Many plants and animals utilize the pigment melanin, capable of regulating a wide range of molecular interactions and metabolic processes, as a defense against any type of non-self(Golkar et al


 

ISJ 9: 153-162, 2012                                                                  ISSN 1824-307X 
 
 

RESEARCH REPORT 
 
Amyloid/Melanin distinctive mark in invertebrate immunity 
 
A Grimaldi1, R Girardello1, D Malagoli2, P Falabella3, G Tettamanti1, R Valvassori1, E Ottaviani2, 
M de Eguileor1 
 
1Department of Biotechnology and Life Science, University of Insubria, Via J. H. Dunant 3, 21100 Varese, Italy 
2Department of Life Sciences, University of Modena and Reggio Emilia, Via Campi 213/D,41125 Modena, Italy 
3Dipartimento di Biologia, Difesa e Biotecnologie Agro-Forestali, University of Basilicata, via dell’Ateneo Lucano 
10, 85100 Potenza, Italy 

 
 

   Accepted September 14, 2012 
 

Abstract 
Protostomes and Deuterostomes show the same nexus between melanin production, and amyloid 

fibril production, i.e., the presence of melanin is indissolubly linked to amyloid scaffold that, in turn, is 
conditioned by the redox status/cytoplasmic pH modification, pro-protein cleavage presence, 
adrenocorticotropin hormone (ACTH), melanocyte-stimulating hormone (α-MSH), and neutral 
endopeptidase (NEP) overexpressions. These events represent the crucial component of immune 
response in invertebrates, while in vertebrates these series of occurrences could be interpreted as a 
modest and very restricted innate immune response. On the whole, it emerges that the mechanisms 
involving amyloid fibrils/pigment synthesis in phylogenetically distant metazoan (viz, cnidaria, 
molluscs, annelids, insects, ascidians and vertebrates) are evolutionary conserved. Furthermore, our 
data show the relationship between immune and neuroendocrine systems in amyloid/melanin 
synthesis. Indeed the process is closely associated to ACTH-α-MSH production, and their role in 
stress responses leading to pigment production reflects and confirms again their ancient phylogeny. 
 
Key Words: amyloid fibrils; melanin; ACTH, α-MSH; neutral endopeptidase; invertebrate immunity 

 
 
Introduction 

 
Living organisms produce melanin as defense 

system against attack, harm, or injury coming from 
any type of non-self (Golkar et al., 1993; Nappi and 
Ottaviani, 2000; Petes et al., 2003; Nappi, 2010). In 
vertebrates, it is well known that the biopigment 
shows its main quality neutralizing the potentially 
deleterious effects of sunlight (Edelstein, 1977). 
Melanin production is considered a widespread 
event, and becomes essential in those invertebrates 
where invaders such as parasites or fungi, are 
rapidly isolated and sequestered in a capsule made 
of pigment and hemocytes (Carton et al., 2008). In 
general melanin, acting as scavenger of reactive 
oxygen species (ROS), defends cells/tissues from 
the toxic effects of free radicals and it is manifested, 
from invertebrates up to man, in the areas of tissue 
repair, during regeneration process and in response 
to pathogens (de Eguileor et al. 2000; Gourdon et 
al., 2001; Ballarin et al., 2002; Nappi and 
Christensen, 2005; Lewis and Pollard, 2006; Nappi, 
___________________________________________________________________________ 

 
Corresponding author: 
Annalisa Grimaldi 
Department of Biotechnology and Life Science 
University of Insubria 
Via J. H. Dunant 3, 21100 Varese, Italy 
E-mail: annalisa.grimaldi@uninsubria.it 

 
 
2010; Palmer et al., 2011). Melanin biosynthesis is 
due to the activation of prophenol oxidase (pro-PO) 
system present in cell and/or in body fluids. The 
pro-PO activating system is best understood in 
crayfish Pacifasticus leniusculus (Söderhäll and 
Smith, 1986), in silkworm Bombyx mori (Ashida and 
Yoshida, 1990; Yasuhara et al., 1995), in 
Drosophila melanogaster (Nappi and Vass, 1993; 
Fujimoto et al., 1995), in Echinoderms such as 
Holoturia tubulosa (Roch et al., 1992), in Ascidians 
(Jhoanson and Söderhäll, 1989; Cammarata and 
Parrinello, 2009; Ballarin, 2012), and in 
Cephalochordates (Pang et al., 2004). Specifically 
about biopigment synthesis, in Cnidaria several 
papers show that pathogens or any kind of 
stressors, induces a localized melanization in sea 
fan corals. The pigment production is due to an 
augment of amebocyte melanosome production 
and to pro-PO activity in the tissues (Petes et al., 
2003; Mydlarz et al., 2008). In Annelida 
(oligochaets and polychaets), several authors 
(Porchet-Henneret, 1987; Valembois et al., 1988; 
Porchet-Henneret and Vernet, 1992; Beschin et al., 
1998; Fyffe et al., 1999; Adamowicz, 2005; 
Prochazkova et al., 2006) describe the efficient 
activation of both pro-PO cascade in coelomic fluid 
and in a subpopulation of granulocytes, with the final 

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Fig. 1 Circulating hemocytes from insect and ascidian. (A-B) Light microscopy: May Grünwald-Giemsa technique 
(the circulating cell cytoplasm is differently stained in relation to their cytoplasmic pH) (pink mark indicates acid 
pH); (C-D) Light microscopy: Masson-Fontana technique: silver staining demonstrates melanin deposition 
(arrowheads); (E-H) Fluorescence and light microscopy: thioflavine T (E-F) and Congo red (G-H) staining 
recognize amyloid or amyloid-like structures; (J-K) Fluorescence microscopy: Immunocytochemical evidence of 
furin-like protein (in red), nuclei in blue are stained with DAPI. 

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result of massive melanin production. An extensive 
literature is reported about humoral factors and 
cellular responses in molluscs, against any type of 
non-self (Ottaviani and Cossarizza, 1990; Ottaviani, 
2006; Novoa et al., 2011). In particular, a 
classification of hemocytes has been proposed 
according to their different function in immune 
responses with particular regard to melanin 
synthesis (Ottaviani, 1983; Ottaviani and Franchini, 
1988; Ottaviani et al., 1990, 1993; Matricon-
Gondran and Letocart, 1999; Gorbushin and 
Iakovleva, 2006; Jiravanichpaisal et al., 2006; 
Koropatnick et al., 2007; Mahilini and Rajendran, 
2008; Venier et al., 2011). Moreover, several 
authors have described in mollusc bivalves and 
gastropod granulocytes, involved in wound repair or 
internal defense, the presence of cytoplasm 
membrane-limited granules containing “filamentous 
matrix” with acid phosphatase activity of unknown 
function (Giamberini et al., 1996; Matricon-Gondran 
and Letocart, 1999). Induced 
melanization/encapsulation against non-self is well 
known also in arthropods (Söderhäll and Smith, 
1986; Ashida et al., 1990; Hoffman and Reichart, 
2002; Martin et al., 2007; Gallo et al., 2011). With 
reference to insects, two cell types are responsible 
for the entrapment of the invaders and this process 
is generally accompanied by the phenoloxidase 
activity inducing the formation of the melanotic 
material. These events have been well-studied in 
insect host/parasitoid model (Heliothis 
virescens/Toxoneuron nigriceps) (Ferrarese et al., 
2005; Falabella et al., 2012; Grimaldi et al., 2012). 

The same implications are also evident in 
Deuterostomes such as Echinoderms (sea urchin, 
holoturians) (Canicattì and Seymour, 1991) and 
Tunicates (sea squirts) (Shirae et al. 2002; Hirose, 
2003; Ballarin, 2008; Cammarata and Parrinello, 
2009; Ballarin, 2012). In Tunicates, morula cells are 
able to recognize the presence of foreign elements 
and release phenoloxidase which induces melanin 
formation (Hirose, 2003; Ballarin et al., 2005; 
Ballarin, 2012). In mammals, melanocytes are cells 
with the main function in synthesizing and 
packaging the brown pigments in melanosomes to 
protect the skin against ultraviolet radiation (UV). As 
previously mentioned, we have demonstrated that in 
the insect H. virescens larvae, during the earliest 
phase of the parasitization, melanin was packaged 
due to the production of large amount of amyloid 
fibrils, sharing these linked events with vertebrates, 
where, as suggested by Fowler and coworkers 
(2006), amyloid fibrils template and accelerate the 
formation of pigment. The principal divergence in 
melanization process of insects and vertebrates is 
that it takes place in specific cell types (granulocytes 
and melanocytes, respectively), but in insect cells 
the phenomenon is faint in respect to that observed 
in the hemocel, where large amount of pigment are 
derived from sieric pro-PO system reactions. On the 
contrary, in vertebrates, the massive melanin 
synthesis occurs intracellularly, i.e., in melanosomal 
organelles (Grimaldi et al., 2012). 

On the basis of our previous data (Falabella et 
al., 2012; Grimaldi et al., 2012) and the evidence 
(previously mentioned), we surmize that 
protostomes and deuterostomes, show the same 

nexus between melanin production, and amyloid 
fibril production, i.e., the presence of melanin is 
indissolubly linked to amyloid scaffold that, in turn, is 
due to a combined redox status/cytoplasmic pH 
modification, pro-protein cleavage presence, 
adrenocorticotropin hormone (ACTH), melanocyte-
stimulating hormone (α-MSH), and neutral 
endopeptidase (NEP) overexpressions. Thus, in the 
present paper, using a variety of techniques we 
confirm our hypothesis. 
 
Materials and Methods 
 
Hemocytes extraction and culture 

Hemocytes from several species stimulated 
with LPS injection (Helix pomatia, Heliothis 
virescens, Ciona intestinalis) were collected by 
centrifuging the circulating fluid at 400 g per 7 min at 
4 °C. The pellet washed with MEAD-PBS solution 
(1:1). The hemocytes were resuspended in 
complete medium (Grace’s medium, FBS 10 %, 
antibiotic-antimicotic solution 1 %, SIGMA) and 
were plated at concentration of 1x106 cells/ml into 
24-well culture plates. The B16-F10 murine 
melanoma cell line (derived from C57BL/6J mouse, 
D/D) was a generous gift from Prof. Douglas 
Noonan (University of Insubria, VA, Italy). B16-F10 
murine melanoma cells were cultured for 24 h in 
DMEM and 10 % FBS and then changed to DMEM 
and 2 % FBS for additional 48 h. Cells were plated 
on glass coverslips in 35-mm-diameter Petri dishes 
containing the appropriate medium as described 
above. Coverslips were washed with phosphate-
buffered saline (PBS), pH 7.2, and the cells fixed 
with 2 % paraformaldehyde containing 0.3 % Triton 
X-100 for 10 min at 37 °C, followed by washing 
three times with PBS. Cells were blocked with 2 % 
bovine serum albumin (BSA) and 5 % goat serum in 
PBS for 1 h at room temperature or overnight at 4 
°C and then incubated for 4 h at room temperature 
in primary antibody diluted in blocking solution. 
 
Circulating cells from stimulated Hirudo medicinalis 

Ten leeches were stimulated, at the level of the 
80th superficial metamere, with injection of Matrigel 
(MG) (BD Biosciences, Mississauga, Canada) (300 
μl) added with LPS. According to Grimaldi and 
coworker (2008) after 1 week MG implants were 
harvested from the animals, minced in small pieces 
using sterilized razor blades and mechanically 
dissociated with a micropipette in 400 μl of tissue 
culture. Cells were plated, cultured, maintained at 
20 °C and examined histologically and 
immunocytochemically after 3 days from seeding. 
All cultures were performed in quadruplicate and 
processed as previously described. 

 
Light microscopy, transmission electron microscopy 
(TEM) (standard procedure)  

For routine TEM, collected circulating cells 
were fixed with 2 % glutaraldehyde in 0.1 M Na-
cacodylate buffer (pH 7.2) for 2 h. The pellet 
washed in 0.1 M Na-cacodylate buffer (pH 7.2), was 
post-fixed at 4 °C for 2 h with 1 % osmic acid in 
cacodylate buffer (pH 7.2). After standard 
dehydration in ethanol series, samples were 
embedded in an Epon-Araldite 812 mixture and 

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Fig. 2 Immunocytochemical characterization of insect/ascidian circulating hemocytes. (A-F) Fluorescence 
microscopy: expression of ACTH (A-B); NEP (C-D); α-MSH (E-F). 
 
 
 
 
sectioned with a Reichert Ultracut S ultratome 
(Leica, Nussloch, Germany). Semithin sections 
were stained by conventional methods (crystal violet 
and basic fuchsin), and with May Grünwald-Giemsa 
staining. Differential May Grünwald-Giemsa staining 
depends on cytoplasmic pH (alkaline pH increases 
blue and acid pH pink or reddish tinge in the stained 
specimens), therefore is useful for a gross-
identification of cells showing an increased reactive 
oxygen species production. Pictures visualized on a 
microscope Olympus BH2 (Olympus, Tokyo, Japan) 
were acquired with a DS-5M-L1 Nikon digital 
camera system. Thin sections were stained by 
uranyl acetate and lead citrate and observed with a 
Jeol 1010 electron microscope (Jeol, Tokyo, Japan). 

 
Amyloid fibrils detection  

Amyloid or amyloid-like structures can be 
recognized by different techniques. Amyloid fibrils 
exhibit strong affinity towards the dye Congo red 

and thioflavine T (Sipe and Cohen, 2000). Congo 
red and thioflavine T staining were performed 
according to Grimaldi et al. (2012). Specific 
fluorescence was visualized on a fluorescence 
microscope Olympus BH2 through a filter set 
(excitation wavelength of 465 nm emission). Images 
were acquired with a DS-5M-L1 Nikon digital 
camera system.  
 
Melanin detection  

Following the manufacturer’s protocol (Bio-
Optica, Milan, Italy), the Masson-Fontana silver 
stain was employed to demonstrate melanin 
deposition. Experiments were performed in 
triplicate. 

 
Indirect immunofluorescence staining 

Cryosections were treated for 30 min with PBS 
containing 2 % BSA before the primary antibody 
incubation (4 °C over night). The presence of ACTH, 

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Fig. 3 Comparison between protostome and deuterostome. Cytoplasmic pH condition (Giemsa staining), melanin 
presence (Masson-Fontana technique), amyloid fibrils production (Thioflavine-T and Congo red staining), and 
furin expression are similar in H. medicinalis (Annelid), H. pomatia (Mollusc), H. virescens (Insect), C.intestinalis 
(Ascidian) circulating cells, and B16-F10 murine melanoma cell line (as positive control). 
 
 
 
 
 
and its cleavage product α-MSH) (responsible for 
stimulation, production and release of melanin), due 
to NEP activity, and furin were assessed using the 
following primary antibodies: anti-human ACTH 
polyclonal antibody (1:50 dilution, SIGMA, Saint 
Louis, MO, USA); anti-human α-MSH polyclonal 
antibody (1:50 dilution, SIGMA); anti-CD10/CALLA 
(NEP) monoclonal antibody (clone 56C6, diluted 
1:50, Thermo Scientific, Freemont, CA, USA), anti-
furin polyclonal antibody (1:50 dilution, Santa Cruz 
Biothecnology, Santa Cruz, CA, USA). Incubations 
with suitable secondary antibodies conjugated with 
tetramethylrhodamine (TRITC) (1:200 dilution, 
Jackson, Immuno Research Laboratories, West 
Grove, Pennsylvania, USA) were performed for 1h 
in a dark moist chamber. Nuclei were eventually 
stained with 4’,6-diamidino-2-phenylindole (DAPI, 
SIGMA, Italy). The PBS buffer used for washing 

steps and antibody dilutions contained 2 % bovine 
serum albumin (BSA). In control samples, primary 
antibodies were omitted, and samples were treated 
with BSA-containing PBS. Nuclei were stained by 
incubating for 15 min with 4-6-Diamidino-2-
Phenylindole (DAPI, 0.1 g/ml in PBS). Coverslips 
were mounted in Vectashield mounting medium for 
fluorescence (Vector Laboratories, Burlingame, CA, 
USA); slides were observed on Olympus BH2 
microscope (Olympus, Tokyo, Japan). Data were 
recorded with a DS-5M-L1 digital camera system 
(Nikon, Tokyo, Japan). Images were combined with 
Adobe Photoshop® (Adobe Systems Inc., USA). 
 
Results 

 
Starting from our previous data about the link 

between the melanin synthesis and the production 

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of amyloid fibrils, that template the pigment in 
activated insect H. virescens hemocytes (Falabella 
et al., 2012; Grimaldi et al., 2012), here we 
described and characterized the morpho-functional 
events linked to the production of amyloid fibrillar 
material in relation to melanin and the concomitant 
events that take place in the immune cells under 
stress condition in different invertebrates 
(Protostome and Deuterostome). For instance in 
insects and ascidians, melanin production (Figs 1 
C, D) and amyloid fibril assemblage (Figs 1 E-H) [in 
the reticulum cisternae (Grimaldi et al., 2012)] are 
always sustained by several conditions such as an 
overproduction of ROS responsible of pH variation 
(as previously validated by enzyme inhibition) (Figs 
1 A, B), the presence of a furin-like proprotein 
convertase cleavage (Figs 1 J, K) that it is well 
known liberates a fibrillogenic fragment in 
melanosomal biogenesis (Berson et al., 2003). In 
addition we have observed that during this 
amyloid/pigment productive phase, a cross-talk 
between immune and endocrine systems occurs. 
These intercommunications are mediated by 
neuromodulators with the activation of stress-
sensoring circuits to produce and release 
molecules such as ACTH and α-MSH (Figs 2 A-F). 
This scenario of cytoplasmic pH condition, amyloid 
fibrils production/melanin synthesis, and interaction 
between immune and neuroendocrine system 
(ACTH-α-MSH presence) as reported by Grimaldi 
and coworkers (2012) is validated also in different 
invertebrates (Fig. 3). On the whole, our data, here 
presented, and regarding H. medicinalis (Annelid), 
H. pomatia (Mollusc), H. virescens (Insect), C. 
intestinalis (Ascidian), and B16-F10 murine 
melanoma cell line (as positive control) were added 
to available data present in literature, and 
summarized respectively in the Figure 3 and Table 
1. 
 
Discussion 

 
Extensive and deep studies were carried out 

on the multiple biochemical and morphological 
aspects involved in the protective responses of the 
immune system in invertebrates. Among the 
various potent weapons typical of a innate defense 
there are pro-PO system and granulocyte 
activations (Söderhäll and Smith, 1986; Jhoanson 
and Söderhäll, 1989; Ashida and Yoshida, 1990; 
Roch et al., 1992; Nappi and Vass, 1993; Fujimoto 
et al., 1995; Yasuhara et al., 1995; Pang et al., 
2004; Cammarata and Parrinello, 2009; Ballarin, 
2012). The humoral pro-PO system that according 
to several authors corresponds in function to the 
activated complement (Söderhäll, 1982; Johansson 
and Söderhäll, 1989) was recorded in several taxa 
but it is not the most important defense mechanism 
for all invertebrates (Smith and Söderhäll, 1991; 
Nappi and Ottaviani, 2000; Cerenius and Söderhäll, 
2004; Cerenius et al., 2008; Cammarata and 
Parrinello, 2009; Ballarin, 2012). Independently 
from their phylogenetic position, invertebrates can 
produce melanin especially by humoral system or 
specifically by cellular population. In the first case 
massive production of melanin is due to the activity 
of pro-PO system that can be coupled with a 

cellular response lesser involved in pigment 
production. In the second one, the melanin 
synthesis is confined in cell where is concentrated 
in organules, the melanosomes. In any case it is 
interesting to underline that the production of 
melanin is always supported by the formation of 
amyloid fibrils, as well as the concurrent events, 
such as ACTH production, NEP increment and α-
MSH formation. 

In several invertebrates, such as arthropods, 
melanin is massively produced in body cavity 
especially as pro-PO system product, while amyloid 
fibrils production is due to exocytosis of circulating 
cells (named in different ways as granulocytes or 
amebocytes) that are able to produce a huge 
amount of amyloid fibrils that adhere to the non-self 
driving the pigment accumulation close to the 
invaders, avoiding the toxic melanin dispersion in 
hemocelic environment (Ferrarese et al., 2005; 
Falabella et al., 2012; Grimaldi et al., 2012). In other 
invertebrates and vertebrates there is a coupled 
productive system (melanin/amyloid fibrils) 
concentrated in a specific cell type, the melanocytes 
where melanin on amyloid fibrils is stocked in 
melanosomes (Fowler et al., 2006). After 
stimulation, invertebrate cells engaged in melanin 
production, degranulate and their products flow 
close to the non-self or, as in vertebrates, the 
melanocytes convey towards superficial surface the 
pigment that are utilized as protection against UV. 

Summarizing it is interesting to highlight that 
melanin employment is always coupled, from 
invertebrates up to man, with a physiological 
production of amyloid fibrils. The trade-off in utilizing 
the coupled system amyloid/melanin may shift from 
the possibility to have two separated producers 
(humoral pro-PO system for melanin, and 
granulocytes for amyloid fibrils) as recorded in 
insects, echinoderms and ascidians with the 
following assemblage of the two products, up to a 
single cellular producer of both products (pigment 
and amyloid fibrils) as in coelenterates, annelids, 
molluscs and vertebrates. 

An additional striking aspect (in the previously 
mentioned taxa) refers to the cells involved in the 
production of amyloid fibrils that after cytoplasmic 
accumulation, are exocytozed to sustain melanin 
production. These cells, belonging to freely 
circulating hemocytes, show the same phenotype 
with a nucleus localized in central position, 
surrounded by large reticulum cisternae filled with 
fibrillar material, spatially organized in respect to a 
central electrondense core (Xing et al., 2008; 
Grimaldi et al., 2012). 

All these features and related processes 
involved in amyloid fibrils/melanin synthesis in 
animal phylogenetically distant (viz., cnidaria, 
molluscs, annelids, insects, ascidians and 
vertebrates) could be interpreted as evolutionary 
conserved. These shared innate immune responses 
could be interpreted in invertebrates as a basic 
event, constituting an integral component of 
immunity, independently deriving from a mix of 
cellular and humoral or from exclusive cellular 
responses, while in vertebrate could be interpreted 
as a modest and very restricted event of innate 
immunity. Indeed, in vertebrates the multiple and 

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Table 1 
 

 Giemsa Melanin Amyloid Furin ACTH α-MSH References 

   Thioflavine T Congo red     

Cnidarians  ***      (1, 2) 

Annelids         

Polichets  ***      (3, 4) 

Oligochets  ***      (5-8) 

Hirudineans *** *** *** *** *** *** *** (here) (9,10) 

Molluscs *** *** *** ***  *** *** (here) (11-25) 

Arthropods         

Crustaceans  ***   *** *** *** (26-28) 

Insects *** *** *** *** *** *** *** (here) (29-33) 

Echinoderms *** *** ***     (34) 

Tunicates *** *** *** *** *** *** *** (here) (35-38) 

Cephalochordates  ***      (39) 

Vertebrates *** *** *** *** *** *** *** (here) (40) 

 
 
Available data present in literature has been summarized. 
Cnidarians: (1, 2) Petes et al., 2003; Mydlarz et al., 2008. Annelids: (3) Porchet-Henneret and Verner, 1992; (4-
10) Porchet-Henneret et al., 1987; Beschin et al., 1998; Fyffe et al., 1999; de Eguileor et al., 2000; Adamowicz, 
2005; Prochazkova et al., 2006; Grimaldi et al., 2008. Molluscs: (11-25) Ottaviani and Cossarizza, 1990; 
Ottaviani, 1983, 2006; Ottaviani et al., 1990, 1993; Gourdon et al., 1993; Giamberini et al., 1996; Ottaviani and 
Franchini, 1998; Matricon-Gondra, 1999; Gorbushin et al., 2007; Koropatnick et al., 2007; Martin et al., 2007; 
Mahilini et al., 2008; Novoa et al., 2011. Arthropods: (26-32) Söderhäll and Smith, 1986; Johansson and 
Söderhäll, 1989; Nappi and Vass, 1993; Hoffman and Reichart, 2002; Ferrarese et al., 2005; Gallo et al., 2011; 
Falabella et al., 2012; Grimaldi et al., 2012. Ehinoderms: (34) Canicattì and Seymur, 1991. Tunicates: (35) 
Ballarin, 2008; (36) Ballarin, 2012; (37) Cammarata and Parrinello, 2009; (38) Hirose, 2003. Cephalochordates: 
(39) Pang et al., 2004. Vertebrates: (40) Fowler et al., 2006 
 
 
 
 
 
multifaceted responses belonging to acquired 
immunity can mask the basic innate responses due 
to the presence of numerous modulate answers 
against the non-self leading to a precise 
discrimination of individual pathogenic species. 
Another aspect that must be considered is the 
evidence of bidirectional messages between immune 

and neuroendocrine system. Thus amyloid/melanin 
production is close associated to ACTH/α-MSH 
production, emerging here as molecule 
overexpression. Their presence and function related 
to stress responses leading to pigment production 
reflect and confirm their ancient phylogeny (Wilder, 
1995; Ottaviani and Franceschi, 1996). 

 159



 

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