CCDC183 is essential for cytoplasmic invagination around the flagellum during spermiogenesis and male fertility.

Sperm flagellum plays a crucial role in male fertility. Here, we generated Ccdc183 knockout mice using the CRISPR/Cas9 system to reveal the protein function of the testis-specific protein CCDC183 in spermiogenesis. We demonstrated that the absence of CCDC183 causes male infertility with morphological and motility defects in spermatozoa. Owing to the lack of CCDC183, centrioles after elongation of axonemal microtubules do not connect the cell surface and nucleus during spermiogenesis, which causes subsequent loss of cytoplasmic invagination around the flagellum. As a result, the flagellar compartment does not form properly and cytosol-exposed axonemal microtubules collapse during spermiogenesis. In addition, ectopic localization of accessory structures, such as the fibrous sheath and outer dense fibers, and abnormal head shape as a result of abnormal sculpting by the manchette are observed in Ccdc183 knockout spermatids. Our results indicate that CCDC183 plays an essential role in cytoplasmic invagination around the flagellum to form functional spermatozoa during spermiogenesis.

TSKS localizes to nuage in spermatids and regulates cytoplasmic elimination during spermiation.

Spermatozoa have a streamlined shape to swim through the oviduct to fertilize oocytes. To become svelte spermatozoa, spermatid cytoplasm must be eliminated in several steps including sperm release, which is part of spermiation. Although this process has been well observed, the molecular mechanisms that underlie it remain unclear. In male germ cells, there are membraneless organelles called nuage, which are observed by electron microscopy in various forms of dense material. Reticulated body (RB) and chromatoid body remnant (CR) are two types of nuage in spermatids, but the functions of both are unknown. Using CRISPR/Cas9 technology, we deleted the entire coding sequence of testis-specific serine kinase substrate (TSKS) in mice and demonstrate that TSKS is essential for male fertility through the formation of both RB and CR, prominent sites of TSKS localization. Due to the lack of TSKS-derived nuage (TDN), the cytoplasmic contents cannot be eliminated from spermatid cytoplasm in Tsks knockout mice, resulting in excess residual cytoplasm with an abundance of cytoplasmic materials and inducing an apoptotic response. In addition, ectopic expression of TSKS in cells results in formation of amorphous nuage-like structures; dephosphorylation of TSKS helps to induce nuage, while phosphorylation of TSKS blocks the formation. Our results indicate that TSKS and TDN are essential for spermiation and male fertility by eliminating cytoplasmic contents from the spermatid cytoplasm.

ARMC12 regulates spatiotemporal mitochondrial dynamics during spermiogenesis and is required for male fertility.

The mammalian sperm midpiece has a unique double-helical structure called the mitochondrial sheath that wraps tightly around the axoneme. Despite the remarkable organization of the mitochondrial sheath, the molecular mechanisms involved in mitochondrial sheath formation are unclear. In the process of screening testis-enriched genes for functions in mice, we identified armadillo repeat-containing 12 (ARMC12) as an essential protein for mitochondrial sheath formation. Here, we engineered Armc12-null mice, FLAG-tagged Armc12 knock-in mice, and TBC1 domain family member 21 (Tbc1d21)-null mice to define the functions of ARMC12 in mitochondrial sheath formation in vivo. We discovered that absence of ARMC12 causes abnormal mitochondrial coiling along the flagellum, resulting in reduced sperm motility and male sterility. During spermiogenesis, sperm mitochondria in Armc12-null mice cannot elongate properly at the mitochondrial interlocking step which disrupts abnormal mitochondrial coiling. ARMC12 is a mitochondrial peripheral membrane protein and functions as an adherence factor between mitochondria in cultured cells. ARMC12 in testicular germ cells interacts with mitochondrial proteins MIC60, VDAC2, and VDAC3 as well as TBC1D21 and GK2, which are required for mitochondrial sheath formation. We also observed that TBC1D21 is essential for the interaction between ARMC12 and VDAC proteins in vivo. These results indicate that ARMC12 uses integral mitochondrial membrane proteins VDAC2 and VDAC3 as scaffolds to link mitochondria and works cooperatively with TBC1D21. Thus, our studies have revealed that ARMC12 regulates spatiotemporal mitochondrial dynamics to form the mitochondrial sheath through cooperative interactions with several proteins on the sperm mitochondrial surface.

SPATA33 localizes calcineurin to the mitochondria and regulates sperm motility in mice.

Calcineurin is a calcium-dependent phosphatase that plays roles in a variety of biological processes including immune responses. In spermatozoa, there is a testis-enriched calcineurin composed of PPP3CC and PPP3R2 (sperm calcineurin) that is essential for sperm motility and male fertility. Because sperm calcineurin has been proposed as a target for reversible male contraceptives, identifying proteins that interact with sperm calcineurin widens the choice for developing specific inhibitors. Here, by screening the calcineurin-interacting PxIxIT consensus motif in silico and analyzing the function of candidate proteins through the generation of gene-modified mice, we discovered that SPATA33 interacts with sperm calcineurin via a PQIIIT sequence. Spata33 knockout mice exhibit reduced sperm motility because of an inflexible midpiece, leading to impaired male fertility, which phenocopies Ppp3cc and Ppp3r2 knockout mice. Further analysis reveals that sperm calcineurin disappears from the mitochondria in the Spata33 knockout testis. In addition, immunoprecipitation analysis indicates that sperm calcineurin interacts with not only SPATA33 but also the mitochondrial protein VDAC2. These results indicate that SPATA33 localizes calcineurin to the mitochondria and regulates sperm motility.

IRGC1, a testis-enriched immunity related GTPase, is important for fibrous sheath integrity and sperm motility in mice.

Immunity-related GTPases (IRGs), also known as p47 GTPases, are a family of interferon-inducible proteins that play roles in immunity defense against intracellular pathogens. Although the molecular functions of IRGs have been well studied, the function of the family member, IRGC1, remains unclear. IRGC1 is unique among IRGs because its expression is not induced by interferon and it is expressed predominantly in the testis. Further, IRGC1 is well conserved in mammals unlike other IRGs. Here, we knocked out (KO) Irgc1 in mice using the CRISPR/Cas9 system and found that the fertility of Irgc1 KO males was severely impaired because of abnormal sperm motility. Further analyses with a transmission electron microscope revealed that the fibrous sheath (FS), an accessory structure of the sperm tail, was disorganized in Irgc1 KO mice. In addition, IRGC1 was detected in the sperm tail and fractionated with FS proteins. These results suggest that IRGC1 is a component of the FS and is involved in the correct formation of the FS.

Bi-allelic DNAH8 Variants Lead to Multiple Morphological Abnormalities of the Sperm Flagella and Primary Male Infertility.

Sperm malformation is a direct factor for male infertility. Multiple morphological abnormalities of the flagella (MMAF), a severe form of asthenoteratozoospermia, are characterized by immotile spermatozoa with malformed and/or absent flagella in the ejaculate. Previous studies indicated genetic heterogeneity in MMAF. To further define genetic factors underlying MMAF, we performed whole-exome sequencing in a cohort of 90 Chinese MMAF-affected men. Two cases (2.2%) were identified as carrying bi-allelic missense DNAH8 variants, variants which were either absent or rare in the control human population and were predicted to be deleterious by multiple bioinformatic tools. Re-analysis of exome data from a second cohort of 167 MMAF-affected men from France, Iran, and North Africa permitted the identification of an additional male carrying a DNAH8 homozygous frameshift variant. DNAH8 encodes a dynein axonemal heavy-chain component that is expressed preferentially in the testis. Hematoxylin-eosin staining and electron microscopy analyses of the spermatozoa from men harboring bi-allelic DNAH8 variants showed a highly aberrant morphology and ultrastructure of the sperm flagella. Immunofluorescence assays performed on the spermatozoa from men harboring bi-allelic DNAH8 variants revealed the absent or markedly reduced staining of DNAH8 and its associated protein DNAH17. Dnah8-knockout male mice also presented typical MMAF phenotypes and sterility. Interestingly, intracytoplasmic sperm injections using the spermatozoa from Dnah8-knockout male mice resulted in good pregnancy outcomes. Collectively, our experimental observations from humans and mice demonstrate that DNAH8 is essential for sperm flagellar formation and that bi-allelic deleterious DNAH8 variants lead to male infertility with MMAF.

FAM209 associates with DPY19L2, and is required for sperm acrosome biogenesis and fertility in mice.

Infertility afflicts up to 15% of couples globally each year with men a contributing factor in 50% of these cases. Globozoospermia is a rare condition found in infertile men, which is characterized by defective acrosome biogenesis leading to the production of round-headed sperm. Here, we report that family with sequence similarity 209 (Fam209) is required for acrosome biogenesis in mouse sperm. FAM209 is a small transmembrane protein conserved among mammals. Loss of Fam209 results in fertility defects that are secondary to abnormalities in acrosome biogenesis during spermiogenesis, reminiscent of globozoospermia. Analysis of the FAM209 proteome identified DPY19L2, whose human orthologue is involved in the majority of globozoospermia cases. Although mutations in human and mouse Dpy19l2 have been shown to cause globozoospermia, no in vivo interacting partners of DPY19L2 have been identified until now. FAM209 colocalizes with DPY19L2 at the inner nuclear membrane to maintain the developing acrosome. Here, we identified FAM209 as the first interacting partner of DPY19L2, and the second protein that is essential for acrosome biogenesis that localizes to the inner nuclear membrane.

LRRC23 is a conserved component of the radial spoke that is necessary for sperm motility and male fertility in mice.

Cilia and flagella are ancient structures that achieve controlled motor functions through the coordinated interaction based on microtubules and some attached projections. Radial spokes (RSs) facilitate the beating motion of these organelles by mediating signal transduction between dyneins and a central pair (CP) of singlet microtubules. RS complex isolation from Chlamydomonas axonemes enabled the detection of 23 radial spoke proteins (RSP1-RSP23), although the roles of some radial spoke proteins remain unknown. Recently, RSP15 has been reported to be bound to the stalk of RS2, but its homolog in mammals has not been identified. Herein, we show that Lrrc23 is an evolutionarily conserved testis-enriched gene encoding an RSP15 homolog in mice. We found that LRRC23 localizes to the RS complex within murine sperm flagella and interacts with RSPH3A and RSPH3B. The knockout of Lrrc23 resulted in male infertility due to RS disorganization and impaired motility in murine spermatozoa, whereas the ciliary beating was not significantly affected. These data indicate that LRRC23 is a key regulator that underpins the integrity of the RS complex within the flagella of mammalian spermatozoa, whereas it is dispensable in cilia. This article has an associated First Person interview with the first author of the paper.

Nexin-Dynein regulatory complex component DRC7 but not FBXL13 is required for sperm flagellum formation and male fertility in mice.

Flagella and cilia are evolutionarily conserved cellular organelles. Abnormal formation or motility of these organelles in humans causes several syndromic diseases termed ciliopathies. The central component of flagella and cilia is the axoneme that is composed of the '9+2' microtubule arrangement, dynein arms, radial spokes, and the Nexin-Dynein Regulatory Complex (N-DRC). The N-DRC is localized between doublet microtubules and has been extensively studied in the unicellular flagellate Chlamydomonas. Recently, it has been reported that TCTE1 (DRC5), a component of the N-DRC, is essential for proper sperm motility and male fertility in mice. Further, TCTE1 has been shown to interact with FBXL13 (DRC6) and DRC7; however, functional roles of FBXL13 and DRC7 in mammals have not been elucidated. Here we show that Fbxl13 and Drc7 expression are testes-enriched in mice. Although Fbxl13 knockout (KO) mice did not show any obvious phenotypes, Drc7 KO male mice were infertile due to their short immotile spermatozoa. In Drc7 KO spermatids, the axoneme is disorganized and the '9+2' microtubule arrangement was difficult to detect. Further, other N-DRC components fail to incorporate into the flagellum without DRC7. These results indicate that Drc7, but not Fbxl13, is essential for the correct assembly of the N-DRC and flagella.

Evaluation of the efficacy of creatine chemical exchange saturation transfer imaging in assessing testicular maturity.

Purpose: Microscopic testicular sperm extraction is the most effective treatment for NOA, but the sperm retrieval rate is low and depends on testicular maturity. However, there are limited useful tests to assess testicular maturity. Chemical exchange saturation transfer (CEST) imaging is a new magnetic resonance imaging (MRI) technique that can image the distribution of trace substances in vivo. We focused on the potential role of creatine (Cr) in testes and hypothesized that Cr-CEST could indicate intratesticular spermatogenesis. Methods: We performed Cr-CEST by using 7T MRI on wild-type C57B6/J mice and several types of male infertility models such as Sertoli-cell only (SCO) (Kitw/Kitwv), maturation arrest (MA) (Zfp541 knockout mouse and Kctd19 knockout mouse), and teratozoospermia (Tbc1d21 knockout mouse). After performing Cr-CEST, histological analysis was performed. Results: The SCO and MA models showed decreased CEST signal intensity (p < 0.05), while no reduction was observed in the teratozoospermia model (p = 1.0). CEST signal intensity increased as the spermatogenesis stage progressed from the SCO model to the MA and teratozoospermia models. Furthermore, CEST signal intensity was reduced in 4-week-old wild-type mice with immature testes (p < 0.05). Conclusions: This study suggests that Cr-CEST evaluates intratesticular spermatogenesis noninvasively and provides a new therapeutic strategy for treating male infertility.

TULP2 deletion mice exhibit abnormal outer dense fiber structure and male infertility.

Purpose: Tulp2 (tubby-like protein 2) is a member of the tubby protein family and expressed predominantly in mouse testis. Recently, it was reported that Tulp2 knockout (KO) mice exhibited disrupted sperm tail morphology; however, it remains to be determined how TULP2 deletion causes abnormal tail formation. Methods: The authors analyzed male fertility, sperm morphology, and motility of two Tulp2 KO mouse lines that were generated using the conventional method that utilizes homologous recombination in embryonic stem (ES) cells as well as the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system. Furthermore, the authors observed the spermatogenesis of Tulp2 KO mice in more detail using scanning and transmission electron microscopy (SEM and TEM). Results: Both mouse lines of Tulp2 KO exhibited male infertility, abnormal tail morphology, and impaired sperm motility. No overt abnormalities were found in the formation of the mitochondrial sheath in Tulp2 KO mice using the freeze-fracture method with SEM. In contrast, abnormal outer dense fiber (ODF) structure was observed in Tulp2 KO testis with TEM. Conclusions: TULP2 may play roles in the correct formation and/or maintenance of ODF, which may lead to abnormal tail morphology, impaired sperm motility, and male infertility.

Testis-enriched kinesin KIF9 is important for progressive motility in mouse spermatozoa.

Kinesin is a molecular motor that moves along microtubules. Kinesin family member 9 (KIF9) is evolutionarily conserved and expressed strongly in mouse testis. In the unicellular flagellate Chlamydomonas, KLP1 (ortholog of KIF9) is localized to the central pair microtubules of the axoneme and regulates flagellar motility. In contrast, the function of KIF9 remains unclear in mammals. Here, we mutated KIF9 in mice using the CRISPR/Cas9 system. Kif9 mutated mice exhibit impaired sperm motility and subfertility. Further analysis reveals that the flagella lacking KIF9 showed an asymmetric waveform pattern, which leads to a circular motion of spermatozoa. In spermatozoa that lack the central pair protein HYDIN, KIF9 was not detected by immunofluorescence and immunoblot analysis. These results suggest that KIF9 is associated with the central pair microtubules and regulates flagellar motility in mice.

RSPH6A is required for sperm flagellum formation and male fertility in mice.

The flagellum is an evolutionarily conserved appendage used for sensing and locomotion. Its backbone is the axoneme and a component of the axoneme is the radial spoke (RS), a protein complex implicated in flagellar motility regulation. Numerous diseases occur if the axoneme is improperly formed, such as primary ciliary dyskinesia (PCD) and infertility. Radial spoke head 6 homolog A (RSPH6A) is an ortholog of Chlamydomonas RSP6 in the RS head and is evolutionarily conserved. While some RS head proteins have been linked to PCD, little is known about RSPH6A. Here, we show that mouse RSPH6A is testis-enriched and localized in the flagellum. Rsph6a knockout (KO) male mice are infertile as a result of their short immotile spermatozoa. Observation of the KO testis indicates that the axoneme can elongate but is disrupted before accessory structures are formed. Manchette removal is also impaired in the KO testis. Further, RSPH9, another radial spoke protein, disappeared in the Rsph6a KO flagella. These data indicate that RSPH6A is essential for sperm flagellar assembly and male fertility in mice.This article has an associated First Person interview with the first author of the paper.

Tmprss12 is required for sperm motility and uterotubal junction migration in mice†.

Spermatozoa are produced in the testis but gain their fertilizing ability during epididymal migration. This necessary step in sperm maturation includes posttranslational modification of sperm membrane proteins that includes protein processing by proteases. However, the molecular mechanism underpinning this epididymal sperm maturation remains unknown. In this study, we focused on transmembrane serine protease 12 (Tmprss12). Based on multi-tissue expression analysis by PCR, Tmprss12 was specifically expressed in the testis, and its expression started on day 10 postpartum, corresponding to the stage of zygotene spermatocytes. TMPRSS12 was detected in the acrosomal region of spermatozoa by immunostaining. To reveal the physiological function of TMPRSS12, we generated two knockout (KO) mouse lines using the CRISPR/Cas9 system. Both indel and large deletion lines were male sterile showing that TMPRSS12 is essential for male fertility. Although KO males exhibited normal spermatogenesis and sperm morphology, ejaculated spermatozoa failed to migrate from the uterus to the oviduct. Further analysis revealed that a disintegrin and metalloprotease 3 (ADAM3), an essential protein on the sperm membrane surface that is required for sperm migration, was disrupted in KO spermatozoa. Moreover, we found that KO spermatozoa showed reduced sperm motility via computer-assisted sperm analysis, resulting in a low fertilization rate in vitro. Taken together, these data indicate that TMPRSS12 has dual functions in regulating sperm motility and ADAM3-related sperm migration to the oviduct. Because Tmprss12 is conserved among mammals, including humans, our results may explain some genetic cases of idiopathic male infertility, and TMPRSS12 and its downstream cascade may be novel targets for contraception.

CRISPR/Cas9-mediated genome-edited mice reveal 10 testis-enriched genes are dispensable for male fecundity.

As the world population continues to increase to unsustainable levels, the importance of birth control and the development of new contraceptives are emerging. To date, male contraceptive options have been lagging behind those available to women, and those few options available are not satisfactory to everyone. To solve this problem, we have been searching for new candidate target proteins for non-hormonal contraceptives. Testis-specific proteins are appealing targets for male contraceptives because they are more likely to be involved in male reproduction and their targeting by small molecules is predicted to have no on-target harmful effects on other organs. Using in silico analysis, we identified Erich2, Glt6d1, Prss58, Slfnl1, Sppl2c, Stpg3, Tex33, and Tex36 as testis-abundant genes in both mouse and human. The genes, 4930402F06Rik and 4930568D16Rik, are testis-abundant paralogs of Glt6d1 that we also discovered in mice but not in human, and were also included in our studies to eliminate the potential compensation. We generated knockout (KO) mouse lines of all listed genes using the CRISPR/Cas9 system. Analysis of all of the individual KO mouse lines as well as Glt6d1/4930402F06Rik/4930568D16Rik TKO mouse lines revealed that they are male fertile with no observable defects in reproductive organs, suggesting that these 10 genes are not required for male fertility nor play redundant roles in the case of the 3 Glt6D1 paralogs. Further studies are needed to uncover protein function(s), but in vivo functional screening using the CRISPR/Cas9 system is a fast and accurate way to find genes essential for male fertility, which may apply to studies of genes expressed elsewhere. In this study, although we could not find any potential protein targets for non-hormonal male contraceptives, our findings help to streamline efforts to find and focus on only the essential genes.

CRISPR/Cas9-mediated genome editing reveals 30 testis-enriched genes dispensable for male fertility in mice†.

More than 1000 genes are predicted to be predominantly expressed in mouse testis, yet many of them remain unstudied in terms of their roles in spermatogenesis and sperm function and their essentiality in male reproduction. Since individually indispensable factors can provide important implications for the diagnosis of genetically related idiopathic male infertility and may serve as candidate targets for the development of nonhormonal male contraceptives, our laboratories continuously analyze the functions of testis-enriched genes in vivo by generating knockout mouse lines using the CRISPR/Cas9 system. The dispensability of genes in male reproduction is easily determined by examining the fecundity of knockout males. During our large-scale screening of essential factors, we knocked out 30 genes that have a strong bias of expression in the testis and are mostly conserved in mammalian species including human. Fertility tests reveal that the mutant males exhibited normal fecundity, suggesting these genes are individually dispensable for male reproduction. Since such functionally redundant genes are of diminished biological and clinical significance, we believe that it is crucial to disseminate this list of genes, along with their phenotypic information, to the scientific community to avoid unnecessary expenditure of time and research funds and duplication of efforts by other laboratories.

Glycerol kinase 2 is essential for proper arrangement of crescent-like mitochondria to form the mitochondrial sheath during mouse spermatogenesis.

The mitochondrial sheath is composed of mitochondria that coil tightly around the midpiece of sperm flagellum. These mitochondria are recruited from the cytoplasm to the flagellum late in spermatogenesis. Initially, recruited mitochondria are spherical-shaped but then elongate laterally to become crescent-like in shape. Subsequently, crescent-like mitochondria elongate continuously to coil tightly around the flagellum. Recently, disorganization of the mitochondrial sheath was reported in Glycerol kinase 2 (Gk2) disrupted mice. To analyze the disorganization of the mitochondrial sheath further, we generated Gk2-deficient mice using the CRISPR/Cas9 system and observed sperm mitochondria in testis using a freeze-fracture method with scanning electron microscopy. Gk2-disrupted spermatids show abnormal localization of crescent-like mitochondria, in spite of the initial proper alignment of spherical mitochondria around the flagellum, which causes abnormal mitochondrial sheath formation leading to exposure of the outer dense fibers. These results indicate that GK2 is essential for proper arrangement of crescent-like mitochondria to form the mitochondrial sheath during mouse spermatogenesis.

Distribution of Karyotypes of the Cryptocercus punctulatus Species Complex (Blattodea: Cryptocercidae) in Great Smoky Mountains National Park.

During the period between 1999 and 2006, wood-feeding cockroaches in the Cryptocercus punctulatus Scudder species complex were collected throughout Great Smoky Mountains National Park, USA. The chromosome numbers of insects from 59 sites were determined, and phylogenetic analyses were performed based on mitochondrial COII and nuclear ITS2 DNA. The distribution of the three male karyotypes found in the park (2n = 37, 39, and 45) is mapped and discussed in relation to recent disturbances and glacial history. Clades of the three karyotype groups meet near the ridgeline separating North Carolina from Tennessee in the center of the park, suggesting that these may have originated from separate lower elevation refugia after the last glacial maximum. The timing of divergence and a significant correlation between elevation difference and genetic distance in two of the clades supports this hypothesis. The ecological role of the cockroaches in the park is discussed.

Polymer networks with bicontinuous gradient morphologies resulting from the competition between phase separation and photopolymerization.

Poly(ethyl acrylate)/poly(methyl methacrylate) (PEA/PMMA) polymer networks (IPNs) with spatially graded bicontinuous morphology were designed and controlled by taking advantage of the spinodal decomposition process induced by photopolymerization of the MMA monomer. Spatial gradients of the quench depth, induced by the gradients of light intensity, were generated along the path of the excitation light travelling through the mixture. Bicontinuous structures with uniaxial gradient of characteristic length scales were obtained by two different methods: simply irradiating the mixture with strong light intensity along the Z-direction and using the so-called computer-assisted irradiation (CAI) method with moderate intensity to generate the light intensity gradient exclusively in the XY plane. These experimental results suggest that the combination of these two irradiation methods could provide polymer materials with biaxially co-continuous gradient morphology. An analysis method using the concept of spatial correlation function was developed to analyze the time-evolution of these graded structures. The experimental results obtained in this study suggest a promising method to design gradient polymers in the bulk state (3D) as well as on the surface (2D) by taking advantage of photopolymerization.

Cellulolytic protist numbers rise and fall dramatically in termite queens and kings during colony foundation.

Among the best-known examples of mutualistic symbioses is that between lower termites and the cellulolytic flagellate protists in their hindguts. Although the symbiosis in worker termites has attracted much attention, there have been only a few studies of protists in other castes. We have performed the first examination of protist population dynamics in queens and kings during termite colony foundation. Protist numbers, as well as measurements of hindgut and reproductive tissue sizes, were undertaken at five time points over 400 days in incipient colonies of Reticulitermes speratus, as well as in other castes of mature colonies of this species. We found that protist numbers increased dramatically in both queens and kings during the first 50 days of colony foundation but began to decrease by day 100, eventually disappearing by day 400. Hindgut width followed a pattern similar to that of protist numbers, while ovary and testis widths increased significantly only at day 400. Kings were found to contain higher numbers of protists than queens in incipient colonies, which may be linked to higher levels of nutrient transfer from kings to queens than vice versa, as is known in some other termite species. Protists were found to be abundant in soldiers from mature colonies but absent in neotenics. This probably reflects feeding of soldiers by workers via proctodeal trophallaxis and of reproductives via stomodeal trophallaxis. The results reveal the dynamic nature of protist numbers during colony foundation and highlight the trade-offs that exist between reproduction and parental care during this critical phase of the termite life cycle.