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Sublethal effects of malathion insecticide on growth of the freshwater crab Poppiana dentata (Randall, 1840) (Decapoda: Trichodactylidae)

Abstract

Pesticides can interfere with various aspects of growth and the normal molt cycle of a crustacean. Poppiana dentata (Randall, 1840), an indigenous crab species, spends most of its life cycle in, and proximal to, benthic sediments in which pesticide residues can reside. This study sought to assess the sublethal effects of a locally-used, commercial malathion insecticide on growth aspects of P. dentata. Juvenile crabs were obtained from berried females collected in northwest Trinidad. Young crabs were placed in a control (insecticide-free) treatment and an exposure treatment involving continuous exposure to the malathion insecticide, at 10 µg/L concentration over five months (n = 4 crabs/treatment). Carapace width (CW), length (CL) and intermolt period were recorded and used to derive size increment, specific growth rate (SGR), growth curves and logistic equations. Malathion-exposed crabs exhibited irregular patterns in SGR and size increment. Exposed crabs also exhibited a delay in molting and longer intermolt periods, compared to the control crabs (p < 0.05). Breakpoint (17.5 mm CW) and maximum size (CW = 25.77 (1+exp (1.500-0.056t))-1) for exposed crabs were relatively smaller than those of the control (22.11 mm CW; CW = 34.30 (1+exp (1.774-0.035t))-1). Findings indicate that sublethal exposure to malathion insecticide altered growth patterns in P. dentata, some of which can influence maturity and later cascade into secondary consequences for local populations.

Keywords
Endpoint; organophosphate pesticide; specific growth rate; Trichodactylidae

INTRODUCTION

Growth is a key endpoint for evaluating the effects of both endogenous and exogenous factors. Growth is characterized as a discontinuous process for crustaceans, whereby increases in size take place when the rigid, calcified cuticle is shed during ecdysis or molting. The release of ecdysteroid (e.g., ecdysone) into circulation by the paired Y-organs is usually associated with the onset of molting, but neurohormones produced in the X-organ-sinus gland complex (e.g. molt-inhibiting hormone (MIH) and crustacean hyperglycemic hormone (CHH)), regulate ecdysteroid release and synthesis by the Y-organs (Dell et al., 1999Dell, S.; Sedlmeier, D.; Böcking, D. and Dauphin-Villemant, C. 1999. Ecdysteroid biosynthesis in crayfish Y-organs: feedback regulation by circulating ecdysteroids. Archives of Insect Biochemistry and Physiology, 41: 148-155. ; Nakatsuji et al., 2000Nakatsuji, T.; Keino, H.; Tamura, K.; Yoshimura, S.; Kawakami, T.; Aimoto, S. and Sonobe, H. 2000. Changes in the Amounts of the Molt-Inhibiting Hormone in Sinus Glands during the Molt Cycle of the American Crayfish, Procambarus clarkii. Zoological Science, 17: 1129-1136.; Chung, 2010Chung, J.S. 2010. Hemolymph ecdysteroids during the last three molt cycles of the blue crab, Callinectes sapidus: quantitative and qualitative analyses and regulation. Archives of Insect Biochemistry and Physiology, 73: 1-13. ). This interplay of hormones along intricate pathways, coupled with limb regeneration, control the molting cycle in crustaceans.

Sublethal effects of toxicants can compromise an organism’s fitness through impairing essential traits, including growth (Connell et al., 1999Connell, D.; Lam, P.; Richardson, B. and Wu, R. 1999. Introduction to Ecotoxicology. Oxford, Blackwell Science, 180p.). Growth can also be indirectly impacted through modification of energy allocation and sudden metabolic demands, brought on by the stress responses of an organism exposed to a toxicant. Xenobiotics, in particular, can interfere with the normal intermolt period (number of days between two successive molts) of the molt cycle of a crustacean, along with negatively influencing size increments and growth rates. Xenobiotic compounds are considered ‘foreign compounds’ which are not normally present in the environment, but rather introduced and/or produced by human activities (Connell et al., 1999Connell, D.; Lam, P.; Richardson, B. and Wu, R. 1999. Introduction to Ecotoxicology. Oxford, Blackwell Science, 180p.). Even at sublethal levels, toxicants like pesticides, can increase respiratory energy demands for estuarine crustaceans while consequently lowering the energy reserved for new tissues, as well as growth efficiency rates for juvenile development (Negro et al., 2011Negro, C.L.; Senkman, L.E.; Montagna, M.C. and Collins, P.A. 2011. Freshwater Decapods and Pesticides: An Unavoidable Relation in the Modern World. p. 197-226. In: M. Stoytcheva (ed), Pesticides in the Modern World: Risks and Benefits. Rijeka, InTech. ). These toxicants can even impair the foraging ability of organisms, compromising energy acquisition, and by extension, growth.

Alteration of growth in life stages sensitive to toxicants can be useful as early warning indicators for detrimental effects in aquatic populations (Negro et al., 2011Negro, C.L.; Senkman, L.E.; Montagna, M.C. and Collins, P.A. 2011. Freshwater Decapods and Pesticides: An Unavoidable Relation in the Modern World. p. 197-226. In: M. Stoytcheva (ed), Pesticides in the Modern World: Risks and Benefits. Rijeka, InTech. ). Disruption in growth and development at an earlier, vulnerable stage of life in an organism (e.g., juvenile stage) can eventually lead to a succession of deleterious consequences, extending from the individual to population and community levels. This is because growth is essentially linked to other crucial aspects like reproduction and fitness. Juvenile development is a fundamental phase in the lifespan of a species, such that this stage, along with reproduction, represents common endpoints used to evaluate interspecific vulnerability, in relation to chronic toxicity (Amiard-Triquet and Amiard, 2013Amiard-Triquet, C. and Amiard, J.-C. 2013. Introduction. p. 1-14. In: C. Amiard-Triquet; J.-C. Amiard and P. S. Rainbow (eds), Ecological Biomarkers Indicators of Ecotoxicological Effects, Boca Raton, CRC Press. ). Examining how a toxicant modifies the typical growth pattern in the juvenile phase can provide in-depth understanding of the long term, ecological risks faced in adulthood. Various studies have looked at the impacts of organophosphate (OP) insecticides on embryo and juvenile stages of freshwater decapods. In all cases OPs were shown to disrupt the normal growth of the life stage. These studies record the effects of sublethal and lethal concentrations of chlorpyrifos-based insecticide on growth and survival of the freshwater prawn, Palaemonetes argentinus Nobili, 1901 (Montagna and Collins, 2007Montagna, M.C. and Collins, P.A. 2007. Survival and growth of Palaemonetes argentinus (Decapoda; Caridea) exposed to insecticides with chlorpyrifos and endosulfan as active element. Archives of Environmental Contamination and Toxicology, 53: 371-378. ), chronic exposure of a commercial chlorpyrifos formulation on growth of the trichodactylid crab, Trichodactylus borellianus Nobili, 1896 (Montagna, 2010Montagna, M.C. 2010. Toxicity of chlorpyrifos as active element of a commercial formulate on juvenile crab Trichodactylus borellianus. Natura Neotropicalis, 1: 31-43.), Diazinon 60EC exposure on young-of-the-year and juveniles of two crayfish species, Pacifastacus leniusculus (Dana, 1852) and Orconectes limosus (Rafinesque, 1817) (Buřič et al., 2013Buřič, M.; Kouba, A.; Máchová, J.; Mahovská, I. and Kozák, P. 2013. Toxicity of the organophosphate pesticide diazinon to crayfish of differing age. International Journal of Environmental Science and Technology, 10: 607-610. ) and sublethal concentrations of chlorpyrifos on embryonic hatching success, hatching time and post-hatching survival of the burrowing crab, Zilchiopsis collastinensis (Pretzmann, 1968) (Negro et al., 2014Negro, C.L.; Senkman, L.E.; Marino, F.; Lorenzatti, E. and Collins, P.A. 2014. Effects of chlorpyrifos and endosulfan on different life stages of the freshwater burrowing crab Zilchiopsis collastinensis P.: protective role of chorion. Bulletin of Environmental Contamination and Toxicology, 92: 625-630.).

Assessing the effects of non-discriminate biocides, like OP insecticides, on growth can be beneficial in terms of understanding the long-term consequences faced by non-target populations. Freshwater, non-target species are particularly vulnerable to impacts of insecticides and require high research priority since contamination of their aquatic habitats can easily occur due to extensive spraying from nearby agricultural lands or via community vector control. OP pesticides represent popular biocides for combating insects, due to their cost-effectiveness, high toxicity toward pests and relatively quick environmental degradation. OPs, such as malathion, encompass the top insecticides for governmental and commercial use (Fulton and Key, 2001Fulton, M.H. and Key, P.B. 2001. Acetylcholinesterase inhibition in estuarine fish and invertebrates as an indicator of organophosphorus insecticide exposure and effects. Environmental Toxicology and Chemistry, 20: 37-45. ). It is commonly applied as part of vector control measures for combating mosquito-borne diseases in many tropical countries, including in Trinidad and Tobago. A total of 15 pesticide products are registered for use in Trinidad and Tobago and contain malathion as the active ingredient (AI) (Ministry of Health, 2020Ministry of Health. 2020. List of registered agricultural pesticides 2020 revised. p. 1-4. Chemistry, Food and Drugs Division, Port of Spain, Trinidad and Tobago.). An active ingredient refers to the chemical constituent contained in a pesticide product that controls pests and is usually listed by its name and percentage by weight on the product label (USEPA, 2019USEPA (United States Environmental Protection Agency). 2019. Basic Information about Pesticide Ingredients. Available at Available at https://www.epa.gov/ingredients-used-pesticide-products/basic-information-about-pesticide-ingredients . Accessed on 25 January 2021.
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). Malathion is an organophosphorus compound that represents an anticholinesterase insecticide, such that the mode of action is inhibition of the neurotransmission enzyme, acetylcholinesterase (Bookhout and Costlow, 1976Bookhout, C.G. and Costlow, J.D. 1976. Effects of mirex, methoxychlor, and malathion on development of crabs. Florida, United States Environmental Protection Agency, 102p.). The latter has been designated as a feasible biomarker for indicating exposure to an OP insecticide. Despite its higher environmental biodegradability in relation to organochlorine pesticides, it still remains a highly toxic chemical for both target and non-target biota. A consequence of its biodegradable nature would be the need for repeated applications of the chemical and the development of insect pest resistance; both of which would incite higher malathion concentrations during application and introduce greater risk to non-target biota. Early research attributes malathion to delayed, late offspring development of brachyurans, such as for the mud crab, Rhithropanopeus harrisii (Gould, 1841) and blue crab, Callinectes sapidus Rathbun, 1896 (Bookhout and Costlow, 1976Bookhout, C.G. and Costlow, J.D. 1976. Effects of mirex, methoxychlor, and malathion on development of crabs. Florida, United States Environmental Protection Agency, 102p.). The authors noted a delayed zoeal development and lengthening of the time period between hatching and the first crab stage, with increasing concentrations of malathion.

To date, there is no information on the effects of OP pesticides on the freshwater crab species, native to Trinidad. Poppiana dentata (Randall, 1840) is one of only three freshwater, indigenous crab species reported for Trinidad (Singh et al., 2020aSingh, D.S.; Alkins-Koo, M.;. Rostant, L.V and Mohammed, A. 2020a. Heart rate responses to different temperatures in juvenile Poppiana dentata. Brazilian Journal of Biology, 80: 30-38. ) and its lifecycle is spent mainly in the freshwater environment, living and feeding in close proximity to the water and benthic sediments where pesticides can reside. Organophosphates, like malathion, are toxic to freshwater species (Pham, 2017Pham, B.; Miranda, A.; Allinson, G. and Nugegoda, D. 2017. Evaluating the non-lethal effects of organophosphorous and carbamate insecticides on the yabby (Cherax destructor) using cholinesterase (AChE, BChE), Glutathione S-Transferase and ATPase as biomarkers. Ecotoxicology and Environmental Safety, 143: 283-288.), so there is a need for investigating the exposure risk faced by non-target biota, residing in contaminated aquatic habitats. Therefore, the goal of this study was to conduct such an assessment, by examining the chronic effects of a commercial malathion insecticide, at sublethal concentration, on the growth of juvenile P. dentata crabs under laboratory conditions. Growth patterns of crabs in this bioassay were evaluated by: (1) specific growth rate (SGR) and (2) breakpoint sizes, growth curves and logistic equations derived from growth components of intermolt period and size, as measured by carapace width (CW) and carapace length (CL).

MATERIAL AND METHODS

Acquisition of crabs and experimental design

This study comprised juvenile crabs obtained from wild stock females. Berried females of P. dentata (Family Trichodactylidae) were collected from Bamboo, in the northwest of Trinidad (10°37’49.2”N 61°25’51.2”W). This site is a freshwater waterway that drains into the Caroni River, located in the northwestern region of Trinidad. Mesh traps, with mesh size of 3.18 mm, were deployed along three accessible points along the waterways, approximately 50 m apart. Each site in which the captured P. dentata resided was shown to have low or minimal biocide presence, as affirmed by negative test results derived from prior qualitative (presence/absence) testing of water from the collection points (in situ), using rapid pesticide detection Agri-Screen® Tickets (Neogen® 2019). Berried females (n = 2) were removed from the traps and their species identification confirmed using the species key of Magalhães and Türkay (2008Magalhães, C. and Türkay, M. 2008. Taxonomy of the Neotropical freshwater crab family Trichodactylidae, IV. The genera Dilocarcinus and Poppiana (Crustacea, Decapoda, Trichodactylidae). Senckenbergiana biologica, 88: 185-215.).

The growth period for this bioassay was selected as five months, since growth beyond this point would have been possibly influenced by natural reproductive maturation processes. This period was selected using a prior baseline study on P. dentata, which yielded breakpoint sizes at structural reproductive maturity of 16.84 mm CW (female) and 28.40 mm CW (male). The 5-month period was selected based on the length of time for male crabs to reach a size of 28.75 ± 2.98 mm CW (Singh et al., 2020bSingh, D.S.; Alkins-Koo, M.;. Rostant, L.V and Mohammed, A. 2020b. Baseline growth of the Trinidad freshwater crab Poppiana dentata (Randall, 1840) under laboratory conditions. Brazilian Journal of Biology, 81: 377-386. ).

The commercial malathion insecticide was chosen for this study as it is extensively used in local vector control and agricultural applications and was assumed to be prevalent in indigenous aquatic environments. Malathion is one of the pesticides that has been involved in dengue prevention and vector control strategies for the Aedes aegypti (Linnaeus, 1762) mosquito throughout Caribbean countries, for approximately 20 to 30 years (Rawlins, 1998Rawlins, S.C. 1998. Spatial distribution of insecticide resistance in Caribbean populations of Aedes aegypti and its significance. Pan American Journal of Public Health, 4: 243-251. ). In addition, previous work assessed sublethal concentrations (0.1, 1 and 10 µg/L) of the same malathion insecticide on acetylcholinesterase (AChE) activity in P. dentata, revealing significantly high inhibitions (> 70 %; p < 0.05) across tissues for 10 µg/L (Singh, unpublished data). Sublethal effects of a toxicant can still adversely impair organism fitness and imply a change in crucial physiological processes, such as growth (Newman and Unger, 2003Newman, M.C. and Unger, M.A. 2003. Fundamentals of Ecotoxicology, 2nd Edition. Boca Raton, Lewis Publishers, 432p.). It was, therefore, reasonable to hypothesize that growth in P. dentata would be affected by this OP and at the normal working concentration.

Chemical description and preparation

The organophosphate insecticide used in this bioassay is commercially available as an emulsifiable concentrate (EC) formulation, Fyanon® (CHEMINOVA), containing 57 % malathion as the active ingredient (AI). This insecticide product is state registered for use (locally) and is available for purchase through many local commercial retailers by agricultural and domestic users. The Material Safety Data Sheet (MSDS) for the insecticide was obtained from the distributing commercial company from which the pesticide was purchased. The original concentration of malathion contained within the total formulation volume (listed by the manufacturer’s MSDS) was then computed. From this, the quantity of commercial formulation that had to be added to the test solution, to maintain a 10 μg/L concentration of malathion in the test water, was determined. Dilution of the original formulation was then performed using the relevant volumes and according to application directions on the product label. Dilution was carried out with distilled water, in order to prepare the exposure test solutions at 10 µg/L concentration. Diluting the pesticide with water (versus an organic solvent like acetone) according to the preparation guidelines resulted in a more practical test solution that was comparable to solutions generally prepared by pesticide users, and by extension, what would typically be introduced into the habitats of P. dentata. The same concentration was analyzed prior to the experiment, using Gas Chromatography-Electron Capture Detector (Shimadzu GC 2010). This affirmed that the concentration of malathion contained within the insecticide formulation was maintained and was close to the nominal concentration in the test water (retention time: 7.473 minutes; concentration: 9.36 µg/L). It should also be noted that any reference to malathion in this study results, refers explicitly to effects caused by the commercial formulation of the malathion insecticide and not the analytical grade form of malathion.

Experimental conditions and growth measurements

Newly hatched crabs were selected and assigned to two treatment groups; a control group (n = 4 crabs) and an exposure group (n = 4 crabs). The latter group is referred to as the malathion-exposed cohort or exposure cohort throughout this work. It should be noted that any newly hatched crabs with missing or lost appendages were not used in this study. Therefore, the number of hatched crabs that met these selection criteria, as well as the material resources (lab infrastructure and resources) that were available prior to initiation of the experiment, both limited the replicate number and insecticide concentrations that could be evaluated. In addition, the number of replicates within the designated laboratory space was limited, to ensure ethical conditions for the animals were met.

The growth patterns associated with the control cohort were taken to represent growth under normal conditions and were monitored synchronously with the exposure cohort, under the same physiochemical and photoperiod conditions. All replicate crabs were of the same age. The malathion-exposed cohort were of initial size 2.96 ± 0.29 mm CW, 2.29 ± 0.10 mm CL (mean ± SD) and those of the control group were 3.04 ± 0.36 mm CW, 2.60 ± 0.22 mm CL. Each crab was reared in a glass aquarium (length 8 inches × width 6 inches × height 6 inches) containing 1600 mL of laboratory prepared, de-chlorinated water. Crabs were observed daily over a 5-month period, under laboratory conditions of dissolved oxygen (DO) at 6.6 ± 0.1 mg/L, pH at 7.60 ± 0.36, temperature at 26.1 ± 0.1 °C and a photoperiod of 12 h light:12 h dark. Previous monitoring at the specimen collection site revealed similar temperatures (mean ± SD: 26.1 ± 0.3 °C; range: 25.8-26.4 °C), pH (7.59 ± 0.31; 6.79-7.93) and DO concentration (6.1 ± 0.9 mg/L; 6.02-7.84 mg/L). The photoperiod was chosen since it is analogous to the diurnal cycle that occurs throughout most of the year in Trinidad.

The newly hatched crabs were allowed to acclimate to laboratory conditions for a period of six to seven days, during which they were allowed to undergo their first three molts under the same pesticide-free growing conditions as those of the control. After their third molt and hardening of their exoskeletons, crabs of the exposure cohort were then subject to continual exposure of 10 µg/L malathion insecticide for the remainder of the 5-month period. Any variations in growth during the pesticide exposure phase, therefore, would have been due to the toxicant, and not from acclimation to the laboratory conditions. In addition, this initial period in which growth is typically highest, allowed the crabs to grow unhindered in pesticide-free conditions, and by extension, ensured that the crabs were healthy and well acclimated.

A renewal of freshly prepared (10 µg/L malathion insecticide) test solution was performed every two days for the exposure cohort. The quantity of insecticide formulation was added to the test solution, such that malathion was maintained at a 10 μg/L concentration. Therefore, the cohort was exposed to a fairly continuous concentration of the insecticide malathion (at 10 µg/L) in the test water, at this renewal rate. Montagna and Collins (2007Montagna, M.C. and Collins, P.A. 2007. Survival and growth of Palaemonetes argentinus (Decapoda; Caridea) exposed to insecticides with chlorpyrifos and endosulfan as active element. Archives of Environmental Contamination and Toxicology, 53: 371-378. ) applied a similar renewal regime when assessing the effects of another OP insecticide (Terminator Ciagro 48 % chlorpyrifos) on juvenile Pa. argentinus freshwater prawns. The authors applied a renewal rate of 70 % of the test solution in each container, every two days. In our experiment water renewal was done for the control cohort with laboratory-prepared (pesticide-free) water only. The water in each test aquarium was also monitored weekly, for temperature, DO and pH to ensure that water quality remained constant, so as to avoid varying the malathion concentration.

The feeding regime entailed crabs being fed daily, using a varied diet of Hikari crab cuisine pellets (Kyorin Food Industries Co. Ltd.) and segments of the aquatic waterweed, Egeria densa. The latter represents a common weed species generally found throughout habitats of P. dentata (personal observation). The regime involved crabs of CW < 5 mm being fed 2 g of pellets and 2 g of E. densa segments, CW ≥ 5-15 mm were fed 5 g of the pellets and 4 g of E. densa segments, CW > 15-25 mm were fed 10 g of pellets and 6 g of E. densa plant segments, and CW > 25 mm were fed 24 g of pellets and 6 g of E. densa segments. This diet and regime were considered suitable for proper growth since it was similarly applied in the prior, baseline growth study (Singh et al., 2020bSingh, D.S.; Alkins-Koo, M.;. Rostant, L.V and Mohammed, A. 2020b. Baseline growth of the Trinidad freshwater crab Poppiana dentata (Randall, 1840) under laboratory conditions. Brazilian Journal of Biology, 81: 377-386. ) in which crabs were observed to exhibit normal growth and behavior. Removal of uneaten food and feces was performed along with renewal of test water. It should be noted that there were no occurrences of mortality throughout the monitoring period.

Carapace width (CW) and carapace length (CL) were measured from the molted exoskeletons or exuviae, in order to avoid stress by handling molted crabs. A digital caliper (VINCA DCLA) was used to measure to the nearest hundredth of a millimeter. CW was measured as the maximum width of the carapace and CL as the distance between the median notch of the frontal margin and the posterior margin of the carapace. General observations were also made on feeding when they consumed their exuviae molt and the appearance of their exoskeletons following molting. These observations were taken during their early growth and served as qualitative indications of how fast crabs of the exposure and control cohorts hardened and advanced through their molt cycles.

Any size increase was computed as the difference between the CW or CL measurements (mm) between two consecutive molts. Size increments for each molt were then expressed as percentages of the corresponding premolt CW or CL. This represented the percent size increment, or the extent to which the CW or CL postmolt exceeded that of the respective premolt (Hartnoll, 1982Hartnoll, R.G. 1982. Growth. p. 111-196. In: D.E. Bliss and L.G. Abele (eds), The Biology of Crustacea. Embryology, Morphology, and Genetics, Vol. 2. New York, Academic Press.). The intermolt period was recorded as the number of days between two successive molts, with the actual day of ecdysis not included in this period. Breakpoint analysis of CW differences was used to estimate structural maturity for each cohort, using scatter plots created in R (R Development Core Team, 2016R Development Core Team. 2016. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna. Available at https://www.r-project.org/. Accessed on 30 June 2018.) and the package ‘strucchange’ (Zeileis et al., 2002Zeileis, A.; Leisch, F.; Hornik, K. and Kleiber, C. 2002. Strucchange: an R Package for testing for structural change in linear regression models. Journal of Statistical Software, 7: 1-38. ). Specific growth rate (SGR) was calculated for each molt from the relevant CW or CL size increment and intermolt period, using the method from Romano and Zeng (2006Romano, N. and Zeng, C. 2006. The effects of salinity on the survival, growth and hemolymph osmolality of early juvenile blue swimmer crabs, Portunus pelagicus. Aquaculture, 260: 151-162. ). Monthly SGR was also calculated using the same procedure. The mean size class (in terms of CW) and cumulative intermolt period were also derived for each molt and used to plot separate growth curves for the exposure and control groups. Variation in CW size (mm) over time (cumulative intermolt period) was also fitted to a standard logistic equation, similar to that used by Jin et al. (2001Jin, G.; Li, Z. and Xie, P. 2001. The growth patterns of juvenile and precocious Chinese mitten crabs, Eriocheir sinensis (Decapoda, Grapsidae), stocked in freshwater lakes of China. Crustaceana, 74: 261-273.). Moreover, variation in CW served as the dimensional change used to estimate structural maturity for this study.

Data analysis

All statistical analyses and graphical depictions were done using R version 3.3.1 (R Development Core Team, 2016R Development Core Team. 2016. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna. Available at https://www.r-project.org/. Accessed on 30 June 2018.). An alpha level of 0.05 was used for all statistical tests. A number of statistical procedures require datasets to come from normal populations and have homogeneity of variances (Zar, 1999Zar, J.H. 1999. Biostatistical analysis, 4th edition. New Jersey, Prentice Hall, 663p.). The current datasets were found to be non-normal (Shapiro-Wilk test; p < 0.05), with unequal variances or heteroscedasticity (Levene’s test; p < 0.05). Accordingly, non-parametric tests were used. Specifically, the Spearman rank correlation coefficient was used to assess the relationship between carapace dimension and intermolt period. The Mann-Whitney U test was used to detect differences between the control and exposure cohorts, in terms of CL, CW, intermolt period, size increment and SGR. The Kruskal-Wallis and post hoc Dunn’s test were also used for pairwise comparisons of SGR of different molts as well as for SGR of different months. The package ‘dunn.test’ (Dinno, 2014Dinno, A. 2014. Dunn’s test of multiple comparisons using rank sums. R Foundation for Statistical Computing. Available at Available at https://mran.microsoft.com/snapshot/2014-09-26/web/packages/dunn.test/index.html . Accessed on 30 June 2018.
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) was used for conducting both of these tests.

RESULTS

Initial sizes of crabs at the start of monitoring were similar. Juvenile P. dentata crabs of the malathion-exposed group were initially 2.96 ± 0.29 mm CW, 2.29 ± 0.10 mm CL at the start of the monitoring period. This was similar to the control group, which had an initial size of 3.04 ± 0.36 mm CW, 2.60 ± 0.22 mm CL. A general observation was made on the extent of crabs feeding, such that those of the control cohort consistently ate all food provided with little to no items remaining in their aquaria. However, the malathion-exposed crabs were noted to eat only a portion of their total pellets and plant segments administered, while the uneaten remainders were removed in each water renewal. Juvenile crabs of the control treatment were observed to consume their molted exuviae within a few hours of their initial molts. This growth behavior was similarly noted for the exposure cohort but only for their first three molts during the pesticide-free period, prior to their contact with 10 µg/L malathion insecticide. The exoskeletons of both control and exposure cohorts changed from translucent to opaque within 24 to 48 hours of molting. The postmolt consumption behavior and fast transitioning of the cuticle appearance indicated a typical and rapid hardening of the exoskeleton. This behavior changed for the control crabs, as they grew older (> 15 mm CW), whereby consumption of exuviae and opacity of the exoskeletons took place within 1 to 3 days after molting. However, juveniles of the exposure treatment were observed to take relatively longer to consume their exuviae (4 ± 2 days), during their exposure to the malathion insecticide.

Within the 5-month study period, all P. dentata crabs that were exposed to 10 µg/L malathion insecticide (n = 4) only reached their eighth molt, by the fourth month of the monitoring period. A total of 32 molts were recorded for this group over the five months period and molt events halted thereafter; with this cohort failing to attain their ninth and tenth molts (Tab. 1). In contrast, those of the control cohort (n = 4) experienced a total of 40 molts, each crab having undergone 10 molt events by the fifth month.

Table 1.
Mean size increase for carapace width (CW) and carapace length (CL) with standard deviations (SD), along with percentage size increments for control and malathion-exposed cohorts of Poppiana dentata crabs.

Generally, CW increased with intermolt period for both cohorts (Fig. 1) and this was congruent with CW being significantly correlated with intermolt period for the malathion-exposed (rho = 0.89, p < 0.05) and control (rho = 0.99, p < 0.05) groups. This was also reflected in the size class distributions for control and malathion-exposed crabs, where mean intermolt period increased with mean CW size class (Fig. 1A, 1B). However, patterns in size increment for exposed crabs were different from the control, following exposure to the malathion insecticide. Initially (pre-exposure), CW and CL size increases and respective percent increments of the exposure cohort were similar to those of the control cohort (Tab. 1), however, this was only observed for the initial three molts, prior to insecticide exposure (pesticide-free period). Size increases of juveniles within this pre-exposure period followed a corresponding trend to that of control crabs, in which high percent increments were associated with initial molts, namely 38 % for CW for the first molt and 38 % CL for the second molt (Tab. 1). Likewise, CW and CL size increments of 44 % and 32 %, for control crabs were the highest for the first molt and second molts, respectively (Tab. 1).

Figure 1.
Growth aspects for Poppiana dentata in relation to: mean intermolt period and CW size classes of control (A) and malathion-exposed (B) crabs over the 5-month period; intermolt period and carapace width (CW) for control (C) and malathion-exposed (D) crabs. Error bars in A and B plots represent the standard errors for the intermolt periods. The dashed line in B separates pre-exposure (no pesticide) conditions for the malathion-exposed cohort and exposure to the malathion insecticide at 10 µg/L concentration. Breakpoints in C and D are denoted by segmented lines for the respective cohort.

Following exposure to the insecticide (after the third molt), inconsistent patterns in CW and CL size increase were noted for the subsequent five molts of malathion-exposed crabs. Moreover, maximum percent CL (56 %) and CW (50 %) size increments occurred around the fourth and fifth molts for these crabs (Tab. 1). This contrasted with the pattern observed for the control cohort whereas the crabs grew older, their dimensional growth slowed down and the intermolt period became longer toward the end of five months (42 ± 6 days). Consequently, their minimum CW (19 %) and CL (17-19 %) percent increments were also associated with the later molts, at around the ninth or tenth molt, toward the end of the five months (Tab. 1).

Despite the irregularity in size increase for malathion-exposed crabs, differences in CW, CL and associated size increments were not significant between the exposure group and the control group (CW: W = 29, p > 0.05; CL: W = 30, p > 0.05; CW size increment: W = 24, p > 0.05; CL size increment: W = 604, p > 0.05). However, intermolt periods for the malathion-exposed cohort were substantially different from those of the control cohort (χ 2 = 6.821, p < 0.05). The exposed cohort also showed signs of a delay in molting and an extension in intermolt period upon exposure to the malathion insecticide; as evidenced by the lack of molt events after the eighth molt (Fig. 1B).

With regards to breakpoint determination and scatter plots, a segmented relationship was noted between CW and intermolt period for both cohorts (Fig. 1C, D). Breakpoint for the exposure cohort was 17.5 mm CW, at 13 days intermolt period, with the overlapping region of the slopes coinciding with a range of 14.74 to 17.5 mm CW and 12 to 13 days intermolt period. This size range was below the breakpoint for the control group (22.11 mm CW; 18 days intermolt period). This disparity was also noted in the growth equations and growth curves. The fitted logistic equation for the control cohort is expressed as CW = 34.30 (1+exp (1.774-0.035t))-1, however, dissimilar components were noted for the malathion-exposed cohort, expressed as CW = 25.77 (1+exp (1.500-0.056t))-1. Growth curves also reflected the variation in dimensional growth between cohorts (Fig. 2A, B). Generally, fast growth of CW was seen early in the monitoring period but eventually slowed down toward the end. While this trend was noted across the two cohorts, CW growth for the exposure group reached asymptote before 100 days at around 25 mm CW (Fig. 2B); a much earlier time and smaller size compared to that of the control at around 150 days and a CW of 35 mm (Fig. 2A).

Figure 2.
Growth curves for Poppiana dentata in relation to: variation in CW size (mm) over time (cumulative intermolt period) for control (A) and malathion-exposed (B) crabs over the 5-month period; mean specific growth rate (SGR) for control (C) and malathion-exposed (D) crabs for the same 5-month period. Error bars in C and D represent the standard errors for SGR. The dashed line in D separates the initial three molts under pre-exposure (no pesticide) conditions for the malathion-exposed cohort and those molts experienced during exposure to the malathion insecticide at 10 µg/L concentration.

The patterns in SGR for the malathion-exposed group were only like the control group during the pre-exposure or pesticide-free period. This was evident from a high and similar SGR for the first molt of the control (CW = 0.23 ± 0.07 mm/day; CL = 0.24 ± 0.09 mm/day) and the exposure group, prior to malathion exposure (CW = 0.25 ± 0.01 mm/day; CL = 0.22 ± 0.09 mm/day). The initial CW SGR was also significantly higher than that of the second molt for both cohorts (control: Z = 2.451, p < 0.05; malathion-exposed: Z = 2.861, p < 0.05) (Fig. 2C, D).

Following exposure to the malathion insecticide, differences in CW SGR between control and malathion-exposed groups were not significant (W = 29, p > 0.05). Likewise, variations in CL SGR were not significantly different between control and malathion-exposed groups (W = 31, p > 0.05). Despite these minimal differences, the trend in CW SGR for the malathion-exposed group was noted to deviate from the control group. A sudden increase in SGR was noted for the exposed group at their fourth molt and this coincided with the time at which these crabs began their exposure to the malathion insecticide (Fig. 2D).

CW SGR also differed across the months for malathion-exposed crabs (χ 2 = 18.396, p < 0.05) and was the highest in the first month, and significantly greater than the second (Z = 2.703, p < 0.05) and subsequent months. This corresponded with the highest CW percent increment (50 %) of the exposure group (Tab. 1) occurring within the initial month, as well as this group experiencing up to five molts events within the first month. This was similar to crabs of the control cohort, where their CW SGR for the first month was also considerably higher than that of the second (control: Z = 2.333, p < 0.05) and subsequent months. Additionally, the control crabs experienced three to six molt events within their first month. These molts of the control cohort were also associated with the highest CW percent increment (44 %) (Tab. 1). CL SGR also differed significantly across molts and months for the malathion-exposed group (across molts: χ 2 = 5.355, p < 0.05; monthly: χ 2 = 21.451, p < 0.05). This was dissimilar to the pattern exhibited by the control crabs, where CL SGR differences across molts and months were not significant (across molts: χ 2 = 5.355, p > 0.05; monthly: χ 2 = 6.132, p > 0.05).

DISCUSSION

Exposure to the malathion insecticide appears to alter the molting behavior and growth patterns of P. dentata crabs. While the new exoskeletons of molted malathion-exposed crabs transitioned in appearance, similar to the control group, juveniles of the former treatment were observed to take relatively longer to consume their exuviae (4 ± 2 days), following exposure to the malathion insecticide. This could have been due to the bioconcentration of malathion within the exoskeleton, thereby decreasing palatability. Though this was only qualitatively assessed, it still indicates alteration in molting behavior for juveniles in terms of their normal prompt feeding on their molted exoskeleton. This can delay assimilation of calcium from the molted shell, prolong the completion of recalcification and exoskeleton hardening, and by extension, increase juvenile vulnerability. Rapid hardening of the brachyuran exoskeleton after ecdysis can be facilitated by prompt ingestion of the molted, calcified shell. This exuvia contains a considerably amount of calcium (30-70 %) and this can be re-ingested during postmolt and used for restoring calcium deficits (Wheatly, 1999Wheatly, M.G. 1999. Calcium homeostasis in Crustacea: The evolving role of branchial, renal, digestive and hypodermal epithelia. Journal of Experimental Zoology, 283: 620-640. ). Therefore, rapid hardening conveys the advantage of efficient growth, through rapid turnover of molt cycles, while reducing juvenile susceptibility to predators and intraspecific cannibalism.

Patterns in CW and CL size increment also appear to be altered from exposure to the malathion insecticide. In addition, exposed crabs show signs of a delay in molting and an extension in intermolt period upon exposure; as evidenced by the lack of molting events after the eighth molt. The delay in the molting cycle is expected since pesticides, among other pollutants, have been known to inhibit molting (Rodríguez et al., 2007Rodríguez, E.; Medesani, D.A. and Fingerman, M. 2007. Endocrine disruption in crustaceans due to pollutants: a review. Comparative Biochemistry and Physiology. Part A Molecular and Integrative Physiology, 146: 661-671. ). Insecticides, in particular, have been reported to delay molting in decapods as Buchanan et al. (1970Buchanan, D.V.; Millemann, R.E. and Stewart, N.E. 1970. Effects of the insecticide Sevin on various stages of the Dungeness crab, Cancer magister. Journal of the Fisheries Board of Canada, 27: 93-104.) observed a delay in molting for young (zoeae stage) Cancer magister Dana, 1852, resulting from exposure to a carbamate insecticide (Sevin) at concentrations as low as 0.1 µg/L. Disruption to the normal molt cycle from toxicant exposure can be attributed to disturbances to the paired Y-organs, and neurosecretory structures, such as the X-organ and sinus gland, thereby resulting in altered levels of MIH and other neurohormones (Montagna and Collins, 2005Montagna, M.C. and Collins, P.A. 2005. Toxicity of glyphosate upon the freshwater prawn Palaemonetes argentinus. Nauplius, 13: 149-157.; 2007Montagna, M.C. and Collins, P.A. 2007. Survival and growth of Palaemonetes argentinus (Decapoda; Caridea) exposed to insecticides with chlorpyrifos and endosulfan as active element. Archives of Environmental Contamination and Toxicology, 53: 371-378. ; Negro et al., 2011Negro, C.L.; Senkman, L.E.; Montagna, M.C. and Collins, P.A. 2011. Freshwater Decapods and Pesticides: An Unavoidable Relation in the Modern World. p. 197-226. In: M. Stoytcheva (ed), Pesticides in the Modern World: Risks and Benefits. Rijeka, InTech. ). This was demonstrated in the effects of 24 h exposure to heptachlor (organochlorine) of larvae of the marine lobster, Homarus americanus H. Milne Edwards, 1837, whereby the suspension in the onset of molting was correlated with significant reductions in ecdysteroid levels present in circulation (Snyder and Mulder, 2001Snyder, M.J. and Mulder, E.P. 2001. Environmental endocrine disruption in decapod crustacean larvae: hormone titers, cytochrome P450, and stress protein responses to heptachlor exposure. Aquatic Toxicology, 55: 177-190. ). Increased levels of cytochrome P450 dependent enzymes were also observed in H. americanus and considered to be upregulated by ecdysteroid hormones, alluding to the connection between these detoxifying enzymes and molting; as well as the possibility that their induction was induced by the heptachlor toxicant (Snyder and Mulder, 2001Snyder, M.J. and Mulder, E.P. 2001. Environmental endocrine disruption in decapod crustacean larvae: hormone titers, cytochrome P450, and stress protein responses to heptachlor exposure. Aquatic Toxicology, 55: 177-190. ). Aside from growth hormonal disturbances, the malathion insecticide could also disrupt energy homeostasis of P. dentata, upon exposure, slowing growth and possibly affecting reproduction. Biotransformation of pollutants, such as pesticides, typically involves ATP consumption, leading to higher energy consumption and deviation of energy from normal growth.

The lack of the ninth and tenth molts in the exposure cohort was especially suggestive of longer intermolt periods, for these molts, and subsequent ones beyond the end of the 5-month monitoring period. These later intermolt periods would have been longer than the corresponding molt periods of the control group (ninth molt: 29 ± 3 days; tenth molt: 42 ± 3 days) and that reported for a baseline group (ninth molt: 33 ± 11 days; tenth molt: 41 ± 8 days) from a previous study (Singh et al., 2020bSingh, D.S.; Alkins-Koo, M.;. Rostant, L.V and Mohammed, A. 2020b. Baseline growth of the Trinidad freshwater crab Poppiana dentata (Randall, 1840) under laboratory conditions. Brazilian Journal of Biology, 81: 377-386. ). The intermolt period of freshwater decapods can be quite sensitive to pesticides; as highlighted by the longer intermolt periods for juveniles of another trichodactylid, T. borellianus, following a 4-month exposure to sublethal concentrations (0.62, 1.25 and 2.50 µg/L) of a chlorpyrifos insecticide, Terminator Ciagro® (Montagna, 2010Montagna, M.C. 2010. Toxicity of chlorpyrifos as active element of a commercial formulate on juvenile crab Trichodactylus borellianus. Natura Neotropicalis, 1: 31-43.), and the irregular intermolt periods of the freshwater prawn, Pa. argentinus, from exposure to low concentrations of chlorpyrifos (0.005-0.022 µg/L) and endosulfan (0.122-0.488 µg/L) insecticides (Montagna and Collins, 2007Montagna, M.C. and Collins, P.A. 2007. Survival and growth of Palaemonetes argentinus (Decapoda; Caridea) exposed to insecticides with chlorpyrifos and endosulfan as active element. Archives of Environmental Contamination and Toxicology, 53: 371-378. ). Juveniles of the same prawn species have been reported to lengthen their intermolt period after two molts, in response to exposure to the Roundup® herbicide (Montagna and Collins, 2005Montagna, M.C. and Collins, P.A. 2007. Survival and growth of Palaemonetes argentinus (Decapoda; Caridea) exposed to insecticides with chlorpyrifos and endosulfan as active element. Archives of Environmental Contamination and Toxicology, 53: 371-378. ), as well as from exposure to sublethal concentrations of a cypermethrin commercial insecticide (Collins and Cappello, 2006Collins, P.A. and Cappello, S. 2006. Cypermethrin toxicity to aquatic life: bioassays for the freshwater prawn Palaemonetes argentinus. Archives of Environmental Contamination and Toxicology, 51: 79-85. ). Similarly, the intermolt period lengthened across successive molts of the P. dentata exposure cohort.

The breakpoint for the P. dentata exposure cohort occurs at relatively smaller values (17.5 mm CW, at 13 days intermolt period) than those of the control cohort (22.11 mm CW; 18 days intermolt period), along with a smaller size at which structural maturity (14.74 to 17.5 mm) was reached. This is also reflected in CW growth approaching asymptote at a much smaller size and a smaller maximum size (25.77 mm CW). Relative to the control group, these reduced maximum and structural maturity sizes of the exposure group may indicate a constraint on the lifespan of these crabs, caused by continuous exposure to the OP insecticide. Xenobiotics, in particular, have been known to affect growth of decapods through their molting events, causing adults to maintain smaller sizes than the average conspecific (Negro et al., 2011Negro, C.L.; Senkman, L.E.; Montagna, M.C. and Collins, P.A. 2011. Freshwater Decapods and Pesticides: An Unavoidable Relation in the Modern World. p. 197-226. In: M. Stoytcheva (ed), Pesticides in the Modern World: Risks and Benefits. Rijeka, InTech. ). This indirectly has consequences for reproduction success, in terms of mate selection and capacity for bearing young, as well as for longevity. However, further examination of P. dentata under similar toxicant conditions, over a longer test period, is needed to confirm this.

Following exposure to the malathion insecticide, patterns of CW and CL SGR for P. dentata became irregular, with a noticeable elevation in rates at the fourth molt, in relation to CW (Fig. 2D). Insecticides can modify dimensional growth rates, as evident by the overall negative or null growth rate for CL of Pa. argentinus exposed to a cypermethrin insecticide, at correspondingly low concentrations of 0.0001, 0.001 and 0.01 µg/L (Collins and Cappello, 2006Collins, P.A. and Cappello, S. 2006. Cypermethrin toxicity to aquatic life: bioassays for the freshwater prawn Palaemonetes argentinus. Archives of Environmental Contamination and Toxicology, 51: 79-85. ). Alterations in dimensional growth and intermolt period can be attributed to toxicant disturbances of the neurohormonal system of the X-organ-sinus gland complex (Negro et al., 2011Negro, C.L.; Senkman, L.E.; Montagna, M.C. and Collins, P.A. 2011. Freshwater Decapods and Pesticides: An Unavoidable Relation in the Modern World. p. 197-226. In: M. Stoytcheva (ed), Pesticides in the Modern World: Risks and Benefits. Rijeka, InTech. ) and/or by disruption of energy homeostasis and increased energy expenditure for survival (versus using the energy for growth).

Growth of an organism involves feeding, assimilation and energy expenditure, but exposure to toxicants can compromise all these processes, through modification of energy allocation and sudden metabolic demands associated with detoxification and stress responses. For instance, the decline in size increased per molt and extended intermolt period for T. borellianus exposed to chlorpyrifos insecticide were attributed to reduced levels of proteins generally used for tissue growth and repair (Montagna, 2010Montagna, M.C. 2010. Toxicity of chlorpyrifos as active element of a commercial formulate on juvenile crab Trichodactylus borellianus. Natura Neotropicalis, 1: 31-43.). Stress responses from OP exposure, like neurotoxicity, can ultimately induce a shift in the normal energy allocations, to facilitate possible coping mechanisms of the affected organism. OPs are neurotoxic and act by inhibiting the acetylcholinesterase (AChE) enzyme, thereby resulting in accumulation of acetylcholine (enzyme substrate), disruption in regular neurotransmission across neuromuscular junctions, and followed by continuous muscular contractions (Bookhout and Costlow, 1976Bookhout, C.G. and Costlow, J.D. 1976. Effects of mirex, methoxychlor, and malathion on development of crabs. Florida, United States Environmental Protection Agency, 102p.; Fulton and Key, 2001Fulton, M.H. and Key, P.B. 2001. Acetylcholinesterase inhibition in estuarine fish and invertebrates as an indicator of organophosphorus insecticide exposure and effects. Environmental Toxicology and Chemistry, 20: 37-45. ; Mulchandani et al., 2001Mulchandani, A.; Chen, W.; Mulchandani, P.; Wang, J. and Rogers, K.R. 2001. Biosensors for direct determination of organophosphate pesticides. Biosensors and Bioelectronics, 16: 225-230. ; Rickwood and Galloway, 2004Rickwood, C.J. and Galloway, T.S. 2004. Acetylcholinesterase inhibition as a biomarker of adverse effect. A study of Mytilus edulis exposed to the priority pollutant chlorfenvinphos. Aquatic Toxicology, 67: 45-56. ; Bolton-Warberg et al., 2007Bolton-Warberg, M.; Coen, L.D. and Weinstein, J.E. 2007. Acute toxicity and acetylcholinesterase inhibition in grass shrimp (Palaemonetes pugio) and oysters (Crassostrea virginica) exposed to the organophosphate dichlorvos: laboratory and field studies. Archives of Environmental Contamination and Toxicology, 52: 207-216. ; Lionetto et al., 2011Lionetto, M.G.; Caricato, R.; Calisi, A. and Schettino, T. 2011. Acetylcholinesterase inhibition as a relevant biomarker in environmental biomonitoring: new insights and perspectives. p. 87-115. In: J.E. Visser (ed), Ecotoxicology Around the Globe. New York, Nova Science Publishers Inc.). This neurotoxicity was previously demonstrated for P. dentata, where significant AChE inhibition resulted from exposures to sublethal concentrations of the same malathion insecticide (Singh, unpublished data).

A reduction in overall energy availability for both somatic and gonadal growth can also result from impaired feeding in an organism experiencing toxic stress. Based on delayed consumption of exuviae and observations on the unconsumed food that was removed during water renewal, it seems that feeding was reduced in P. dentata under malathion insecticide exposure (as opposed to the control cohort in which little to no food remained after feeding events). This reduced feeding would ultimately reduce the available energy for these crabs to undergo normal growth. A portion of the total pellets and plant segments were consumed by the exposure group, each time these were provided, and so it cannot be fully verified that feeding was impaired.

The responses of P. dentata in this study highlight that their growth is sensitive to malathion insecticide and even at sublethal levels this OP biocide can significantly modify the molting cycle. Disruption in energy homeostasis and the hormonal endocrine system involved in molting may have occurred in exposed P. dentata, delaying the intermolt period and cycle for later molts. However, this remains quantitatively unconfirmed and requires further investigation into metabolism energetics, and levels of MIH, ecdysteroids and other related growth hormones in crabs exposed to malathion insecticide. Malathion can be broken down to its toxic intermediate, malaoxon, through hydrolysis, and the latter is known to be more toxic than the parent compound, despite its fast ability to degrade (Wendel and Smee, 2009Wendel, C.M. and Smee, D.L. 2009. Ambient Malathion concentrations modify behavior and increase mortality in blue crabs. Marine Ecology Progress Series, 392: 157-165. ). It is reasonable to infer that commercial malathion insecticide, at sublethal levels, can alter growth patterns of P. dentata juveniles and subsequently influence their maturity. Consequently, the presence of malathion insecticide in the aquatic habitats of P. dentata could indirectly impact reproduction in indigenous populations, through altered energy investment for growth and reproductive processes (e.g., oocyte/sperm development), reduced success for smaller males in mate competition and limited egg carrying capacity of smaller females.

ACKNOWLEDGMENTS

The authors extend their deepest thanks to Ms. Raquel Khan Ali and Dr. Faisal Mohammed for their laboratory assistance, the technical staff at the Department of Life Sciences (Faculty of Science and Technology, The University of the West Indies St. Augustine) and Mr. Rakesh Bhukal for invaluable field support. Additional gratitude is extended to the farmers of Bamboo Settlement for granting access to the sample site.

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  • Compliance with ethical standards

    Ethical sanction was granted by the Campus Ethics Committee of The University of the West Indies St. Augustine (Trinidad and Tobago) for conducting research on Poppiana dentata.

Publication Dates

  • Publication in this collection
    16 June 2021
  • Date of issue
    2021

History

  • Received
    23 Oct 2020
  • Accepted
    24 Feb 2021
Sociedade Brasileira de Carcinologia Instituto de Biociências, UNESP, Campus Botucatu, Rua Professor Doutor Antônio Celso Wagner Zanin, 250 , Botucatu, SP, 18618-689 - Botucatu - SP - Brazil
E-mail: editor.nauplius@gmail.com