning the regulation of fluid and salt balance. Because insects no longer have these organs after hatching, and because the most intensively studied species, Drosophila, belongs to a group of insects that no longer develops pleuropods, they have largely escaped the attention of insect biologists.

Developments in genetic technology now make it to possible to study the function of pleuropods in new ways. We have used large scale sequencing of dissected pleuropods from locusts to identify the complete set of genes that these unique organs express. By comparing these genes with those expressed in other organs we will gain insight into the likely physiological role of pleuropods. We will eliminate the function of likely key genes to test these hypotheses. We will also eliminate the development of pleuropods altogether by inactivating the "master control gene" that specifies them, and examine the effect on the physiology of the resulting embryos.

We shall also carry out similar experiments on collembolan hexapods, representatives of an important group of soil organisms. These close relatives of the insects, colloquially known as springtails, retain throughout life a major organ, the ventral tube, that develops from the same embryonic structures as the insect pleuropods. These organs are known to be important for water and salt balance in springtails, many of which are very sensitive to the humidity of the environment. We have developed a species of collembolan as a laboratory model for gene manipulation, and have shown that we can generate collembolans that lack ventral tubes. These die a short while after hatching. We have generated a list of genes expressed in this species, and will test whether the genes expressed in the ventral tube are related to those expressed in the insect pleuropods, and so assess whether the physiology of the ventral tubes is related to that of pleuropods. We will study the effect on the organisms of removing ventral tube function.

Finally, the "master control gene" that specifies pleuropods and ventral tubes is much better known for other well studied roles during development. It is of fundamental interest to understand how this one gene, called Ultrabithorax, can do such different things as transform the patterning of wings and legs in the thorax, and specify a secretory/excretory organ in another part of the body. We shall investigate how the different roles of this gene depend on the timing, amount and precise structure of the products that it makes, and on the combination of other genes with which it works.

Technical Summary

Most insects develop a pair of specialised organs during embryogenesis, the pleuropods, which appear to be active in secretion or excretion. In grasshoppers pleuropods have been shown to be necessary for digesting the serosal cuticle to allow hatching, but how in molecular terms they function is not known.

Pleuropods are the serial homologues of legs, developing from the ventral appendage buds of the first abdominal segment. In Collembolans, the appendages of the first abdominal segment develop into the ventral tube, a structure that persists throughout life and is known to be important for osmoregulation. We propose that the collembolan ventral tube and insect pleuropod are homologous organs.

We have generated a transcriptome sequence from dissected pleuropods of locusts, and for comparison from embryonic third legs dissected at the same stage. We have also generated an embryonic transcriptome from a collembolan.

We will identify genes expressed specifically in pleuropods, and assess to what extent the genes expressed support existing hypotheses of pleuropod function (e.g. secretion of hatching enzyme, endocrine function, osmoregulatory role). We will test whether genes expressed specifically in insect pleuropods are also expressed in the collembolan ventral tube.

We will generate embryos that lack pleuropods (surgically, and by knock down of Ubx gene function), and investigate what defects they develop. We will determine the effect of knocking down specific pleuropod expressed genes, and compare this with the effect of removing pleuropods entirely. We shall also seek to establish simple physiological assays for pleuropod function to test for a secretory or osmoregulatory role.

Pleuropods and the ventral tube are specified by the Hox gene Ubx. This role of Ubx has never been studied. Using the beetle Tribolium, we will investigate why Ubx specifies pleuropods in A1, but modified wings and legs in the thorax.

Planned Impact

Insects are the most successful and biodiverse group of animals on earth. They have major economic impact on man through their effects on agriculture and food storage, and they are among the most significant of disease vectors. The practical economic value of an understanding of insect physiology is very clear from the history of work on insect hormones, leading to hormone analogue insecticides, and more recent work, for example on insect olfaction leading to an understanding of insect repellent action.

We cannot predict a direct impact of our work on applied insect biology, but we believe a strong case can be made that this proposal addresses an area of insect physiology that has been neglected unjustifiably. For comparison, a large amount of work on the post embryonic excretory organs of insects, the Malpighian tubules, has not only provided important insight into excretory processes generally, but has also been essential for understanding specific adaptation of insects to biological niches of major economic importance, such as that of plant sap feeding aphids.

By choosing to work initially on the African plague locust as a model for our transcriptome work, and for physiological assays on pleuropod function, we ensure that any functional insight will be directly relevant to potential strategies for locust control. One process that is affected by pleuropod function in these eggs - the digestion of the serosal (egg) cuticle - is specific to lower insect groups. Such a cuticle is not found in the eggs of many insects of economic value (flies, bees, butterflies). It is therefore likely that blocking this function would have rather specific insecticidal effects.

Springtails (collembolans) are abundant components of the soil fauna, likely to play a major role in nutrient recycling. They are also valuable as toxicological indicators (see for example Van Straalen, N.M. & Feder, M.E. (2012). Environmental Science & Technology 46: 3-9.) Until recently, the amount of molecular physiological and genetic work on collembolans has been very limited, not least because of the lack of well established laboratory models for this sort of work, and the lack of basic information about such understudied groups. Technological advance has radically changed this situation - it is now realistic to consider genome and/or transcriptome sequencing as a first step to studying any new organism, and these data are now available for our chosen model species. At the same time, it is essential to have a study species that is amenable to convenient laboratory culture and gene manipulation. Our demonstration that the springtail Orchesella cincta can be reared conveniently on a laboratory grown diet, that embryos can be prepared for in situ examination of gene expression, and that gene knockdown by RNAi works well, has already attracted attention from the ecotoxicology community. We believe that O. cincta is now a species of choice for molecular work. We anticipate that our work on this species will lead to further "technology transfer", helping to bring techniques now well established among developmental geneticists to a wider community of biologists with diverse interests, including in applied fields.

From existing work it seems that the organ we propose to study in springtails - the ventral tube - is a key component of the osmotic and excretory regulatory systems that adapt them to life in the soil. As this organ is not found (at least postembryonically) in the major groups of well studied insects, we remain almost completely ignorant of how it functions. Information on the physiological mechanisms and molecular processes utilised by the ventral tube must be a prerequisite for any understanding of how increasing salinity, reduced humidity or toxic contamination may affect these key components of the soil ecosystem. Our work represents a step in that direction.
Description We have developed tools to manipulate gene expression in Collembolan hexapods, and studied the role of genes controlling the specification of different segments in the abdomen of these insects.

We have sequenced the embryonic transciptome of the African plague locust, Schistocerca gregaria, which provides a new resource for those wishing to work on any aspect of locust physiology or development.

We have collected data on gene expression from the pleuropods and limb buds of locusts, by next generation sequencing of samples from nine timepoints from the early development of these organs until their final differentaition and degeneration.

We have assembled all of this data and generated a reference set of transcripts that aim to represent each gene just once.

We carried out an initial analysis of these transcriptome data, to determine those genes that are overexpressed during pleuropod development. This analysis provided evidence that the active pleuropodial cells express multiple genes for proteins known to be important for digestion of insect cuticle. This provides genetic support for the hypothesis previously proposed by Slifer (1937) that the pleuropodia secrete a substance dissolving the serosal cuticle (egg envelope) to enable the larva to hatch. The data also provide some evidence that the pleuropods function in osmoregulation, a hypothesis that was based on electron microscopical observations carried out before as well as during our own study. Additionally, we found that the pleuropods are enriched in genes for some proteins involved in immune defence, including lysozymes. This work is nopw published
We have carried out an electron microscopy study of pleuropod development, determining when the pleuropods differentiate into organs ressembling transport epithelia, and showing that they appear to be active as secretory organs from mid embryogenesis onwards. This work is now published.
We have sequenced RNA samples from dissected ventral tubes of the springtail Orchesella in order to identify the genes specifically expressed in these organs; we also sequenced RNA samples from whole bodies and legs as controls. Initial analysis of these samples was unsatisfactory, but
re-analysis using a better assembly of the transcriptome revealed that the main function of the collophore is most likely water and ion transport. We identified specific genes for transporters functioning in the organs, including Na ,K ATPase, V-ATPase, NKA, ion channels, water channels etc.). qPCR analysis on selected genes supports the role of ventral tube in active transport (e.g., upregulation of carbonic anhydrases and Na+,K+ATPase).
Exploitation Route The Schistocerca whole embryo transcriptome and appendage time series that we have generated will be valuable resources for all those working with this locust pest, because there are currently no public genome or comprehensive transcriptome data for this species.
The datasets generated and analysed during the study are available in the NCBI repository: BioProject ID PRJNA524786 (the reference transcriptome 1009
described in this paper is the first version, GHHP01000000) and GSE128394.
Sectors Agriculture, Food and Drink,Environment

Title Developmental transcriptome for locust embryonic development 
Description Staged RNA samples have been collected and sequenced for timepoints throughout embryogenesis for samples of whole locust embryos (Schistocerca gregaria), pleuropods and dissected limb buds. While the pleuropod samples will be of interest primarily for our research, the whole embryo samples and the limb bud samples that serve as our controls will be valuable for other researchers. The samples have been analysed to assemble a reference transcriptome (Unigene set) 
Type Of Material Database/Collection of data 
Year Produced 2019 
Provided To Others? Yes  
Impact The locust transcriptomne is available on the NCBI repository (BioProject PRJNA524786; BBSRC is acknowledged in the project). 
Title RNAseq analysis for the Collembolan Orchesella cincta 
Description RNA samples from whole bodies, legs and ventral tubes were collected and sequenced. Our preliminary expression analysis (provided by our collaborating lab in Amsterdam) did not yield satisfactory results, likely because the transcriptome to which the reads were mapped was poorly assembled. Subsequent to the end date of the grant, Barbora Konopova established a new collaboration with bioinformatician Montserrat Torres Oliva (at IKMB, Kiel, Germany), who re-mapped the reads onto a new version of the Orchesella transcriptome. This provided more satisfactory results, confirming the quality of our samples and the sequencing process. BK is applying for further funds to map our samples onto the full genome (now available) , and to validate them by PCR. 
Type Of Material Database/Collection of data 
Year Produced 2018 
Provided To Others? No  
Impact Pubic release and any subsequent Impact is not expected until the re-analysis is completed 
Description Collaboration with Montserrat Torres Oliva, IKMB, Kiel, Germany 
Organisation University of Kiel
Country Germany 
Sector Academic/University 
PI Contribution We provided transcriptome samples from the springtail Orchesella cincta
Collaborator Contribution Dr Torres Oliva provided bioinformatics expertise to reanalyse these samples
Impact None yet
Start Year 2018
Description Vrije Universiteit Amsterdam 
Organisation Free University of Amsterdam
Country Netherlands 
Sector Academic/University 
PI Contribution Training in techniques to manipulate collembolan embryos and to perform RNAi. Transcriptome data for collembolan embryos
Collaborator Contribution Genome sequence data for the collembolan Orchesella cincta Information on culture and laboratory maintenance of collembolan species
Impact None
Start Year 2012
Description "Nature of Life" lecture, Amsterdam 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact broad lecture for the students and researchers at the Free University of Amsterdam to introduce them developmental genetics and our research.
Year(s) Of Engagement Activity 2014
Description Seminar by researcher co-investigator B. Konopova at the Department of Ecological Science, Vrije University of Amsterdam, Amsterdam, NL. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Talk entitled "Pleuropods of insect embryos and abdominal appendages of springtails" for ecology researchers and students to introduce them to our genetic- and development-focused project.
Year(s) Of Engagement Activity 2015
Description Talk by researcher co-investigator Barbora Konopova at the University of Roehampton, London, UK. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact Talk for researchers specialising on springtail ecology to introduce them to genetic manipulation in the springtail Orchesella
Year(s) Of Engagement Activity 2013