A mismatch in the expression of cell surface molecules induces tissue-intrinsic defense against aberrant cells.

Tissue-intrinsic error correction enables epithelial cells to detect abnormal neighboring cells and facilitate their removal from the tissue. One of these pathways, "interface surveillance," is triggered by cells with aberrant developmental and cell-fate-patterning pathways. It remains unknown which molecular mechanisms provide cells with the ability to compare fate between neighboring cells. We demonstrate that Drosophila imaginal discs express an array of cell surface molecules previously implicated in neuronal axon guidance processes. They include members of the Robo, Teneurin, Ephrin, Toll-like, or atypical cadherin families. Importantly, a mismatch in expression levels of these cell surface molecules between adjacent cells is sufficient to induce interface surveillance, indicating that differences in expression levels between neighboring cells, rather than their absolute expression levels, are crucial. Specifically, a mismatch in Robo2 and Robo3, but not Robo1, induces enrichment of actin, myosin II, and Ena/Vasp, as well as activation of JNK and apoptosis at clonal interfaces. Moreover, Robo2 can induce interface surveillance independently of its cytosolic domain and without the need for the Robo-ligand Slit. The expression of Robo2 and other cell surface molecules, such as Teneurins or the Ephrin receptor is regulated by fate-patterning pathways intrinsic and extrinsic to the wing disc, as well as by expression of oncogenic RasV12. Combined, we demonstrate that neighboring cells respond to a mismatch in surface code patterns mediated by specific transmembrane proteins and reveal a novel function for these cell surface proteins in cell fate recognition and removal of aberrant cells during development and homeostasis of epithelial tissues.

The anti-aging effect of vitamin D and vitamin D receptor in Drosophila midgut.

Adult stem cells are pivotal for maintaining tissue homeostasis, and their functional decline is linked to aging and its associated diseases, influenced by the niche cells' environment. Age- and cancer-related reduction of vitamin D and its receptor levels are well documented in human clinical studies. However, the mechanisms through which the vitamin D/vitamin D receptor pathway contributes to anti-aging and extends life expectancy are not well understood. In this study, we aimed to determine the protective role of the vitamin D/vitamin D receptor pathway in differentiated enterocytes (ECs) during intestinal stem cell (ISC) aging. By utilizing a well- established Drosophila midgut model for stem cell aging biology, we revealed that vitamin D receptor knockdown in ECs induced ISC proliferation, EC death, ISC aging, and enteroendocrine cell differentiation. Additionally, age- and oxidative stress-induced increases in ISC proliferation and centrosome amplification were reduced by vitamin D treatment. Our findings suggest a direct evidence of the anti-aging role of the vitamin D/vitamin D receptor pathway and provides insights into the molecular mechanisms underlying healthy aging in Drosophila.

Continuous, long-term crawling behavior characterized by a robotic transport system.

Detailed descriptions of behavior provide critical insight into the structure and function of nervous systems. In Drosophila larvae and many other systems, short behavioral experiments have been successful in characterizing rapid responses to a range of stimuli at the population level. However, the lack of long-term continuous observation makes it difficult to dissect comprehensive behavioral dynamics of individual animals and how behavior (and therefore the nervous system) develops over time. To allow for long-term continuous observations in individual fly larvae, we have engineered a robotic instrument that automatically tracks and transports larvae throughout an arena. The flexibility and reliability of its design enables controlled stimulus delivery and continuous measurement over developmental time scales, yielding an unprecedented level of detailed locomotion data. We utilize the new system's capabilities to perform continuous observation of exploratory search behavior over a duration of 6 hr with and without a thermal gradient present, and in a single larva for over 30 hr. Long-term free-roaming behavior and analogous short-term experiments show similar dynamics that take place at the beginning of each experiment. Finally, characterization of larval thermotaxis in individuals reveals a bimodal distribution in navigation efficiency, identifying distinct phenotypes that are obfuscated when only analyzing population averages.

Pigment-dispersing factor is present in circadian clock neurons of pea aphids and may mediate photoperiodic signalling to insulin-producing cells.

The neuropeptide pigment-dispersing factor (PDF) plays a pivotal role in the circadian clock of most Ecdysozoa and is additionally involved in the timing of seasonal responses of several photoperiodic species. The pea aphid, Acyrthosiphon pisum, is a paradigmatic photoperiodic species with an annual life cycle tightly coupled to the seasonal changes in day length. Nevertheless, PDF could not be identified in A. pisum so far. In the present study, we identified a PDF-coding gene that has undergone significant changes in the otherwise highly conserved insect C-terminal amino acid sequence. A newly generated aphid-specific PDF antibody stained four neurons in each hemisphere of the aphid brain that co-express the clock protein Period and have projections to the pars lateralis that are highly plastic and change their appearance in a daily and seasonal manner, resembling those of the fruit fly PDF neurons. Most intriguingly, the PDF terminals overlap with dendrites of the insulin-like peptide (ILP) positive neurosecretory cells in the pars intercerebralis and with putative terminals of Cryptochrome (CRY) positive clock neurons. Since ILP has been previously shown to be crucial for seasonal adaptations and CRY might serve as a circadian photoreceptor vital for measuring day length, our results suggest that PDF plays a critical role in aphid seasonal timing.

On the origin of appetite: GLWamide in jellyfish represents an ancestral satiety neuropeptide.

Food intake is regulated by internal state. This function is mediated by hormones and neuropeptides, which are best characterized in popular model species. However, the evolutionary origins of such feeding-regulating neuropeptides are poorly understood. We used the jellyfish Cladonema to address this question. Our combined transcriptomic, behavioral, and anatomical approaches identified GLWamide as a feeding-suppressing peptide that selectively inhibits tentacle contraction in this jellyfish. In the fruit fly Drosophila, myoinhibitory peptide (MIP) is a related satiety peptide. Surprisingly, we found that GLWamide and MIP were fully interchangeable in these evolutionarily distant species for feeding suppression. Our results suggest that the satiety signaling systems of diverse animals share an ancient origin.

Resveratrol and Sir2 Reverse Sleep and Memory Defects Induced by Amyloid Precursor Protein.

Resveratrol (RES), a natural polyphenolic phytochemical, has been suggested as a putative anti-aging molecule for the prevention and treatment of Alzheimer's disease (AD) by the activation of sirtuin 1 (Sirt1/Sir2). In this study, we tested the effects of RES and Sirt1/Sir2 on sleep and courtship memory in a Drosophila model by overexpression of amyloid precursor protein (APP), whose duplications and mutations cause familial AD. We found a mild but significant transcriptional increase of Drosophila Sir2 (dSir2) by RES supplementation for up to 17 days in APP flies, but not for 7 days. RES and dSir2 almost completely reversed the sleep and memory deficits in APP flies. We further demonstrated that dSir2 acts as a sleep promotor in Drosophila neurons. Interestingly, RES increased sleep in the absence of dSir2 in dSir2-null mutants, and RES further enhanced sleep when dSir2 was either overexpressed or knocked down in APP flies. Finally, we showed that Aß aggregates in APP flies were reduced by RES and dSir2, probably via inhibiting Drosophila ß-secretase (dBACE). Our data suggest that RES rescues the APP-induced behavioral deficits and Aß burden largely, but not exclusively, via dSir2.

Nanobodies combined with DNA-PAINT super-resolution reveal a staggered titin nanoarchitecture in flight muscles.

Sarcomeres are the force-producing units of all striated muscles. Their nanoarchitecture critically depends on the large titin protein, which in vertebrates spans from the sarcomeric Z-disc to the M-band and hence links actin and myosin filaments stably together. This ensures sarcomeric integrity and determines the length of vertebrate sarcomeres. However, the instructive role of titins for sarcomeric architecture outside of vertebrates is not as well understood. Here, we used a series of nanobodies, the Drosophila titin nanobody toolbox, recognising specific domains of the two Drosophila titin homologs Sallimus and Projectin to determine their precise location in intact flight muscles. By combining nanobodies with DNA-PAINT super-resolution microscopy, we found that, similar to vertebrate titin, Sallimus bridges across the flight muscle I-band, whereas Projectin is located at the beginning of the A-band. Interestingly, the ends of both proteins overlap at the I-band/A-band border, revealing a staggered organisation of the two Drosophila titin homologs. This architecture may help to stably anchor Sallimus at the myosin filament and hence ensure efficient force transduction during flight.

From ants to humans, the muscles that set an organism in motion are formed of bundles of fiber-like cells which can shorten and lengthen at will. At the microscopic level, changes in muscle cell lengths are underpinned by contractile filaments formed of multiple repeats of a basic unit, known as the sarcomere. Each unit is bookended by intricate 'Z-discs' and features an 'M-band' in its center. Three protein types give a sarcomere its ability to shorten and expand at will: two types of filaments (myosin and actin), which can slide on one another; and a spring-like molecule known as titin, which ensures that the unit does not fall apart by mechanically connecting myosin and actin. More specifically, actin filaments are anchored to the Z-discs and extend towards the M-band, while myosin filaments are centered around the M-band and extend towards the Z-discs. As myosin and actin slide alongside each other, the overlap between the two types of filaments increases or decreases and the whole unit changes its length. In vertebrates, one gigantic molecule of titin spans from the Z-disc to the M-band, linking together actin and myosin filaments and determining the length of the sarcomere. In insects and other invertebrates, however, this single molecule is replaced by two titin proteins known as Projectin and Sallimus. Understanding how these titins work together remains unclear and difficult to study. Traditional approaches are unable to precisely label titin in an environment teaming with other molecules, and they cannot offer the nanometer resolution required to dissect sarcomere organization. As a response, Schueder, Mangeol et al. combined super-resolution microscopy and a new toolbox of labelling molecules known as nanobodies to track the position of Sallimus and Projectin in the flight muscles of fruit flies. These experiments revealed that the two proteins are arranged in tandem along the length of the sarcomere, forming a structure that measures about 350 nm. Sallimus is anchored in the Z-disc and it runs alongside actin until it reaches the end of a myosin filament; there, it overlaps with Projectin for about 10 nm. Projectin then stretches for 250 nm along the length of the beginning myosin filament. These findings confirm the importance of titin in dictating the length of a sarcomere; they suggest that, in invertebrates, this role is split between two proteins, each possibly ruling over a section of the sarcomere. In addition, the work by Schueder, Mangeol et al. demonstrate the value of combining nanobodies and super-resolution microscopy to study complex structures in tissues.

Polyphasic circadian neural circuits drive differential activities in multiple downstream rhythmic centers.

Circadian clocks align various behaviors such as locomotor activity, sleep/wake, feeding, and mating to times of day that are most adaptive. How rhythmic information in pacemaker circuits is translated to neuronal outputs is not well understood. Here, we used brain-wide, 24-h in vivo calcium imaging in the Drosophila brain and searched for circadian rhythmic activity among identified clusters of dopaminergic (DA) and peptidergic neurosecretory (NS) neurons. Such rhythms were widespread and imposed by the PERIOD-dependent clock activity within the ∼150-cell circadian pacemaker network. The rhythms displayed either a morning (M), evening (E), or mid-day (MD) phase. Different subgroups of circadian pacemakers imposed neural activity rhythms onto different downstream non-clock neurons. Outputs from the canonical M and E pacemakers converged to regulate DA-PPM3 and DA-PAL neurons. E pacemakers regulate the evening-active DA-PPL1 neurons. In addition to these canonical M and E oscillators, we present evidence for a third dedicated phase occurring at mid-day: the l-LNv pacemakers present the MD activity peak, and they regulate the MD-active DA-PPM1/2 neurons and three distinct NS cell types. Thus, the Drosophila circadian pacemaker network is a polyphasic rhythm generator. It presents dedicated M, E, and MD phases that are functionally transduced as neuronal outputs to organize diverse daily activity patterns in downstream circuits.

Attachment and detachment of cortical myosin regulates cell junction exchange during cell rearrangement in the Drosophila wing epithelium.

Epithelial cells remodel cell adhesion and change their neighbors to shape a tissue. This cellular rearrangement proceeds in three steps: the shrinkage of a junction, exchange of junctions, and elongation of the newly generated junction. Herein, by combining live imaging and physical modeling, we showed that the formation of myosin-II (myo-II) cables around the cell vertices underlies the exchange of junctions in the Drosophila wing epithelium. The local and transient detachment of myo-II from the cell cortex is regulated by the LIM domain-containing protein Jub and the tricellular septate junction protein M6. Moreover, we found that M6 shifts to the adherens junction plane on jub RNAi and that Jub is persistently retained at reconnecting junctions in m6 RNAi cells. This interplay between Jub and M6 can depend on the junction length and thereby couples the detachment of cortical myo-II cables and the shrinkage/elongation of the junction during cell rearrangement. Furthermore, we developed a mechanical model based on the wetting theory and clarified how the physical properties of myo-II cables are integrated with the junction geometry to induce the transition between the attached and detached states and support the unidirectionality of cell rearrangement. Collectively, this study elucidates the orchestration of geometry, mechanics, and signaling for exchanging junctions.

Damage-induced regeneration of the intestinal stem cell pool through enteroblast mitosis in the Drosophila midgut.

Many adult tissues and organs including the intestine rely on resident stem cells to maintain homeostasis and regeneration. In mammals, the progenies of intestinal stem cells (ISCs) can dedifferentiate to generate ISCs upon ablation of resident stem cells. However, whether and how mature tissue cells generate ISCs under physiological conditions remains unknown. Here, we show that infection of the Drosophila melanogaster intestine with pathogenic bacteria induces entry of enteroblasts (EBs), which are ISC progenies, into the mitotic cycle through upregulation of epidermal growth factor receptor (EGFR)-Ras signaling. We also show that ectopic activation of EGFR-Ras signaling in EBs is sufficient to drive enteroblast mitosis cell autonomously. Furthermore, we find that the dividing enteroblasts do not gain ISC identity as a prerequisite to divide, and the regenerative ISCs are produced through EB mitosis. Taken together, our work uncovers a new role for EGFR-Ras signaling in driving EB mitosis and replenishing the ISC pool during fly intestinal regeneration, which may have important implications for tissue homeostasis and tumorigenesis in vertebrates.

Ets21C sustains a pro-regenerative transcriptional program in blastema cells of Drosophila imaginal discs.

An important unanswered question in regenerative biology is to what extent regeneration is accomplished by the reactivation of gene regulatory networks used during development versus the activation of regeneration-specific transcriptional programs. Following damage, Drosophila imaginal discs, the larval precursors of adult structures, can regenerate missing portions by localized proliferation of damage-adjacent tissue. Using single-cell transcriptomics in regenerating wing discs, we have obtained a comprehensive view of the transcriptome of regenerating discs and identified two regeneration-specific cell populations within the blastema, Blastema1 and Blastema2. Collectively, these cells upregulate multiple genes encoding secreted proteins that promote regeneration including Pvf1, upd3, asperous, Mmp1, and the maturation delaying factor Ilp8. Expression of the transcription factor Ets21C is restricted to this regenerative secretory zone; it is not expressed in undamaged discs. Ets21C expression is activated by the JNK/AP-1 pathway, and it can function in a type 1 coherent feedforward loop with AP-1 to sustain expression of downstream genes. Without Ets21C function, the blastema cells fail to maintain the expression of a number of genes, which leads to premature differentiation and severely compromised regeneration. As Ets21C is dispensable for normal development, these observations indicate that Ets21C orchestrates a regeneration-specific gene regulatory network. We have also identified cells resembling both Blastema1 and Blastema2 in scribble tumorous discs. They express the Ets21C-dependent gene regulatory network, and eliminating Ets21C function reduces tumorous growth. Thus, mechanisms that function during regeneration can be co-opted by tumors to promote aberrant growth.

EGFR signaling activates intestinal stem cells by promoting mitochondrial biogenesis and ß-oxidation.

EGFR-RAS-ERK signaling promotes growth and proliferation in many cell types, and genetic hyperactivation of RAS-ERK signaling drives many cancers. Yet, despite intensive study of upstream components in EGFR signal transduction, the identities and functions of downstream effectors in the pathway are poorly understood. In Drosophila intestinal stem cells (ISCs), the transcriptional repressor Capicua (Cic) and its targets, the ETS-type transcriptional activators Pointed (pnt) and Ets21C, are essential downstream effectors of mitogenic EGFR signaling. Here, we show that these factors promote EGFR-dependent metabolic changes that increase ISC mass, mitochondrial growth, and mitochondrial activity. Gene target analysis using RNA and DamID sequencing revealed that Pnt and Ets21C directly upregulate not only DNA replication and cell cycle genes but also genes for oxidative phosphorylation, the TCA cycle, and fatty acid beta-oxidation. Metabolite analysis substantiated these metabolic functions. The mitochondrial transcription factor B2 (mtTFB2), a direct target of Pnt, was required and partially sufficient for EGFR-driven ISC growth, mitochondrial biogenesis, and proliferation. MEK-dependent EGF signaling stimulated mitochondrial biogenesis in human RPE-1 cells, indicating the conservation of these metabolic effects. This work illustrates how EGFR signaling alters metabolism to coordinately activate cell growth and cell division.

Adaptive changes in energy reserves and effects of body melanization on thermal tolerance in Drosophila simulans.

Seasonally polyphenic types have been documented in many Drosophilids, which differ significantly during thermal stress. Although Drosophila simulans is a sibling species to Drosophila melanogaster, both thrive in the temperate and tropical climates, but various climatic factors are expected to impact their distribution and abundance. As a result, D. simulans may use phenotypic plasticity to adapt to colder and drier circumstances in temperate zones, although such studies are less known. In the present study, our main aim was to find a link between adaptive plasticity and thermal tolerance in D. simulans. We characterized two morphs in D. simulans flies based on the abdominal melanization collected from the same locality and season, as this trait is highly associated with the larval developmental conditions. Our results suggested that flies reared from dark and light morph showed significant differences in the basal level of proline, carbohydrates (trehalose, glycogen), and lipids (cuticular lipids and total body lipids) within simulated seasons and morph lineages in D. simulans flies. We further showed that D. simulans reared from dark morph are better adapted to cold conditions, whereas light flies are more adapted to warm conditions. The flies, both from light and dark morph lineages, when reared at 15 °C, showed an increase in the level of total body lipids after acclimation at 0 °C but a decrease in the level of proline and carbohydrates (trehalose, glycogen). Heat acclimation increases glycogen levels in the flies from light morph lineage while decreases trehalose and proline.

Cellular mechanisms underlying adult tissue plasticity in Drosophila.

Adult tissues in Metazoa dynamically remodel their structures in response to environmental challenges including sudden injury, pathogen infection, and nutritional fluctuation, while maintaining quiescence under homoeostatic conditions. This characteristic, hereafter referred to as adult tissue plasticity, can prevent tissue dysfunction and improve the fitness of organisms in continuous and/or severe change of environments. With its relatively simple tissue structures and genetic tools, studies using the fruit fly Drosophila melanogaster have provided insights into molecular mechanisms that control cellular responses, particularly during regeneration and nutrient adaptation. In this review, we present the current understanding of cellular mechanisms, stem cell proliferation, polyploidization, and cell fate plasticity, all of which enable adult tissue plasticity in various Drosophila adult organs including the midgut, the brain, and the gonad, and discuss the organismal strategy in response to environmental changes and future directions of the research.

Molecular and neuronal mechanisms for amino acid taste perception in the Drosophila labellum.

Amino acids are essential nutrients that act as building blocks for protein synthesis. Recent studies in Drosophila have demonstrated that glycine, phenylalanine, and threonine elicit attraction, whereas tryptophan elicits aversion at ecologically relevant concentrations. Here, we demonstrated that eight amino acids, including arginine, glycine, alanine, serine, phenylalanine, threonine, cysteine, and proline, differentially stimulate feeding behavior by activating sweet-sensing gustatory receptor neurons (GRNs) in L-type and S-type sensilla. In turn, this process is mediated by three GRs (GR5a, GR61a, and GR64f), as well as two broadly required ionotropic receptors (IRs), IR25a and IR76b. However, GR5a, GR61a, and GR64f are only required for sensing amino acids in the sweet-sensing GRNs of L-type sensilla. This suggests that amino acid sensing in different type sensilla occurs through dual mechanisms. Furthermore, our findings indicated that ecologically relevant high concentrations of arginine, lysine, proline, valine, tryptophan, isoleucine, and leucine elicit aversive responses via bitter-sensing GRNs, which are mediated by three IRs (IR25a, IR51b, and IR76b). More importantly, our results demonstrate that arginine, lysine, and proline induce biphasic responses in a concentration-dependent manner. Therefore, amino acid detection in Drosophila occurs through two classes of receptors that activate two sets of sensory neurons in physiologically distinct pathways, which ultimately mediates attraction or aversion behaviors.

Myc suppresses male-male courtship in Drosophila.

Despite strong natural selection on species, same-sex sexual attraction is widespread across animals, yet the underlying mechanisms remain elusive. Here, we report that the proto-oncogene Myc is required in dopaminergic neurons to inhibit Drosophila male-male courtship. Loss of Myc, either by mutation or neuro-specific knockdown, induced males' courtship propensity toward other males. Our genetic screen identified DOPA decarboxylase (Ddc) as a downstream target of Myc. While loss of Ddc abrogated Myc depletion-induced male-male courtship, Ddc overexpression sufficed to trigger such behavior. Furthermore, Myc-depleted males exhibited elevated dopamine level in a Ddc-dependent manner, and their male-male courtship was blocked by depleting the dopamine receptor DopR1. Moreover, Myc directly inhibits Ddc transcription by binding to a target site in the Ddc promoter, and deletion of this site by genome editing was sufficient to trigger male-male courtship. Finally, drug-mediated Myc depletion in adult neurons by GeneSwitch technique sufficed to elicit male-male courtship. Thus, this study uncovered a novel function of Myc in preventing Drosophila male-male courtship, and supports the crucial roles of genetic factors in inter-male sexual behavior.

Roles of matrix metalloproteinases in development, immunology, and ovulation in fruit Fly (Drosophila).

Matrix metalloproteinase (MMP), a protease enzyme, participates in proteolytic cleavage of extracellular matrix proteins from Drosophila and mammals. But, recent studies have revealed other physiologically important roles of MMP in Drosophila. MMP contributes to cardioblast movement and distribution of collagen proteins during cardiogenesis in developing Drosophila. Tissue remodeling, especially tracheal development is also maintained by MMP. MMP regulates certain immunological functions in Drosophila such as wound repairing, plasmatocyte assemblage at the injured site of the basement membrane and glial response to axon degeneration in Drosophila nervous system. But, the contribution of MMP to tumor formation and metastasis in Drosophila has made it an interesting topic among researchers. Ovulation and egg laying are also found to be affected positively by MMP in Drosophila.

The Drosophila anterior-posterior axis is polarized by asymmetric myosin activation.

The Drosophila anterior-posterior axis is specified at mid-oogenesis when the Par-1 kinase is recruited to the posterior cortex of the oocyte, where it polarizes the microtubule cytoskeleton to define where the axis determinants, bicoid and oskar mRNAs, localize. This polarity is established in response to an unknown signal from the follicle cells, but how this occurs is unclear. Here we show that the myosin chaperone Unc-45 and non-muscle myosin II (MyoII) are required upstream of Par-1 in polarity establishment. Furthermore, the myosin regulatory light chain (MRLC) is di-phosphorylated at the oocyte posterior in response to the follicle cell signal, inducing longer pulses of myosin contractility at the posterior that may increase cortical tension. Overexpression of MRLC-T21A that cannot be di-phosphorylated or treatment with the myosin light-chain kinase inhibitor ML-7 abolishes Par-1 localization, indicating that the posterior of MRLC di-phosphorylation is essential for both polarity establishment and maintenance. Thus, asymmetric myosin activation polarizes the anterior-posterior axis by recruiting and maintaining Par-1 at the posterior cortex. This raises an intriguing parallel with anterior-posterior axis formation in C. elegans, where MyoII also acts upstream of the PAR proteins to establish polarity, but to localize the anterior PAR proteins rather than Par-1.

Sperm Cyst "Looping": A Developmental Novelty Enabling Extreme Male Ornament Evolution.

Postcopulatory sexual selection is credited as a principal force behind the rapid evolution of reproductive characters, often generating a pattern of correlated evolution between interacting, sex-specific traits. Because the female reproductive tract is the selective environment for sperm, one taxonomically widespread example of this pattern is the co-diversification of sperm length and female sperm-storage organ dimension. In Drosophila, having testes that are longer than the sperm they manufacture was believed to be a universal physiological constraint. Further, the energetic and time costs of developing long testes have been credited with underlying the steep evolutionary allometry of sperm length and constraining sperm length evolution in Drosophila. Here, we report on the discovery of a novel spermatogenic mechanism-sperm cyst looping-that enables males to produce relatively long sperm in short testis. This phenomenon (restricted to members of the saltans and willistoni species groups) begins early during spermatogenesis and is potentially attributable to heterochronic evolution, resulting in growth asynchrony between spermatid tails and the surrounding spermatid and somatic cyst cell membranes. By removing the allometric constraint on sperm length, this evolutionary innovation appears to have enabled males to evolve extremely long sperm for their body mass while evading delays in reproductive maturation time. On the other hand, sperm cyst looping was found to exact a cost by requiring greater total energetic investment in testes and a pronounced reduction in male lifespan. We speculate on the ecological selection pressures underlying the evolutionary origin and maintenance of this unique adaptation.