BACKGROUND
it is commonly acknowledged that insects are capable of producing many eggs in short time intervals.
therefore, oogenesis could be an interesting target for the development of novel strategies for insect population control
<cit> .
the functional unit of the insect ovary is the follicle.
it is formed in the anterior region of the ovariole, in the germarium, and is moved posteriorly to the vitellarium developing during oogenesis.
each follicle consists of an oocyte and the surrounding monolayer of somatic follicular cells which constitute the follicular epithelium
<cit> .
ullmann distinguished nine developmental stages of the oocyte during oogenesis in t. molitor; in stages 1– <dig> oocytes are previtellogenic and in stages 6– <dig> they are vitellogenic
<cit> .
in early vitellogenic oocyte  patency between the follicular cells appears to allow the passage of yolk protein precursors into the oolemma in which they are internalized by endocytosis and deposited as vitellin.
as a consequence, a very rapid increase of the oocyte size follows.
during midvitellogenesis  the follicular epithelium shows maximum patency and yolk deposition, whereas in late vitellogenic oocyte  yolk deposition declines, space between follicle cells disappears and follicle cells begin to secrete chorion.
the oocyte is mature with fully formed chorion and it is expelled from follicle into lateral oviduct
<cit> .
at present three oostatic peptide hormones have been isolated from insect ovaries: trypsin modulating oostatic factor  from aedes aegypti, trypsin-modulating oostatic factor  and neb-colloostatin from neobellieria bullata <cit> .
aea-tmof discovered by borovsky et al.
<cit>  in late vitellogenic ovaries of a. aegypti females as well as neb-tmof isolated by bylemans et al.
<cit>  from vitellogenic ovaries of the grey fleshfly n. bullata, directly inhibit the biosynthesis of trypsin- and chymotrypsin-like enzymes in the epithelial cells of the insect midgut.
this results in the reduction of free amino acids in hemolymph and, in consequence, in the blocking of the biosynthesis of vitellogenin, a protein essential for oocyte growth.
neb-tmof also exerts a strong gonadoinhibitory activity on the ovaries of tenebrio molitor and zophobas atratus beetles
<cit> .
moreover, hua et al.
<cit>  and de loof et al.
<cit>  have shown that neb-tmof also inhibits ecdysone biosynthesis by larval ring glands of the fleshflies.
neb-colloostatin was discovered during the isolation of neb-tmof from the ovary of the grey fleshfly.
this peptide inhibits yolk uptake by previtellogenic oocytes and, contrary to aea-tmof and neb-tmof, it does not inhibit trypsin biosynthesis in the gut or ecdysone biosynthesis by larval ring glands
<cit> .
the gonadoinhibitory properties of neb-colloostatin were confirmed in t. molitor ovaries in which it interferes with vitellogenin production by the fat body as well as with vitellogenin uptake by oocytes through modification of patency
<cit> .
moreover, a novel physiological effect of neb-colloostatin in insects has been recently detected.
injection of neb-colloostatin in physiological concentrations results in significant hemocytotoxicity and a marked increase in apoptotic activity in t. molitor haemocytes
<cit> .
programmed cell death is an active and genetically regulated process eliminating unnecessary or abnormal cells from the organism.
type i programmed cell death, apoptosis, appears to be distinct from autophagic programmed cell death .
apoptotic cell death is mainly characterized by condensation of chromatin at the nuclear membrane, caspase activation, fragmentation of nuclei and cells, and cell fragmentation into apoptotic bodies, whereas organelles are well preserved and autophagocytosis is absent
<cit> .
the activation of caspases, the key mediators of this type of cell death, is responsible for the cleavage of cellular target proteins and, in consequence, the observed morphological changes.
during autophagic cell death, the autodegradation of cytoplasmatic components takes place, and therefore it is thought that autophagy mainly has a cytoprotective role under stress stimuli
<cit> .
follicular atresia, defined as the degeneration of the follicle, generally occurs during normal oogenesis in many animal species
<cit> .
under physiological conditions it plays a significant role in the maturation process during the normal development of eggs and in the removal of abnormal or damaged oocytes before they reach maturity.
in drosophila, giorgi and deri
<cit>  found that some egg chambers degenerate almost exclusively between stages  <dig> and  <dig> of oogenesis.
other studies of dipteran ovarian follicles confirmed the activation of programmed cell death during mid-oogenesis and late-oogenesis.
therefore apoptotic death of both the nurse cells and the follicle cells in polytrophic ovaries may be observed in either the developmental phase or the degenerative phase of dipteran oogenesis
<cit> .
while the nurse cells exhibit the morphological hallmarks of apoptosis, the follicle cells remain remarkably intact and phagocytose the cellular remnants of the nurse cells after they have completed their apoptotic program
<cit> .
however, follicular atresia has been massively induced in response to starvation, malfunction of ecdysone signalling or treatment with chemotherapeutic drugs
<cit> .
pathogen infection can also lead to atresia of the ovarian vitellogenic follicles as recently shown in rodnius prolixus <cit> .
in the present study, we describe neb-colloostatin-induced atresia in the mealworm ovary.
degeneration of t. molitor follicles includes changes in morphology and viability of follicular cells, and oosorption as a consequence of these changes.
RESULTS
neb-colloostatin injection leads to resorption of vitellogenic ovarian follicles in t. molitor
injection of neb-colloostatin  into the hemocoel of t. molitor females during their first reproductive cycle showed that this peptide strongly inhibited ovarian growth and oocyte development .
the terminal oocytes of the peptide-injected females were significantly smaller and many follicles showed resorption in contrast to terminal oocytes of control ovaries that were larger and in midvitellogenesis.
the control follicles possessed oocytes with translucent ooplasm, whereas follicles from the ovaries of females injected with neb-colloostatin showed oocyte ooplasm alterations under the stereomicroscope .
the terminal oocytes are much longer and wider in the control ovary than in that from the peptide injected female  and patency in follicular epithelium is obvious in the control but not in the ovary from the injected female .
apoptosis and autophagy are involved in follicle cell death after neb-colloostatin injection
after morphological observations, the entire ovaries were stained in order to detect peptide-induced changes in the organization of the f-actin cytoskeleton and chromatin, and presence of indicators of cell death of the terminal ovarian follicles.
the first marker, oregon green  <dig> phalloidin, delimited changes in the f-actin cytoskeleton.
in the control follicular cells, a strong fluorescent signal was localized subcortically , and was also detected in the oocyte .
maximum patency of follicular epithelium was visible.
injection of neb-colloostatin caused failure of f-actin organization in these cells.
they lost their regular f-actin staining pattern as a result of depolymerization of f-actin microfilaments.
the application of  <dig> nmole of peptide caused full depolymerization of f-actin, therefore the positive oregon green-phalloidin staining signal was not detected , but a lower dose of the peptide  inhibited intracellular space formation in the follicular epithelium .
the sulphorhodamine derivative of valylalanylaspartic acid fluoromethyl ketone  staining showed that neb-colloostatin induced the activation of caspases in the ovarioles .
in this assay, we observed that the degree of caspase activation depended on the applied hormone dose.
one day after injection of neb-colloostatin, at a dose of  <dig> or  <dig> nmole per insect, caspase activity was detected in all ovaries of the studied females.
injection of 1 nmol of peptide caused caspase activation only in oocytes .
injection of neb-colloostatin at a dose of  <dig> nmole/female activated caspases in the follicle cells, whereas the oocyte was resorbed .
next, we used propidium iodide staining to visualize changes in chromatin organization .
the nuclei of follicular cells from ovarian follicles of females injected with neb-colloostatin  were characterized by highly condensed chromatin redistributed along the nuclear membrane as compared to the control.
moreover, in the nuclei of follicular cells from the peptide-injected females, propidium iodide revealed dna fragmentation events visible as small, red dots, compared to the control nuclei, the morphology of which looked normal.
the mono-dansyl-cadaverin  and the orange acridine staining were used for tracing the induction of autophagic vacuoles and detection of lysosomes in order to assess the type of programmed cell death, i.e.
type i or type ii, caused by the injection of neb-colloostatin .
after neb-colloostatin injection at a dose of 10 nmol of peptide per female, fluorescent and confocal microscopy revealed extensive vacuolization of the cytoplasm of follicular cells indicating degeneration of their contents in an autophagy-like process.
orange acridine staining showed the presence of lysosomes in follicular cells of terminal follicles of peptide-injected females , whereas autophagic vacuoles detected by mdc staining were visible as distinct dot-like blue structures .
neb-colloostatin injection induces changes in the ultrastructure of ovarian follicles
in the t. molitor ovary, the distinct differences in ultrastructure between the control follicles and the follicles of the neb-colloostatin injected females  become visible.
in the control follicles, the follicular epithelium surrounding the terminal oocyte at the 7th  stage of development
<cit>  is composed of the cuboid cells with microvilli directed to the oocyte.
the nucleus of the follicle cells contains quite a number of the small clumps of heterochromatin and several vast irregular nucleoli.
the follicle cells exhibit characteristic morphology of active secretion.
in the basal part of the follicle cells, the cisterns of the rough endoplasmic reticulum  and the golgi complexes are well-developed .
the apical part is rich in the electron dense secretory vesicles, mitochondria and the dilated cisterns of rer .
in the follicles of injected females, the follicle cells surrounding the terminal oocyte show no morphological evidence for synthetic activity.
a part of the follicle cells is still cube-shaped, although their apical surfaces become more or less wrinkled.
the nuclei of these cells usually contain a granular nucleoplasm and a few extensive and irregular nucleoli.
less and smaller, than in the control, heterochromatin clumps are visible.
the cisterns of rer are clearly less developed than in the cells of the control follicles .
in the apical cytoplasm, there are no secretory granules and a few elements of rer and mitochondria are located in the vicinity of the nucleus .
this area is filled with numerous free ribosomes .
after injection of neb-colloostatin, many follicle cells reveal much more advanced changes.
these cells are irregular in shape and their cytoplasm is vacuolated in the varying degrees.
in the nuclei of these cells, chromatin becomes highly condensed .
in cytoplasm, the detected vacuoles are electron-transparent  or include the degradated fragments of cytoplasm .
the cells shrink and gradually lose their connection with the neighboring follicle cells and the basal lamina .
in the control follicle, between the terminal oocyte at the 7th stage of development and the follicle cells, is a narrow cleft filled with the electron dense material, forming a vitelline membrane surrounding the oocyte .
the cortical ooplasm in particular contains numerous yolk granules and lipid droplets.
.
free ribosomes, rough endoplasmic reticulum systems  and stacks of annulate lamellae  are also abundant in the ooplasm.
in the follicle of females injected with neb-colloostatin, the intercellular cleft between the surface of the follicular epithelium and oocyte is wider than in the control follicle .
the cortical region of ooplasm contains fewer and significantly small yolk granules but relatively numerous lipid droplets .
the number of free ribosomes is dramatically reduced.
the rough endoplasmic reticulum is less developed.
it displays vesicles with few ribosomes attached .
stacks of annulate lamellae are not found in the ooplasm.
in the cortical ooplasm, in between the yolk granules are autophagic vacuoles containing materials in various degrees of degradation .
the core of oocyte is usually devoid of yolk granules and the autophagic vacuoles with a highly degraded content are particularly numerous .
discussion
this study describes the process of atresia in ovarian follicles induced by the gonadoinhibitory peptide neb-colloostatin during the first gonadotropic cycle of t. molitor females.
in recent years, several control strategies have been proposed as species-specific and non-polluting methods for reducing or suppressing pest reproduction, especially insect–pathogen interactions or development of the sterile insect technique
<cit> .
as previously reported
<cit> , injection of neb-colloostatin and neb-tmof into t. molitor females causes gonadoinhibitory effects on ovarian development, however, the mode of action of these peptides remains unknown.
wasielewski and rosiński
<cit>  showed that the injection of approximately  <dig> mmole of neb-coollostatin into females on days  <dig>   <dig> and  <dig> of the first reproductive cycle inhibits patency formation, slowing down vitellogenesis and ovarian development, delaying ovulation and ovarian development, and reducing the number of eggs laid.
in t. molitor, part of the inhibitory process of the oocytes growth is manifested by qualitative and quantitative changes in proteins sequestered and synthesized by the ovary.
the authors showed that the injections of neb-colloostatin reduced by about 60% total protein content in ovaries and induced qualitative changes in ovarian protein patterns.
electrophoretic analysis pointed out that neb-colloostatin caused a loss of two polypeptides and a strong reduction of four another.
all these polypeptides were the most abundant in ovaries of the control females.
on the other hand, neb-colloostatin injection increased the concentration of two polypeptides, which were found in very small amounts in ovaries of control females.
however, neb-colloostatin did not cause a significant change in protein composition of the haemolymph
<cit> .
it seems that neb-colloostatin reduces proteins sequestration by ovaries and the peptide action is more specific to ovaries.
in this paper, we described the morphological alterations caused by neb-colloostatin injections in the oocytes and the follicular epithelium of t. molitor egg chambers.
microscopic observation and molecular analysis of the ovarian follicle facilitated a more detailed characterization of the gonadoinhibitory action of this peptide.
first, the doses of peptide injected into females applied in this work were much lower than those of wasielewski and rosiński
<cit> .
this suggests that the peptide is very potent and could be acting at a physiologically relevant concentration.
second, a very important phase of oocyte growth is the formation of intercellular spaces in the follicular epithelium enabling the transport of nutrients to the growing vitellogenic oocytes
<cit> .
our findings show that the direct action of neb-colloostatin on follicular cells resulting in f-actin cytoskeleton disorganization, and in consequence inhibition of patency formation, may prompt peptide-induced follicular atresia.
peptide injections strongly inhibited oocyte development.
next, apoptosis was switched on in affected follicles and, in consequence, resorption of oocyte remnants occurred.
in the t. molitor follicular epithelium, apart from the disorganization of the f-actin cytoskeleton network, other alterations comprised typical features of apoptosis including caspase activation, chromatin condensation and dna fragmentation.
moreover, evidence for a distinct type of physiological cell death, autophagocytosis, was also observed in oocytes and follicular cells.
incorporation of cellular contents into autophagosomes suggests that these cell components can be released into the insect haemolymph as a result of lysosomal enzyme activity.
during autophagy all cellular contents are disintegrated into autophagic vacuoles and destroyed by the cell’s lysosomes
<cit> .
aguirre et al.
<cit>  showed in dipetalogaster maxima that atresia induced by food deprivation was characterized by loss of the ability of oocytes to uptake vitellogenin, partial vitellin proteolysis and activation of cathepsin d-like peptidases.
these authors suggested that cathepsin d-like peptidase is involved in the process of follicle degeneration, probably by promoting early yolk protein degradation, and would also be involved in the oosorption of atretic follicles.
apoptosis can also be detected during extensive autophagocytosis in the course of insect oogenesis.
for example, uchida et al.
<cit>  realized that apoptosis may occur in the epithelial cells of degenerating follicles in the final stages of the atretic process within developing mosquito ovaries.
these authors detected caspase and catepsin-like proteinase activation, as well as the presence of fragmented dna in the epithelial region of the culex pipiens pallens atretic follicles but not in normally developing follicles within the same ovaries.
autophagosomes have also been observed in electron micrographs of degenerating mid-stage follicles from drosophila virilis <cit> .
valentzas and colleagues
<cit>  suggested that apoptosis and autophagy act synergistically to achieve more efficient elimination of degenerated nurse cells and abnormal eggs chambers.
boolmart et al.
<cit>  suggested that autophagocytosis depends on an intact cytoskeleton.
thus intermediates and microfilaments are considered to be essential for the initial formation of autophagosomes, whereas their subsequent fusion with lysosomes depends on microtubuli
<cit> .
in spite of the neb-colloostatin-induced disorganization of the f-actin cytoskeleton network in follicular cells, we observed the occurrence of autophagosomes, however, we cannot explain these data at present.
moreover, electron microscopy of neb-colloostatin-induced changes in the t. molitor ovary showed that the apical cytoplasm of the follicular cells is filled with numerous free ribosomes, suggesting that after peptide injection the follicular cells probably have a higher demand on protein biosynthesis, especially in the perspective of increased formation of the autophagic vacuoles process.
this result shows that the process of polyribosomes formation can be inhibited, indicating the contributing effect of neb-colloostatin.
CONCLUSIONS
in summary, our results show that in t. molitor females, neb-colloostatin injection resulted in atresia of ovarian follicles both the apoptotic and the autophagic mechanism of programmed cell death.
the first observable effect of neb-colloostatin-induced atresia is the disappearance of patency in the follicular epithelium.
this probably inhibits the active uptake of vitellogenins by the oocyte.
induction of apoptosis and autophagy of ovarian follicles may contribute to more efficient removal of atretic follicles during ovarian follicular regression in insects.

 it is commonly acknowledged that insects are capable of producing many eggs in short time intervals. therefore, oogenesis could be an interesting target for the development of novel strategies for insect population control. the functional unit of the insect ovary is the follicle. it is formed in the anterior region of the ovariole, in the germarium, and is moved posteriorly to the vitellarium developing during oogenesis. each follicle consists of an oocyte and the surrounding monolayer of somatic follicular cells which constitute the follicular epithelium. ullmann distinguished nine developmental stages of the oocyte during oogenesis in t. molitor; in stages 1– oocytes are previtellogenic and in stages 6– they are vitellogenic. in early vitellogenic oocyte patency between the follicular cells appears to allow the passage of yolk protein precursors into the oolemma in which they are internalized by endocytosis and deposited as vitellin. as a consequence, a very rapid increase of the oocyte size follows. during midvitellogenesis the follicular epithelium shows maximum patency and yolk deposition, whereas in late vitellogenic oocyte yolk deposition declines, space between follicle cells disappears and follicle cells begin to secrete chorion. the oocyte is mature with fully formed chorion and it is expelled from follicle into lateral oviduct. at present three oostatic peptide hormones have been isolated from insect ovaries: trypsin modulating oostatic factor from aedes aegypti, trypsin-modulating oostatic factor and neb-colloostatin from neobellieria bullata. aea-tmof discovered by borovsky et al. in late vitellogenic ovaries of a. aegypti females as well as neb-tmof isolated by bylemans et al. from vitellogenic ovaries of the grey fleshfly n. bullata, directly inhibit the biosynthesis of trypsin- and chymotrypsin-like enzymes in the epithelial cells of the insect midgut. this results in the reduction of free amino acids in hemolymph and, in consequence, in the blocking of the biosynthesis of vitellogenin, a protein essential for oocyte growth. neb-tmof also exerts a strong gonadoinhibitory activity on the ovaries of tenebrio molitor and zophobas atratus beetles. moreover, hua et al. and de loof et al. have shown that neb-tmof also inhibits ecdysone biosynthesis by larval ring glands of the fleshflies. neb-colloostatin was discovered during the isolation of neb-tmof from the ovary of the grey fleshfly. this peptide inhibits yolk uptake by previtellogenic oocytes and, contrary to aea-tmof and neb-tmof, it does not inhibit trypsin biosynthesis in the gut or ecdysone biosynthesis by larval ring glands. the gonadoinhibitory properties of neb-colloostatin were confirmed in t. molitor ovaries in which it interferes with vitellogenin production by the fat body as well as with vitellogenin uptake by oocytes through modification of patency. moreover, a novel physiological effect of neb-colloostatin in insects has been recently detected. injection of neb-colloostatin in physiological concentrations results in significant hemocytotoxicity and a marked increase in apoptotic activity in t. molitor haemocytes. programmed cell death is an active and genetically regulated process eliminating unnecessary or abnormal cells from the organism. type i programmed cell death, apoptosis, appears to be distinct from autophagic programmed cell death. apoptotic cell death is mainly characterized by condensation of chromatin at the nuclear membrane, caspase activation, fragmentation of nuclei and cells, and cell fragmentation into apoptotic bodies, whereas organelles are well preserved and autophagocytosis is absent. the activation of caspases, the key mediators of this type of cell death, is responsible for the cleavage of cellular target proteins and, in consequence, the observed morphological changes. during autophagic cell death, the autodegradation of cytoplasmatic components takes place, and therefore it is thought that autophagy mainly has a cytoprotective role under stress stimuli. follicular atresia, defined as the degeneration of the follicle, generally occurs during normal oogenesis in many animal species. under physiological conditions it plays a significant role in the maturation process during the normal development of eggs and in the removal of abnormal or damaged oocytes before they reach maturity. in drosophila, giorgi and deri found that some egg chambers degenerate almost exclusively between stages and of oogenesis. other studies of dipteran ovarian follicles confirmed the activation of programmed cell death during mid-oogenesis and late-oogenesis. therefore apoptotic death of both the nurse cells and the follicle cells in polytrophic ovaries may be observed in either the developmental phase or the degenerative phase of dipteran oogenesis. while the nurse cells exhibit the morphological hallmarks of apoptosis, the follicle cells remain remarkably intact and phagocytose the cellular remnants of the nurse cells after they have completed their apoptotic program. however, follicular atresia has been massively induced in response to starvation, malfunction of ecdysone signalling or treatment with chemotherapeutic drugs. pathogen infection can also lead to atresia of the ovarian vitellogenic follicles as recently shown in rodnius prolixus. in the present study, we describe neb-colloostatin-induced atresia in the mealworm ovary. degeneration of t. molitor follicles includes changes in morphology and viability of follicular cells, and oosorption as a consequence of these changes.  neb-colloostatin injection leads to resorption of vitellogenic ovarian follicles in t. molitor injection of neb-colloostatin into the hemocoel of t. molitor females during their first reproductive cycle showed that this peptide strongly inhibited ovarian growth and oocyte development. the terminal oocytes of the peptide-injected females were significantly smaller and many follicles showed resorption in contrast to terminal oocytes of control ovaries that were larger and in midvitellogenesis. the control follicles possessed oocytes with translucent ooplasm, whereas follicles from the ovaries of females injected with neb-colloostatin showed oocyte ooplasm alterations under the stereomicroscope. the terminal oocytes are much longer and wider in the control ovary than in that from the peptide injected female and patency in follicular epithelium is obvious in the control but not in the ovary from the injected female. apoptosis and autophagy are involved in follicle cell death after neb-colloostatin injection after morphological observations, the entire ovaries were stained in order to detect peptide-induced changes in the organization of the f-actin cytoskeleton and chromatin, and presence of indicators of cell death of the terminal ovarian follicles. the first marker, oregon green phalloidin, delimited changes in the f-actin cytoskeleton. in the control follicular cells, a strong fluorescent signal was localized subcortically, and was also detected in the oocyte. maximum patency of follicular epithelium was visible. injection of neb-colloostatin caused failure of f-actin organization in these cells. they lost their regular f-actin staining pattern as a result of depolymerization of f-actin microfilaments. the application of nmole of peptide caused full depolymerization of f-actin, therefore the positive oregon green-phalloidin staining signal was not detected, but a lower dose of the peptide inhibited intracellular space formation in the follicular epithelium. the sulphorhodamine derivative of valylalanylaspartic acid fluoromethyl ketone staining showed that neb-colloostatin induced the activation of caspases in the ovarioles. in this assay, we observed that the degree of caspase activation depended on the applied hormone dose. one day after injection of neb-colloostatin, at a dose of or nmole per insect, caspase activity was detected in all ovaries of the studied females. injection of 1 nmol of peptide caused caspase activation only in oocytes. injection of neb-colloostatin at a dose of nmole/female activated caspases in the follicle cells, whereas the oocyte was resorbed. next, we used propidium iodide staining to visualize changes in chromatin organization. the nuclei of follicular cells from ovarian follicles of females injected with neb-colloostatin were characterized by highly condensed chromatin redistributed along the nuclear membrane as compared to the control. moreover, in the nuclei of follicular cells from the peptide-injected females, propidium iodide revealed dna fragmentation events visible as small, red dots, compared to the control nuclei, the morphology of which looked normal. the mono-dansyl-cadaverin and the orange acridine staining were used for tracing the induction of autophagic vacuoles and detection of lysosomes in order to assess the type of programmed cell death, i.e. type i or type ii, caused by the injection of neb-colloostatin. after neb-colloostatin injection at a dose of 10 nmol of peptide per female, fluorescent and confocal microscopy revealed extensive vacuolization of the cytoplasm of follicular cells indicating degeneration of their contents in an autophagy-like process. orange acridine staining showed the presence of lysosomes in follicular cells of terminal follicles of peptide-injected females, whereas autophagic vacuoles detected by mdc staining were visible as distinct dot-like blue structures. neb-colloostatin injection induces changes in the ultrastructure of ovarian follicles in the t. molitor ovary, the distinct differences in ultrastructure between the control follicles and the follicles of the neb-colloostatin injected females become visible. in the control follicles, the follicular epithelium surrounding the terminal oocyte at the 7th stage of development is composed of the cuboid cells with microvilli directed to the oocyte. the nucleus of the follicle cells contains quite a number of the small clumps of heterochromatin and several vast irregular nucleoli. the follicle cells exhibit characteristic morphology of active secretion. in the basal part of the follicle cells, the cisterns of the rough endoplasmic reticulum and the golgi complexes are well-developed. the apical part is rich in the electron dense secretory vesicles, mitochondria and the dilated cisterns of rer. in the follicles of injected females, the follicle cells surrounding the terminal oocyte show no morphological evidence for synthetic activity. a part of the follicle cells is still cube-shaped, although their apical surfaces become more or less wrinkled. the nuclei of these cells usually contain a granular nucleoplasm and a few extensive and irregular nucleoli. less and smaller, than in the control, heterochromatin clumps are visible. the cisterns of rer are clearly less developed than in the cells of the control follicles. in the apical cytoplasm, there are no secretory granules and a few elements of rer and mitochondria are located in the vicinity of the nucleus. this area is filled with numerous free ribosomes. after injection of neb-colloostatin, many follicle cells reveal much more advanced changes. these cells are irregular in shape and their cytoplasm is vacuolated in the varying degrees. in the nuclei of these cells, chromatin becomes highly condensed. in cytoplasm, the detected vacuoles are electron-transparent or include the degradated fragments of cytoplasm. the cells shrink and gradually lose their connection with the neighboring follicle cells and the basal lamina. in the control follicle, between the terminal oocyte at the 7th stage of development and the follicle cells, is a narrow cleft filled with the electron dense material, forming a vitelline membrane surrounding the oocyte. the cortical ooplasm in particular contains numerous yolk granules and lipid droplets.. free ribosomes, rough endoplasmic reticulum systems and stacks of annulate lamellae are also abundant in the ooplasm. in the follicle of females injected with neb-colloostatin, the intercellular cleft between the surface of the follicular epithelium and oocyte is wider than in the control follicle. the cortical region of ooplasm contains fewer and significantly small yolk granules but relatively numerous lipid droplets. the number of free ribosomes is dramatically reduced. the rough endoplasmic reticulum is less developed. it displays vesicles with few ribosomes attached. stacks of annulate lamellae are not found in the ooplasm. in the cortical ooplasm, in between the yolk granules are autophagic vacuoles containing materials in various degrees of degradation. the core of oocyte is usually devoid of yolk granules and the autophagic vacuoles with a highly degraded content are particularly numerous. discussion this study describes the process of atresia in ovarian follicles induced by the gonadoinhibitory peptide neb-colloostatin during the first gonadotropic cycle of t. molitor females. in recent years, several control strategies have been proposed as species-specific and non-polluting methods for reducing or suppressing pest reproduction, especially insect–pathogen interactions or development of the sterile insect technique. as previously reported, injection of neb-colloostatin and neb-tmof into t. molitor females causes gonadoinhibitory effects on ovarian development, however, the mode of action of these peptides remains unknown. wasielewski and rosiński showed that the injection of approximately mmole of neb-coollostatin into females on days and of the first reproductive cycle inhibits patency formation, slowing down vitellogenesis and ovarian development, delaying ovulation and ovarian development, and reducing the number of eggs laid. in t. molitor, part of the inhibitory process of the oocytes growth is manifested by qualitative and quantitative changes in proteins sequestered and synthesized by the ovary. the authors showed that the injections of neb-colloostatin reduced by about 60% total protein content in ovaries and induced qualitative changes in ovarian protein patterns. electrophoretic analysis pointed out that neb-colloostatin caused a loss of two polypeptides and a strong reduction of four another. all these polypeptides were the most abundant in ovaries of the control females. on the other hand, neb-colloostatin injection increased the concentration of two polypeptides, which were found in very small amounts in ovaries of control females. however, neb-colloostatin did not cause a significant change in protein composition of the haemolymph. it seems that neb-colloostatin reduces proteins sequestration by ovaries and the peptide action is more specific to ovaries. in this paper, we described the morphological alterations caused by neb-colloostatin injections in the oocytes and the follicular epithelium of t. molitor egg chambers. microscopic observation and molecular analysis of the ovarian follicle facilitated a more detailed characterization of the gonadoinhibitory action of this peptide. first, the doses of peptide injected into females applied in this work were much lower than those of wasielewski and rosiński. this suggests that the peptide is very potent and could be acting at a physiologically relevant concentration. second, a very important phase of oocyte growth is the formation of intercellular spaces in the follicular epithelium enabling the transport of nutrients to the growing vitellogenic oocytes. our findings show that the direct action of neb-colloostatin on follicular cells resulting in f-actin cytoskeleton disorganization, and in consequence inhibition of patency formation, may prompt peptide-induced follicular atresia. peptide injections strongly inhibited oocyte development. next, apoptosis was switched on in affected follicles and, in consequence, resorption of oocyte remnants occurred. in the t. molitor follicular epithelium, apart from the disorganization of the f-actin cytoskeleton network, other alterations comprised typical features of apoptosis including caspase activation, chromatin condensation and dna fragmentation. moreover, evidence for a distinct type of physiological cell death, autophagocytosis, was also observed in oocytes and follicular cells. incorporation of cellular contents into autophagosomes suggests that these cell components can be released into the insect haemolymph as a result of lysosomal enzyme activity. during autophagy all cellular contents are disintegrated into autophagic vacuoles and destroyed by the cell’s lysosomes. aguirre et al. showed in dipetalogaster maxima that atresia induced by food deprivation was characterized by loss of the ability of oocytes to uptake vitellogenin, partial vitellin proteolysis and activation of cathepsin d-like peptidases. these authors suggested that cathepsin d-like peptidase is involved in the process of follicle degeneration, probably by promoting early yolk protein degradation, and would also be involved in the oosorption of atretic follicles. apoptosis can also be detected during extensive autophagocytosis in the course of insect oogenesis. for example, uchida et al. realized that apoptosis may occur in the epithelial cells of degenerating follicles in the final stages of the atretic process within developing mosquito ovaries. these authors detected caspase and catepsin-like proteinase activation, as well as the presence of fragmented dna in the epithelial region of the culex pipiens pallens atretic follicles but not in normally developing follicles within the same ovaries. autophagosomes have also been observed in electron micrographs of degenerating mid-stage follicles from drosophila virilis. valentzas and colleagues suggested that apoptosis and autophagy act synergistically to achieve more efficient elimination of degenerated nurse cells and abnormal eggs chambers. boolmart et al. suggested that autophagocytosis depends on an intact cytoskeleton. thus intermediates and microfilaments are considered to be essential for the initial formation of autophagosomes, whereas their subsequent fusion with lysosomes depends on microtubuli. in spite of the neb-colloostatin-induced disorganization of the f-actin cytoskeleton network in follicular cells, we observed the occurrence of autophagosomes, however, we cannot explain these data at present. moreover, electron microscopy of neb-colloostatin-induced changes in the t. molitor ovary showed that the apical cytoplasm of the follicular cells is filled with numerous free ribosomes, suggesting that after peptide injection the follicular cells probably have a higher demand on protein biosynthesis, especially in the perspective of increased formation of the autophagic vacuoles process. this result shows that the process of polyribosomes formation can be inhibited, indicating the contributing effect of neb-colloostatin.  in summary, our results show that in t. molitor females, neb-colloostatin injection resulted in atresia of ovarian follicles both the apoptotic and the autophagic mechanism of programmed cell death. the first observable effect of neb-colloostatin-induced atresia is the disappearance of patency in the follicular epithelium. this probably inhibits the active uptake of vitellogenins by the oocyte. induction of apoptosis and autophagy of ovarian follicles may contribute to more efficient removal of atretic follicles during ovarian follicular regression in insects. 
this peptide inhibits intercellular space formation in follicular epithelium and results in slowed vitellogenesis, delayed ovulation, reduced number of eggs laid and presumably cell death in the terminal follicles. moreover, the apical cytoplasm of follicular cells is filled with numerous free ribosomes, probably indicating a higher demand for protein biosynthesis, especially in preparation for autophagic vacuole formation. we report that injection of nanomolar doses of neb-colloostatin induce ovarian follicle atresia in 4-day old females during their first gonadotropic cycle. the injection of neb-colloostatin into t. molitor females causes gonadoinhibitory effects on ovarian development. neb-colloostatin induces atresia in the mealworm ovary. light microscope observations revealed morphological changes in the ovary: after neb-colloostatin injection the terminal oocytes are significantly smaller and elicit massive follicle resorption, but the control terminal follicles possess translucent ooplasm in oocytes at different stages of vitellogenesis. we observed f-actin cytoskeleton disorganization, induction of caspase activity, changes in chromatin organization and autophagic vacuole formation. confocal and electron microscope examination showed that peptide injection causes changes in the morphology indicating death of follicular cells. degeneration of t. molitor follicles includes changes in morphology and viability of follicular cells, and oosorption as a consequence of these changes. on the other hand, the process of polyribosomes formation is inhibited, indicating the contributing effect of this hormone. a patency is visible in follicular epithelium of the control vitellogenic oocytes, whereas peptide injection inhibits intercellular space formation and, in consequence, inhibits vitellogenesis. however, as does the form of cell death in the terminal follicle, the mode of action of neb-colloostatin remains unknown. we tested neb-colloostatin for a sterilizing effect on females of tenebrio molitor.
 the injection of neb-colloostatin into t. molitor females causes gonadoinhibitory effects on ovarian development. this peptide inhibits intercellular space formation in follicular epithelium and results in slowed vitellogenesis, delayed ovulation, reduced number of eggs laid and presumably cell death in the terminal follicles. however, as does the form of cell death in the terminal follicle, the mode of action of neb-colloostatin remains unknown.  we tested neb-colloostatin for a sterilizing effect on females of tenebrio molitor. we report that injection of nanomolar doses of neb-colloostatin induce ovarian follicle atresia in 4-day old females during their first gonadotropic cycle. light microscope observations revealed morphological changes in the ovary: after neb-colloostatin injection the terminal oocytes are significantly smaller and elicit massive follicle resorption, but the control terminal follicles possess translucent ooplasm in oocytes at different stages of vitellogenesis. a patency is visible in follicular epithelium of the control vitellogenic oocytes, whereas peptide injection inhibits intercellular space formation and, in consequence, inhibits vitellogenesis. confocal and electron microscope examination showed that peptide injection causes changes in the morphology indicating death of follicular cells. we observed f-actin cytoskeleton disorganization, induction of caspase activity, changes in chromatin organization and autophagic vacuole formation. moreover, the apical cytoplasm of follicular cells is filled with numerous free ribosomes, probably indicating a higher demand for protein biosynthesis, especially in preparation for autophagic vacuole formation. on the other hand, the process of polyribosomes formation is inhibited, indicating the contributing effect of this hormone.  neb-colloostatin induces atresia in the mealworm ovary. degeneration of t. molitor follicles includes changes in morphology and viability of follicular cells, and oosorption as a consequence of these changes. insect ovaryneb-colloostatingonadoinhibitory peptidefollicle atresiaapoptosisautophagy 