Control of life, death, and differentiation in cultured midgut cells of the lepidopteran, Heliothis virescens

Summary Differentiated cells in the insect midgut depend on stem cells for renewal. We have immunologically identified Integrin β₁, a promotor of cell-cell adhesion that also induces signals mediating proliferation, differentiation, and apoptosis on the surfaces of culturedHeliothis virescens midgut cells; clusters of immunostained integrin β₁-like material, indicative of activated integrin, were detected on aggregating midgut columnar cells. Growth factor-like peptides (midgut differentiation factors 1 and 2 [MDF1 and MDF2]), isolated from conditioned medium containingManduca sexta midgut cells, may be representative of endogenous midgut signaling molecules. Exposing the cultured midgut cells toBacillus thuringiensis (Bt) toxin caused large numbers of mature differentiated cells to die, but the massive cell death simultaneously induced a 150–200% increase in the numbers of midgut stem and differentiating cells. However, after the toxin was washed out, the proportions of cell types returned to near-control levels within 2 d, indicating endogenous control of cell-population dynamics. MDF1 was detected immunologically in larger numbers of Bt-treated columnar cells than controls, confirming its role in inducing the differentiation of rapidly produced stem cells. However, other insect midgut factors regulating increased proliferation, differentiation, as well as inhibition of proliferation and adjustment of the ratio of cell types, remain to be discovered. Products mentioned in this article are not endorsed by the U.S. Department of Agriculture.     

Cleavage and gastrulation of the dendrobranchiate shrimp Penaeus monodon (Crustacea,Malacostraca, Decapoda)

The cleavage pattern of the black tiger shrimp Penaeus monodon was analyzed from the first division until gastrulation. Observations were based on microscopy combined with the use of fluorescent dyes, histological techniques, and computer based three-dimensional reconstructions. Early cleavage is holoblastic and follows a stereotypic pattern, which largely corresponds to what is known from other dendrobranchiate decapods. However, for the first time in this group, we report the presence of an intracellular structure throughout early development. This intracellular body (icb) marks the lineage of one of the two enlarged and division-delayed mesendoderm cells that initiate gastrulation. The identity of the icb as a natural marker and putative determinant of the germ line and its implications on the establishment of the body axes are discussed. The icb as a landmark reveals that the same stereotypic cell division pattern can lead to different fates of individual cells. Hence, the results of this study permit an additional approach to study the relation between cell lineage pattern and the identity of cell lineages.     

Early embryonic development of the freshwater shrimp Caridina multidentata (Crustacea,Decapoda, Atyidae)

Decapod crustaceans show a great diversity of developmental modes at all levels. In particular, early cleavage varies from total via mixed to superficial modes and from determinate cleavage with a stereotyped pattern to indeterminate cleavage. However, the ground pattern of early decapod development is not clear. To address this problem, we studied the early embryonic development of the caridean shrimp Caridina multidentata with a combination of confocal laser scanning microscopy, scanning electron microscopy, 4D microscopy and 3-D reconstruction software. Despite a yolk-rich egg, the cleavage is holoblastic and shows a distinct pattern of blastomere arrangement, characterized by two interlocking cell bands. This resembles the conditions in dendrobranchiate shrimps, which most likely are the sister group to Pleocyemata to which C. multidentata belongs. Hence, our results offer the possibility to assume total cleavage with blastomeres arranged in two interlocking cell bands as ancestral cleavage mode for Decapoda.     

Pleonal muscle development in the shrimp Penaeus (Litopenaeus) vannamei (Crustacea: Malacostraca: Decapoda: Dendrobranchiata)

Penaeoidean shrimp pleonal muscle is a valuable economic resource worldwide, but little is known of its development during larval stages. The development of pleonal muscle in Penaeus (Litopenaeus) vannamei was studied by rhodamine-phalloidin staining and laser-scanning confocal microscopy. Dorsal pleonal muscle was first evident at the protozoea I stage while ventral pleonal muscle was present by the protozoea II stage. Identifiable ventral pleonal muscles were evident by the protozoea III stage and all ventral muscle types were present in the mysis I. The tail flex response began at the mysis stage and growth of existing pleonal muscles continued. The pleopods formed during the mysis stages, with coxal and basis muscles developed by mysis III. The pleopods became functional beginning with the first post-larval stage. We conclude that the pleonal muscle pattern of P. vannamei larvae is similar to that of adult Penaeus setiferus, and that homologous muscles are present. The major formation of dorsal pleonal muscles occurs during the protozoea II stage, while significant development of ventral pleonal muscles occurs during the protozoea III stage.     

Phylogenetic systematics of the reptantian Decapoda (Crustacea, Malacostraca)

Although the biology of the reptantian Decapoda has been much studied, the last comprehensive review of reptantian systematics was published more than 80 years ago. We have used cladistic methods to reconstruct the phylogenetic system of the reptantian Decapoda. We can show that the Reptantia represent a monophyletic taxon. The classical groups, the 'Palinura', 'Astacura' and 'Anomura' are paraphyletic assemblages. The Polychelida is the sister-group of all other reptantians. The Astacida is not closely related to the Homarida, but is part of a large monophyletic taxon which also includes the Thalassinida, Anomala and Brachyura. The Anomala and Brachyura are sister-groups and the Thalassinida is the sister-group of both of them. Based on our reconstruction of the sister-group relationships within the Reptantia, we discuss alternative hypotheses of reptantian interrelationships, the systematic position of the Reptantia within the decapods, and draw some conclusions concerning the habits and appearance of the reptantian stem species.     

Ultrastructural studies of midgut epithelium formation in Lepisma saccharina L. (Insecta, Zygentoma)

At the end of embryogenesis of Lepisma saccharina L. (Insecta, Zygentoma), when the stomodaeum and proctodaeum are completely formed, the midgut epithelium is replaced by the primary midgut, a yolk mass is surrounded by a cell membrane. Midgut epithelium formation begins in the 1st larval stage. Energids migrate toward the yolk periphery and aggregate just beneath the cell membrane. They are gradually enclosed by cell membrane folds of the primary midgut. Single cells are formed. Succeeding energids join just formed cells. Thus, groups of cells, regenerative cell groups, are formed. Their number gradually increases. The external cells of the regenerative cell groups transform into epithelial cells and their basal regions spread toward the next regenerative cell groups. Epithelial cells of neighboring regenerative cell groups join each other to form the epithelium. At the end of the 2nd larval stage, just before molting, degeneration of newly the formed epithelium begins. Remains of organelles and basal membrane occur between the regenerative cell groups. The new epithelium is formed from the regenerative cell groups, which are now termed stem cells of the midgut epithelium.     

Cell Death in the Epithelia of the Intestine and Hepatopancreas in Neocaridina heteropoda (Crustacea,Malacostraca)

The endodermal region of the digestive system in the freshwater shrimp Neocaridina heteropoda (Crustacea, Malacostraca) consists of a tube-shaped intestine and large hepatopancreas, which is formed by numerous blind-ended tubules. The precise structure and ultrastructure of these regions were presented in our previous studies, while here we focused on the cell death processes and their effect on the functioning of the midgut. We used transmission electron microscopy, light and confocal microscopes to describe and detect cell death, while a quantitative assessment of cells with depolarized mitochondria helped us to establish whether there is the relationship between cell death and the inactivation of mitochondria. Three types of the cell death were observed in the intestine and hepatopancreas–apoptosis, necrosis and autophagy. No differences were observed in the course of these processes in males and females and or in the intestine and hepatopancreas of the shrimp that were examined. Our studies revealed that apoptosis, necrosis and autophagy only involves the fully developed cells of the midgut epithelium that have contact with the midgut lumen–D-cells in the intestine and B- and F-cells in hepatopancreas, while E-cells (midgut stem cells) did not die. A distinct correlation between the accumulation of E-cells and the activation of apoptosis was detected in the anterior region of the intestine, while necrosis was an accidental process. Degenerating organelles, mainly mitochondria were neutralized and eventually, the activation of cell death was prevented in the entire epithelium due to autophagy. Therefore, we state that autophagy plays a role of the survival factor.     

Morphology of the midgut trunk in the penaeid shrimp, Sicyonia ingentis, highlighting novel nuclear pore particles and fixed hemocytes

The morphology of the midgut trunk (MGT) in the penaeid shrimp Sicyonia ingentis was examined by light and scanning and transmission electron microscopy. Although the function of the MGT is poorly understood, it is not involved with the digestion and absorption of nutrients, and it appears to be the surface of a shrimp least protected from penetration by potential pathogens. As described for other decapod crustaceans, the MGT in shrimp is composed of a simple columnar epithelium separated from a layer of connective tissue by a thick basal lamina. Beneath the basal lamina is a previously unreported layer of hemocytes, exclusively of the granulocyte variety, embedded in a matrix continuous with the basal lamina and extending into the connective tissue. This layer was observed in four other species of penaeid shrimp. Granulocytes in circulation can phagocytose and encapsulate foreign material and the granules contain antibacterial molecules, lysosomal enzymes, and prophenoloxidase. We suggest that the granulocytes associated with the basal lamina have matured at this site and are well positioned to fight potential pathogens that have penetrated the epithelial layer of the MGT. A second observation is the presence of clusters of cylinders bound to the nuclear pores of the epithelial cells. The possibility that these clusters are viruses, organelles, or abnormal organelles induced by disease or toxic materials is discussed. These unique particles were observed in S. ingentis but none of the other penaeid shrimp we examined.     

Role of integrin beta1-like protein in proliferation and differentiation of cultured stem cells from midgut of Heliothis virescens

Cultured midgut cells from Heliothis virescens larvae were incubated with anti-human integrin beta1 made in rabbit and then passed over a column of magnetic beads bound to anti-rabbit IgG (MACS, Miltenyi Bergisch Gladbach, Germany). Cells bound to integrin beta1 antibody also bound to the anti-rabbit IgG on the magnetic beads (MACS) and were retained in the column while it remained in the magnetic field. Non-bound cells were eluted at this time. They did not stain with anti-integrin antibody just after elution. Removing the column from the magnetic field allowed cells bound to the beads-integrin beta1 antibody to be eluted. All of these cells stained with human anti-integrin beta1 upon elution. Each cell fraction was cultured in medium for 3 days. During this time, the populations of cells tended to return to heterogeneous staining patterns characteristic of control populations. However, cells that did not stain immediately with anti-integrin beta1 antibody exhibited double the rate of multiplication and 8 times more differentiation than the integrin-antibody positive cells that eluted later, as well as the non-treated control cells. In a second experiment, midgut cells were incubated for 4 days with various titers of human anti-integrin beta1 to block surface integrin beta1-like reactive sites. Stem cells blocked with anti-integrin beta1 antibody during incubation exhibited double the rate of differentiation than non-treated control cells and those showing anti-integrin beta1-positive stain upon elution.     

Degenerative and regenerative processes involved in midgut pseudotumor formation in the stick insect (Carausius morosus)

Spontaneous and experimentally induced pseudotumor formation in Carausius morosus impairs the midgut tissue homeostasis. Spontaneous pseudotumor formation begins by the break down of a single or a small group of columnar cells (CCs) and is followed by the degeneration of neighboring CCs. There are not only marked similarities but also decisive differences between normal dying CCs in healthy specimens and the degeneration of CCs leading to pseudotumors: in both cases, the apical cell parts with the nucleus are extruded into the midgut lumen, but only during of pseudotumor formation an “amorphous substance” originates from the basal parts of the CCs. Hemocytes are attracted to this substance and form a nodule‐like aggregation, which is responsible for the phenotype of pseudotumors. Pseudotumor infestation has also an impact on the midgut nidi, which consist of an intestinal stem cell and several CC progenitor cells. In healthy specimens only one progenitor cell per nidus differentiates at a time, but, several to all progenitor cells differentiate simultaneously in pseudotumor‐infested specimens. Extirpation of the ingluvial ganglion in healthy specimens results in an immediate onset of pseudotumor formation and a dramatic acceleration of pseudotumor growth. Importantly, the ultrastructural characteristics of spontaneous and experimentally induced pseudotumors are identical. This supports the idea that the stomatogastric nervous system plays an integral role in the maintenance of midgut tissue homeostasis. J. Morphol., 2009. © 2009 Wiley‐Liss, Inc.     

The population dynamics of some freshwater carideans (Crustacea: Decapoda) in Hong Kong,with special reference to Neocaridina serrata (Atyidae)

During a 15-month investigation of the population dynamics of three caridean shrimp species in the Lam Tsuen River, New Territories, Hong Kong, the numbers of the commonest species, Neocaridina serrata (Stimpson)(Atyidae), were reduced as water temperatures fell. By contrast, Caridina lanceifrons Yu became more numerous during the winter. The relatively scarce Macrobrachium hainanense (Parisi)(Palaemonidae) tended to be more abundant during the summer, and the abundance of this species was directly correlated with that of N. serrata. The population size of these two species was positively correlated with water temperatures prevailing one and two months prior to the collection of samples, but there was no significant relationship between C. lanceifrons abundance and water temperature.All species exhibited similar growth patterns with an inflection at the attainment of sexual maturity, occurring after the (presumed) 14th moult for N. serrata and in the (presumed) 13th and 18th stadium for C. lanceifrons and M. hainanensis respectively. Ovigerous shrimps of all species were only recorded when water temperatures exceeded 20 °C and the % incidence of ovigerous N. serrata was positively correlated with prevailing temperatures. It is suggested that restriction of freshwater caridean breeding periods by low temperatures may be common in the subtropics while perennial breeding is more likely to be typical of tropical regions.The mean carapace length of N. serrata populations was negatively correlated with prevailing temperatures, but more strongly correlated with temperatures recorded one and two months prior to sampling when gametogenesis may have taken place. Smallest mean sizes were recorded in late summer upon the cessation of juvenile recruitment. N. serrata has a maximum longevity of approximately 12 months and reproduction occurs in the 7th month of life or later. The hatchlings are well developed and clutch size is not related to the size of the brooding female. An annual life cycle, as seen in N. serrata, may be typical of many small freshwater carideans.Department of Zoology, The University of Hong Kong     

Histopathological and ultrastructural changes in the antennal gland,midgut, hepatopancreas,and gill of grass shrimp following exposure to hexavalent chromium

Grass shrimp, Palaemonetes pugio, were exposed for 1 month to subacute concentrations of hexavalent chromium (0.5, 1.0, 2.0, 4.0 ppm) after which the gills, midgut, hepatopancreas, and antennal glands were examined for histopathological and ultrastructural changes. Pathological changes were greatest in the antennal glands, followed by hepatopancreas, gills, and midgut. Severe changes occurred in some shrimp, even at 0.5 ppm chromium. Cells of all tissues frequently had both swollen mitochondria and rough endoplasmic reticulum. Small, spherical or ring-like intranuclear inclusions, possibly indicative of cellular hyperactivity or manifestions of chromium and/or protein complexes, were most prevalent in the hepatopancreas and antennal glands but also occurred in the midgut and gills. Other major degenerative changes in the antennal glands were restricted to the labyrinth and included diminution of basal plasmalemmal infoldings and cytoplasmic density, nuclear hypertrophy followed by widespread nuclear pyknosis and epithelial desquamation. In severely altered hepatopancreas hypertrophy was indicated for the basal laminae, nuclei, and possibly for the nucleoli. There was an apparent reduction in mitotic events and many observed mitotic nuclei were abnormal. Abnormal midgut hypertrophy was present in only 8 of 20 examined shrimp, exposed to 0.5 and 1 ppm chromium. Further, the gills of only 10 of the 40 examined chromium-exposed shrimp possessed abnormal features detectable with ligh microscopy. Ultrastructural analysis of the latter indicated an increase in lysosomes and a decrease in cytoplasmic density. In addition, there was a pronounced diminution in the degree of lamellar, subcuticular plasmalemmal infolding. This latter feature is postulated to be a mechanism for the regulation of chromium influx. Possible explanations for most observed alterations in the above tissues are proposed.     

Structure and Ultrastructure of the Endodermal Region of the Alimentary Tract in the Freshwater Shrimp Neocaridina heteropoda (Crustacea,Malacostraca)

The freshwater shrimp Neocaridina heteropoda (Crustacea, Malacostraca, Decapoda) originates from Asia and is one of the species that is widely available all over the world because it is the most popular shrimp that is bred in aquaria. The structure and the ultrastructure of the midgut have been described using X-ray microtomography, transmission electron microscopy, light and fluorescence microscopes. The endodermal region of the alimentary system in N. heteropoda consists of an intestine and a hepatopancreas. No differences were observed in the structure and ultrastructure of males and females of the shrimp that were examined. The intestine is a tube-shaped organ and the hepatopancreas is composed of two large diverticles that are divided into the blind-end tubules. Hepatopancreatic tubules have three distinct zones – proximal, medial and distal. Among the epithelial cells of the intestine, two types of cells were distinguished – D and E-cells, while three types of cells were observed in the epithelium of the hepatopancreas – F, B and E-cells. Our studies showed that the regionalization in the activity of cells occurs along the length of the hepatopancreatic tubules. The role and ultrastructure of all types of epithelial cells are discussed, with the special emphasis on the function of the E-cells, which are the midgut regenerative cells. Additionally, we present the first report on the existence of an intercellular junction that is connected with the E-cells of Crustacea.     

Microsatellite loci from the pink shrimp Farfantenaeus notialis (Crustacea,Decapoda)

Farfantepenaeus notialis is an important resource for fisheries in Cuba. For this reason and for a sustainable exploitation it is important to study their population structure and genetic variability. We report and characterize microsatellites as genetic markers from this species. Fifteen microsatellite polymerase chain reaction (PCR) primers were designed and tested in some individuals from different populations. Seven pair of primers showed reliable amplification products and five were polymorphic. The allele number ranged from 4 to 33, and the observed and expected heterozygosities were relatively high with values between 0.63 and 0.74 and 0.56 and 0.81, respectively. Departures from Hardy–Weinberg equilibrium were observed for all loci.     

Regenerative cells and the architecture of beetle midgut epithelia

The architectural ground plan of beetle and other insect midguts is represented by a monolayer of epithelial cells arranged in a cylindrical configuration. Proliferation and differentiation of regenerative cells maintain the integrity of this monolayer in the face of continual losses of individual cells through cytoplasmic budding and/or expulsion of entire epithelial cells. Peritrophic membranes have conventionally been considered universal features of insect midguts that function to protect vulnerable microvillar surfaces of the midgut epithelium from abrasion by ingested food; however, peritrophic membranes were found in only a small fraction of the adult beetle species examined in this study. In adult beetles, midgut epithelial cells are continually replaced by cells recruited from populations of mitotic regenerative cells that are interspersed among the differentiated epithelial monolayer. To remain contiguous with the other cells in the midgut monolayer, some of these proliferating populations have adopted evaginated configurations of cells that extend for varying distances from the basal surface of the monolayer. These configurations are referred to as regenerative crypts or pouches and consist of progenitor cells and stem cells. The presence, the relative densities, and the relative lengths of these regenerative pouches vary considerably among families of beetles. Placement of longitudinal muscles of the midgut relative to the proximodistal axes of these regenerative pouches also varies among species of beetles. The presence, the size, and the density of regenerative cell populations are related to 1) feeding habits of adult beetles, 2) presence of peritrophic membranes, and 3) expulsion of entire midgut epithelial cells or fragments of these epithelial cells into midgut lumens. © 2012 Wiley Periodicals, Inc.     

The role of stem cells in midgut growth and regeneration

Summary TheManduca sexta (L.) [Lepidoptera: Sphingidae] andHeliothis virescens (F.) [Lepidoptera: Noctuidae] midguts consist of a pseudostratified epithelium surrounded by striated muscle and tracheae. This epithelium contains goblet, columnar, and basal stem cells. The stem cells are critically important in that they are capable of massive proliferation and differentiation. This growth results in a fourfold enlargement of the midgut at each larval molt. The stem cells are also responsible for limited cell replacement during repair. While the characteristics of the stem cell population vary over the course of an instar, stem cells collected early in an instar and those collected late can start in vitro cultures. Cultures of larval stem, goblet, and columnar cells survive in vitro for several mo through proliferation and differentiation of the stem cells. One of the two polypeptide differentiation factors which have been identified and characterized from the culture medium has now been shown to be present in midgut in vivo. Thus the ability to examine lepidopteran midgut stem cell growth in vitro and in vivo is proving to be effective in determining the basic features of stem cell action and regulation. Mention of any product in this publication does not imply endorsement by the USDA.     

Ultrastructural changes in the midgut epithelium of the first larva of Allacma fusca (Insecta, Collembola, Symphypleona)

Abstract. In the newly hatched larva in Allacma fusca , the midgut epithelium was fully developed and formed by flattened epithelial cells surrounding the yolk mass in the midgut lumen. Immediately after hatching, the first larva began to feed; the migut lumen was filled with the yolk mass and food (mainly algae). Regenerative cells typical of the developing midgut epithelium of many insects were not observed. Initially, midgut cells of the larva were cuboidal but became columnar in shape with distinct regionalization in the distribution of cell organelles. Furthermore, urospherites appeared in the midgut cell cytoplasm, i.e., structures characteristic for the midgut epithelium of insects having no Malpighian tubules. As a result, cells with the capacity for digestion, absorption, and excretion were observed to be completely formed in the first larval stage.     

A spring-matrix model for pigment translocation in the red ovarian chromatophores of the freshwater shrimp Macrobrachium olfersi (Crustacea, Decapoda)

A model for intracellular transport of pigment granules in the red ovarian chromatophores of the freshwater shrimp Macrobrachium olfersi is proposed on the basis of shifts in the equilibrium of resting forces acting on an elastic pigment matrix. The model describes a pigment-transport mechanism in which mechanochemical protein motors like kinesin and myosin alternately stretch and compress a structurally unified, elastic pigment matrix. Quantifiable properties of the spring-matrix obey Hooke's Law during the rapid phases of pigment aggregation and dispersion. The spring-like response of the pigment mass is estimated from previous kinetic experiments on pigment translocation induced by red pigment concentrating hormone, or by the calcium ionophore A23187. Both translocation effectors trigger an initial phase of rapid pigment aggregation, and their removal or washout after complete aggregation produces a phase of rapid pigment dispersion, followed by slow pigment translocation. The rapid-phase kinetics of pigment transport are in reasonable agreement with Hooke's Law, suggesting that such phases represent the release of kinetic energy, probably produced by the mechanochemical protein motors and stored in the form of matrix deformation during the slow phases of translocation. This semiquantitative model should aid in analyzing intracellular transport systems that incorporate an elastic component.     

Ultrastructure and regeneration of midgut epithelial cells in Lithobius forficatus (Chilopoda,Lithobiidae)

Lithobius forficatus (Myriapoda, Chilopoda, Lithobiidae) is a widespread species of centipede that is common across Europe. Its midgut epithelial cells are an important line of defense against toxic substances that originate in food, such as pathogens and metals. Despite this important role, the biology of the midgut epithelium is not well known. Here we describe the ultrastructure of the midgut epithelium, as well as the replacement of degenerated midgut epithelial cells. The midgut epithelium of L. forficatus is composed of digestive, secretory, and regenerative cells. The cytoplasm of digestive cells shows regionalization in organelle distribution, which is consistent with the role of these cells in secretion of enzymes, absorption of nutrients, and accumulation of lipids and glycogen. Secretory cells, which do not reach the luminal surface of the midgut epithelium, possess numerous electron‐dense and electron‐lucent granules and may have an endocrine function. Hemidesmosomes anchor secretory cells to the basal lamina. Regenerative cells play the role of midgut stem cells, as they are able to proliferate and differentiate. Their proliferation occurs in a continuous manner, and their progeny differentiate only into digestive cells. The regeneration of secretory cells was not observed. Mitotic divisions of regenerative cells were confirmed using immunolabeling against BrdU and phosphohistone H3. Hemocytes associate with the midgut epithelium, accumulating between the visceral muscles and beneath the basal lamina of the midgut epithelium. Hemocytes also occur among the digestive cells of the midgut epithelium in animals infected with Rickettsia‐like microorganisms. These hemocytes presumably have an immunoprotective function in the midgut.     

Ultrastructural and histological study of degenerative changes in the antennal glands, hepatopancreas, and midgut of grass shrimp exposed to two dithiocarbamate biocides

The histological and ultrastructural alterations observed in the antennal glands, hepatopancreas, and midgut of grass shrimp exposed to either a 50% potassium dimethyldithiocarbamate biocide (Busan-85; 5–60 ppb) for 14 days, or to a different biocide, composed of 15% sodium dimethyldithiocarbamate and 15% disodium ethylene bisdithiocarbamate (Aquatreat DNM-30), for 3–4 days (60–140 ppb) and 28–35 days (40–120 ppb), were compared and contrasted with the normal morphological features in control shrimp. Only those experimental shrimp that exhibited various degrees of branchial abnormality were examined. Although the alterations in Busan-exposed shrimp were generally more pronounced, the antennal glands of 32 out of 36 experimental shrimp exhibited abnormalities that were manifested primarily as increased secretory activity by the labyrinth cells. In dithiocarbamate-exposed shrimp with “black gills”, the labyrinth epithelium exhibited moderate nuclear hypertrophy, apparent cell sloughing, intense secretory activity, and occasional melanized lesions; alterations in the antennal gland coelomosac included nuclear pyknosis, a general deterioration of podocyte organization, and an unusual increase in hemolymph density adjacent to affected tissues. Although there was an apparent increase in mitotic activity in the hepatopancreatic tubules of shrimp exposed to Aquatreat for 28–35 days, degenerative changes were most frequent and extensive in the hepatopancreas and midgut of dithiocarbamate-exposed shrimp with “black gills”. These observed changes included the diminution of the basal midgut and hepatopancreatic tubular system, moderate midgut hypertrophy, pronounced activity by the hepatopancreatic fixed phagocytes, development of mitochondrial inclusions and megamitochondria, loss of cytoplasmic density, hepatopancreatic nuclear pyknosis, and irreversible degeneration of hepatopancreatic tubule apices. This study suggests that some of the observed abnormal/pathological changes are the indirect consequence of branchial degeneration. A number of possible defensive reactions to dithiocarbamate poisoning, including heterostasis, phagocytosis, encapsulation, and the possible participation of reserve inclusion cells are proposed.