- What is Holometabolous Metamorphosis in Insects?
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- Difference Between Holometabolous and Hemimetabolous Metamorphosis in Insects
- Metamorphosis: a remarkable change
- What is Hemimetabolous Metamorphosis in Insects?
Insect metamorphosis is regulated by ecdysteroids, which induce molts, and juvenile hormone JH , which inhibits metamorphic changes. The molecular action of ecdysteroids has been thoroughly studied, but that of JH is poorly understood, with data currently only being available for holometabolous species, like Drosophila melanogaster and Tribolium castaneum. Kr-h1 is a Zn finger transcription factor whose function as transductor of the antimetamorphic action of JH has recently been demonstrated in D.
Insect metamorphosis has fascinated mankind since the time of Aristotle, some two thousand years ago. Much later, Renaissance entomologists established that post-embryonic changes are most spectacular in insects like beetles, moths and flies, which undergo a dramatic morphological transformation from larva to pupa and adult, a phenomenon nowadays known as holometaboly, which is typical in endopterygote species.
Exopterygote insects, such as locusts and cockroaches, also transform from last nymphal instar to adult, although the transformation is not as radical as the nymphs are relatively similar to the adult stage. However, they undergo qualitative metamorphic changes, such as formation of mature wings and external genitalia, amongst others, in a type of metamorphosis known as hemimetaboly 1 , 2 , 3.
Understanding insect metamorphosis at molecular level is still a challenging mystery because we still only have a few pieces of the puzzle. From an endocrine point of view, metamorphosis is regulated by two kinds of hormones, namely molting hormone, which induces molts, and juvenile hormone JH , which modulates the quality of the molt: to an immature stage when it is present, and to the adult when it is absent; JH therefore plays a crucial repressive role in insect metamorphosis 1 , 4 , 5.
The effect of the commonest molting hormone, namely hydroxyecdysone, is mediated by a cascade of transcription factors and starts upon its binding to the heterodimeric receptor composed by the ecdysone receptor and the ultraspiracle or RXR , both of which belong to the nuclear receptor superfamily.
This activates expression of a hierarchy of transcription factors, such as E75, E78, HR3, HR4 and FTZ-F1, which regulate the genes that underlie the cellular changes associated with molting and metamorphosis 6 , 7.
Most of the reported data on this cascade of transcription factors refer to Drosophila melanogaster , the fruit fly, a holometabolous insect that shows many highly derived characters, which has been the most thoroughly studied species from the point of view of molecular endocrinology 8 , 9.
What is Holometabolous Metamorphosis in Insects?
From this point of view, the most widely studied hemimetabolous model is the German cockroach Blattella germanica , and results indicate that the transcription factors involved in hydroxyecdysone signalling are generally conserved, although the functions of some of them and the precise epistatic relationships between them may differ with respect to D.
Conversely, the molecular mechanisms underlying the action of JH are poorly understood, and our current understanding relies completely on holometabolous models 4. This and other characteristics 4 , 16 suggest that Met plays the role of JH receptor, or is a component of a heterodimeric JH receptor.
Key functional evidence that Met is required for the repressor action of JH on metamorphosis was not obtained in D. Ectopic application of JH prior to the prepupal stage prevents the normal differentiation of the abdominal epidermis, and the bristles that normally occur in the dorsal midline of the adult fly become shorter or simply do not form 24 , Moreover, ectopic expression of Kr-h1 in the abdominal epidermis during metamorphosis causes missing or short dorsal midline bristles, just as in the experiments with JH treatment, thereby suggesting that, in D.
As all functional data on the signalling pathway of JH have been obtained in holometabolous models, research in hemimetabolous species is needed if we aim at elucidating the evolution of insect metamorphosis. In light of this, we studied the function of Kr-h1 in B. Methodologically, our strategy was to knockdown Kr-h1 by RNAi, a technique that has been shown to be highly effective for silencing gene expression in B.
The alignment of the Kr-h1 protein sequences Supplementary Fig. To gain a first insight into the possible involvement of BgKr-h1 in cockroach metamorphosis, we studied its expression pattern in the whole body of females during the three last nymphal instars.
The results Fig. Moreover, expression is not confined to a single or few tissue types, but is ubiquitously distributed amongst practically all tissues, especially the muscle, epidermis pronotum, mesonotum and metanotum samples and ovaries Fig. The results obtained indicate that JH up-regulates BgKr-h1 expression and that the stimulatory effect lasts practically the entire sixth instar nymph, although with decreasing intensity Fig.
A number of JH-treated specimens were left alive until the next molt, and they molted into adultoids with nymphal features Supplementary Fig. Data in b represent a pool of 5 specimens. We approached the study of BgKr-h1 function in B.
Controls received the same dose of unspecific dsRNA dsMock.
These precocious adults lived much longer than the nymph-adult intermediates between 2 and 3 months, as average and did not molt again. Controls were equivalently treated with dsMock. None of these precocious adults molted again.
Hemimetabolous and holometabolous metamorphosis book
In order to test whether precocious metamorphosis could also be provoked in younger instars, we carried out experiments equivalent to those just described but using fourth instar female nymphs. The length of the fourth instar was the same ca. However, the length of the fifth instar in the dsKr-h1-treated specimens was almost twice that for the controls Fig. These six intermediates died between 8 and 10 days after the molt.
After the next molt, all dsKr-h1-treated specimens became precocious adults with imperfectly extended wings Fig. Controls molted to normal sixth instar nymphs.
A few male nymphs, all of which yielded precocious adults after two molts, were inadvertently included in the experiments involving injecting a single dose of dsKr-h1 into fourth instar female nymphs.
This prompted us to test whether males could be more sensitive than females to the silencing effects of RNAi on BgKr-h1.
First, we obtained data regarding BgKr-h1 mRNA levels on selected days of the penultimate and last nymphal instars in males. Our determinations indicated that the expression pattern Fig.
Moreover, the fourth instar had the same length in dsKr-h1-treated and in control males, and all specimens molted normally to the fifth instar, whereas the length of the latter in dsKr-h1-treated males was practically twice that of the controls Fig.
Of note, the posterior margin of tergite 7 was somewhat shorter and notched in these precocious adults, which allowed seeing directly the paired glands of the tergite 8, whereas they are partially hidden by the well developed tergite 7 in normal adults Fig.
Male precocious adults resulting from dsKr-h1 treatments did not molt again. Finally, we carried the same experiments but treating freshly emerged fifth instar male nymphs.
Considering all sequences included in the alignment Supplementary Fig. The similarity is particularly high in the Zn finger domain, where the most complex binding capacity of the molecule resides. The high degree of conservation found in the C 2 H 2 zinc finger domain in all studied species suggests that the Kr-h1 function might have been generally conserved across the insect class.
The discovery of a Kr-h1 homolog in the crustacean D. Our mRNA determinations showed that BgKr-h1 is ubiquitously expressed in different tissues, whereas time-course studies indicated that expression vanishes after the first day of the last instar nymph. Similar decreases of Kr-h1 expression occur between the prepupal and pupal stages in holometabolous models, such as the fly D.
The coincidence of the patterns of BgKr-h1 and that of circulating JH which decreases during the first days of the last nymphal instar 28 in the cockroach B. This hypothesis was assessed in experiments that showed that treatment of last instar nymphs with JH readily induced the re-expression of BgKr-h1.
Difference Between Holometabolous and Hemimetabolous Metamorphosis in Insects
Early works on D. However, more recent work revealed that Kr-h1 expression is induced by JH in the fly D. Of note, when the RNAi was carried out in the fourth nymphal instar, two molts were needed before the occurrence of precocious metamorphosis. Pioneering experiments have shown that metamorphosis can be precociously induced by dissecting out the corpora allata, i.
However, when such allatectomy is performed in very young larvae, one or two additional molts are currently needed before precocious adult features appear More recent studies have shown that depletion of JH through overexpression of JH esterase fails to cause premature pupation in larvae of the lepidopteran Bombyx mori if they are younger than third instar These observations, and those described herein in B. Connected with this issue is the observation showing that the stage previous to precocious metamorphosis in dsKr-h1-treated specimens, i.
Again, this delay could be necessary to reach a minimal critical weight or perhaps more plausibly, given that there are no delays in the fourth nymphal instar to unfold the developmental transition between the nymph and the adult.
RNAi was found to be more efficient in males than in females of B. The experiments with males made clearer that there is a rough correlation between the percentage of transcript decrease after dsKr-h1 treatment and that of precocious adults obtained in the experiment. According to the functional studies performed on D. This suggests that the repressor role of Kr-h1 on metamorphosis is an ancestral condition that has been conserved from hemimetabolous to holometabolous species. Thus, the ancestral role of Kr-h1 can be a useful starting point to study the mechanisms underlying the evolutionary transition from hemimetaboly to holometaboly, which still remain a challenging enigma.
The specimens of B. They were anaesthetized with carbon dioxide prior to injection treatments, dissections and tissue sampling.
The B. A template-free control was included in all batches. The efficiency of this primer set was first validated by constructing a standard curve through four serial dilutions. The detailed procedures for RNAi experiments were as described previously A dsRNA encompassing a bp fragment located between nucleotides and dsKr-h1 , in the Zn finger domain, was designed.
Metamorphosis: a remarkable change
The dsRNAs were prepared as reported previously Control specimens were treated with the same dose and volume of dsMock. Finally, the protein sequences included in the analysis were the following GenBank accession number and annotation details, if needed, in parenthesis. Both authors discussed the results, decided about subsequent experiments and commented on the manuscript. National Center for Biotechnology Information , U.
Sci Rep. Published online Nov Jesus Lozano 1 and Xavier Belles a, 1. Author information Article notes Copyright and License information Disclaimer. Received Sep 19; Accepted Nov 8.
What is Hemimetabolous Metamorphosis in Insects?
All rights reserved. This article has been cited by other articles in PMC. Abstract Insect metamorphosis is regulated by ecdysteroids, which induce molts, and juvenile hormone JH , which inhibits metamorphic changes. Open in a separate window.
Figure 1. Figure 2. Effects of BgKr-h1 depletion in fifth nymphal instar N5 of Blattella germanica females. RNAi in fourth instar female nymphs results in precocious metamorphosis after two molts In order to test whether precocious metamorphosis could also be provoked in younger instars, we carried out experiments equivalent to those just described but using fourth instar female nymphs.
Figure 3. Effects of BgKr-h1 depletion in fourth nymphal instar N4 of Blattella germanica females. Figure 4. Expression of BgKr-h1 and effects of its depletion in Blattella germanica males. Methods Insects The specimens of B.