# Dendrobranchiata Classification Essay

Dendrobranchiata is a suborder of decapod shrimps, commonly known as prawns. There are 540 extant species in seven families, and a fossil record extending back to the Devonian. They differ from related animals, such as Caridea and Stenopodidea, by the branching form of the gills and by the fact that they do not brood their eggs, but release them directly into the water. They may reach a length of over 330 millimetres (13 in) and a mass of 450 grams (1.0 lb), and are widely fished and farmed for human consumption.

## Shrimp and prawns

While Dendrobranchiata and Caridea belong to different suborders of Decapoda, they are very similar in appearance, and in many contexts such as commercial farming and fisheries, they are both often referred to as "shrimp" and "prawn" interchangeably. In the United Kingdom, the word "prawn" is more common on menus than "shrimp", while the opposite is the case in North America. The term "prawn" is also loosely used to describe any large shrimp, especially those that come 15 (or fewer) to the pound (such as "king prawns", yet sometimes known as "jumbo shrimp"). Australia and some other Commonwealth nations follow this British usage to an even greater extent, using the word "prawn" almost exclusively. When Australian comedian Paul Hogan used the phrase, "I'll slip an extra shrimp on the barbie for you" in an American television advertisement,[3] it was intended to make what he was saying easier for his American audience to understand, and was thus a deliberate distortion of what an Australian would typically say.

## Description

Together with other swimming Decapoda, Dendrobranchiata show the "caridoid facies", or shrimp-like form.[4] The body is typically robust, and can be divided into a cephalothorax (head and thorax fused together) and a pleon (abdomen).[4] The body is generally slightly flattened side-to-side.[4] The largest species, Penaeus monodon, can reach a mass of 450 grams (16 oz) and a length of 336 millimetres (13.2 in).[5]

The most conspicuous appendages arising from the head are the antennae. The first pair are biramous (having two flagella), except in Luciferidae, and are relatively small.[6] The second pair can be 2–3 times the length of the body and are always uniramous (having a single flagellum).[6] The mouthparts comprise pairs of mandibles, maxillules and maxillae, arising from the head, and three pairs of maxillipeds, arising from the thorax.[7] A pair of stalked eyes points forwards from the head.[8]

### Thorax

The carapace grows from the thorax to cover the cephalothorax, and extends forwards between the eyes into a rostrum.[8] This is only as long as the stalked eyes in Benthesicymidae, Luciferidae and Sergestidae, but considerably longer in Aristeidae.[8]

As well as the three pairs of maxillipeds, the thorax also bears five pairs of pereiopods, or walking legs; the first three of these end in small claws.[9] The last two pereiopods are absent in Luciferidae and Acetes, but much longer than the preceding pereiopods in Hymenopenaeus and Xiphopenaeus.[10]

The thoracic appendages carry gills, which are protected beneath the carapace.[11] The gills are typically branched, and so resemble trees, lending the group its scientific name, Dendrobranchiata, from the Greek words δένδρον (dendron, tree) and βράγχια (branchia, gill).[12]

### Pleon

The pleon, or abdomen, is similar in length to the cephalothorax.[13] It has six segments, the first five bearing lamellar pleopods, and the last one bearing uropods.[14] The pleopods are biramous, except in Sicyoniidae, where they are uniramous.[10] The uropods and telson collectively form the tail fan; the uropods are not divided by a diaeresis, as they are in many other decapods.[15] The telson is pointed and is usually armed with four pairs of setae or spines.[15]

### Internal anatomy

Most of the musculature of a prawn is used for bending the pleon, and almost all the space in the pleon is filled by muscle.[16] More than 17 muscles operate each of the pleopods, and a further 16 power the tail fan in the rapid backward movement of the caridoid escape reaction.[17] These muscles, collectively, are the meat for which prawns are commercially fished and farmed.[18]

The nervous system of prawns comprises a dorsal brain, and a ventral nerve cord, connected by two commissures around the oesophagus.[19] The chief sensory inputs are visual input from the eyes, chemoreceptors on the antennae and in the mouth, and mechanoreceptors on the antennae and elsewhere.[20]

The digestive system comprises a foregut, a midgut and a hindgut, and is situated dorsally.[21] The foregut begins at the mouth, passes through the oesophagus, and opens into a sac which contains the grinding apparatus of the gastric mill.[21] The hepatopancreas feeds into the midgut, where digestive enzymes are released, and nutrients taken up.[21] The hindgut forms faecal pellets, which are then passed out through the muscular anus.[22]

The circulatory system is based around a compact, triangular heart, which pumps blood into three main arteries.[23]Excretion is carried out through the gills, and by specialised glands located at the base of the antennae, and is mostly in the form of ammonia.[24]

## Life cycle

Prawns may be divided into two groups: those with an open thelycum (female genitalia) and those with a closed thelycum.[25] In the open–thelycum species, mating takes place towards the end of the moulting cycle, and usually at sunset.[26] In closed–thelycum species, mating takes place shortly after moulting, when the exoskeleton is still soft, and usually occurs in the night.[27]Courtship and mating may take up to 3 hours in Penaeus monodon, while in Farfantepenaeus paulensis, mating lasts just 4–5 seconds.[27] Spawning may occur several times during the moulting cycle, and usually occurs at night.[28]

With the exception of Luciferidae, the eggs of prawns are shed directly into the water, rather than being brooded.[29] The eggs hatch into naupliuslarvae, which are followed by zoea larvae (initially protozoea, and later mysis) and then a postlarva, before reaching adulthood.[29] The changes between moults are gradual, and so the development is anamorphic rather than metamorphic.[29]

Uniquely among the Decapoda, the nauplii of Dendrobranchiata are free-swimming.[29] There are five to eight naupliar stages.[30] The earlier stages have three pairs of appendages which are used for locomotion – two pairs of antennae and the mandibles. Later stages also have rudiments of other mouthparts, but the nauplius is unable to feed, and only lasts 24 to 68 hours.[29] The body ends at a two-lobed telson, and the beginnings of a carapace emerge at this stage.[30]

There are typically 5 or 6 zoea stages in Dendrobranchiata, divided into protozoea and mysis.[29] In the protozoea larvae, the antennae are still used for locomotion, but the mandibles become specialised for mastication.[30] All the thoracic somites (body segments) have formed, and a carapace is present, covering part of the thorax.[30] It is smooth in the family Penaeidae, but bears many spines in the family Solenoceridae.[30] The pleon (abdomen) is unsegmented in the first protozoea, and ends in a bilobed telson, which may be used for cleaning other appendages, or for steering.[30] By the second protozoea, segmentation appears on the pleon,[30] and by the third protozoea, which may also be called the metazoea, the uropods have appeared.[31]

By the mysis stages, the pereiopods (thoracic appendages) start to be used instead of the antennae for locomotion.[31] The larva swims backwards, with its tail upwards, spinning slowly as it goes.[31] The carapace covers most of the segments of the thorax, and claws appear on the first three pereiopods.[31] By the last mysis stage, the beginnings of pleopods have appeared on the first five segments of the abdomen.[31]

The post-larva or juvenile stage is characterised by the use of the pleopods for locomotion.[32] The claws become functional, but the gills are still rudimentary.[32] The telson is narrower and only retains traces of its two-lobed development.[32] Through a series of gradual changes over following moults, the animal takes on its adult form.[32]

## Systematics

Dendrobranchiata were traditionally grouped together with Caridea as "Natantia" (the swimming decapoda), as opposed to the Reptantia (the walking decapods). In 1888, Charles Spence Bate recognised the differences in gill morphology, and separated Natantia into Dendrobranchiata, Phyllobranchiata and Trichobranchiata.[33] Recent analyses using cladistics and molecular phylogenetics recognise Dendrobranchiata as the sister group to all other Decapoda, collectively called Pleocyemata.[34]

Before 2010, the earliest known fossil prawns come from rocks in Madagascar of Permo-Triassic age, 250 million years ago.[35][36] In 2010, however, the discovery of Aciculopoda from Famennian–stage rocks in Oklahoma extended the group's fossil record back to 360 million years ago.[37] The best known fossil prawns are from the JurassicSolnhofen limestones from Germany.[36]

Living prawns are divided among seven families, five in the superfamily Penaeoidea, and two in the Sergestoidea,[2] although molecular evidence disagrees with some aspects of the current classifications.[38] Collectively, these include 540 extant species, and nearly 100 exclusively fossil species.[1] A further two families are known only from fossils.[1]

Suborder PenaeoideaRafinesque-Schmaltz, 1815

† AciculopodidaeFeldmann & Schweitzer, 2010 – a single Famennian species, Aciculopoda mapesi[37]
† AegeridaeBurkenroad, 1963 – two Mesozoic genera: Aeger and Acanthochirana[39]
AristeidaeWood-Mason, 1891 – 26 extant species in 9 genera, and one fossil genus [40]
BenthesicymidaeWood-Mason, 1891 – 41 species in 4 genera [40]
† CarpopenaeidaeGarassino, 1994 – two Cretaceous species of Carpopenaeus[41]
PenaeidaeRafinesque-Schmaltz, 1815 – 216 extant species in 26 genera, and several extinct genera, mostly Mesozoic [42]
SicyoniidaeOrtmann, 1898 – 43 species of Sicyonia[43]
SolenoceridaeWood-Mason, 1891 – 81 species in 9 genera [44]

LuciferidaeDe Haan, 1849 – 7 species in 2 genera
SergestidaeDana, 1852 – 90 extant species in six genera, and two extinct monotypic genera [45]

## Distribution

The biodiversity of Dendrobranchiata decreases markedly at increasing latitudes; most species are only found in a region between 40° north and 40° south.[46] Some species may occur at higher latitudes. For instance, Bentheogennema borealis is abundant at 57° north in the Pacific Ocean, while collections of Gennadas kempi have been made as far south as 61° south in the Antarctic Ocean.[46]

## Ecology and behaviour

There is a great deal of ecological variation within the suborder Dendrobranchiata. Some species of Sergestidae live in fresh water, but most prawns are exclusively marine.[32] Species of Sergestidae and Benthesicymidae mostly live in deep water, and Solenoceridae species live offshore, while most Penaeidae species live in shallow inshore waters, and Lucifer is planktonic.[32] Some species burrow in mud on the sea floor during the day and emerge at night to feed.[32]

Prawns are "opportunistic omnivores",[47] and their diet can include a range of food items from fine particles to large organisms. These may include fish, chaetognaths, krill, copepods, radiolarians, phytoplankton, nematocysts, ostracods and detritus.[47] Prawns eat less around the time of ecdysis (moulting), probably because of the softness of the mouthparts, and must eat more than usual to compensate, once ecdysis is complete.[47]

Prawns are an attractive food for predators, with a higher energy content than most other invertebrates.[48] The larvae are prey to comb jellies, jellyfish, chaetognaths, fish and other crustaceans (such as mantis shrimp and crabs), and only a tiny proportion survive.[49] Juveniles are targeted by a number of fish, cephalopods and birds; Litopenaeus vannamei juveniles experience 90% mortality in the 6–12 weeks they spend in Mexican lagoons, and this is thought to be due almost entirely to predation.[49] Adult prawns are less susceptible to predation, but can fall prey to some fish.[50]

## Economic importance

Dendrobranchiata are of huge importance. While in some countries, such as the United States, production is almost entirely through fisheries, other countries have concentrated on aquaculture (shrimp farms), including Ecuador where 95% of production is farmed; some countries produce similar amounts from fisheries and aquaculture, including Mexico, China, India and Indonesia.[51]

Species from the family Aristeidae are important to deep-water fisheries, particularly in the Mediterranean Sea, where Aristaeomorpha foliacea is caught by trawlers.[51] In Brazil, Aristaeomorpha foliacea, Aristaeopsis edwardsiana and Aristeus antillensis are of commercial importance.[51] The shallow-water Penaeidae are of greater importance, however, and the most important species for fisheries is Fenneropenaeus chinensis, with a catch in 2005 of over 100,000 tons.[34]

The most important species for aquaculture are Marsupenaeus japonicus (Kuruma prawn), Fenneropenaeus chinensis (Chinese prawn), Penaeus monodon (giant tiger prawn) and Litopenaeus vannamei (whiteleg prawn).[34]

## References

1. ^ abcDe Grave et al., 2009
2. ^ abMartin & Davis, 2001
3. ^Baker & Bendel, 2007
4. ^ abcTavares & Martin, 2010, p. 100
5. ^Dall, 1990, pp. 3–4
6. ^ abTavares & Martin, 2010, p. 106
7. ^Tavares & Martin, 2010, pp. 106–108
8. ^ abcTavares & Martin, 2010, p. 102
9. ^Tavares & Martin, 2010, pp. 108–110
10. ^ abTavares & Martin, 2010, p. 110
11. ^Tavares & Martin, 2010, pp. 103–105
12. ^Tavares & Martin, 2010, p. 103
13. ^Tavares & Martin, 2010, p. 105
14. ^Tavares & Martin, 2010, pp. 110–111
15. ^ abTavares & Martin, 2010, p. 111
16. ^Tavares & Martin, 2010, p. 113
17. ^Tavares & Martin, 2010, pp. 113–114
18. ^Kanduri & Eckhardt, 2002, p. 42
19. ^Tavares & Martin, 2010, p. 114
20. ^Tavares & Martin, 2010, pp. 116–118
21. ^ abcTavares & Martin, 2010, p. 118
22. ^Tavares & Martin, 2010, pp. 118–119
23. ^Tavares & Martin, 2010, p. 120
24. ^Tavares & Martin, 2010, pp. 120–121
25. ^Tavares & Martin, 2010, p. 125
26. ^Tavares & Martin, 2010, pp. 125–126
27. ^ abTavares & Martin, 2010, p. 126
28. ^Tavares & Martin, 2010, p. 127
29. ^ abcdefTavares & Martin, 2010, p. 130
30. ^ abcdefgTavares & Martin, 2010, p. 131
31. ^ abcdeTavares & Martin, 2010, p. 133
32. ^ abcdefgTavares & Martin, 2010, p. 134
33. ^Tavares & Martin, 2010, p. 99
34. ^ abcTavares & Martin, 2010, p. 137
35. ^Crean, 2004
36. ^ abSchram et al., 2000
37. ^ abFeldmann & Schweitzer, 2010
38. ^Ma et al., 2009
39. ^Tavares & Martin, 2010, p. 151
40. ^ abTavares & Martin, 2010, p. 152
41. ^Tavares & Martin, 2010, pp. 152–153
42. ^Tavares & Martin, 2010, p. 153
43. ^Tavares & Martin, 2010, p. 154
44. ^Tavares & Martin, 2010, p. 155
45. ^Tavares & Martin, 2010, p. 156
46. ^ abTavares & Martin, 2010, p. 145
47. ^ abcTavares & Martin, 2010, p. 135
48. ^Dall, 1990, p. 357
49. ^ abDall, 1990, p. 358
50. ^Dall, 1990, p. 359
51. ^ abcTavares & Martin, 2010, p. 136

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• Bill Baker; Peggy Bendel. "Come and Say G'Day!". Travel Marketing Decisions. Association of Travel Marketing Executives (Summer 2005). Archived from the original(PDF) on November 4, 2007. Retrieved December 21, 2007.
• Robert P. D. Crean (November 14, 2004). "Dendrobranchiata". Order Decapoda. University of Bristol.
• William Dall (1990). The Biology of the Penaeidae. Advances in Marine Biology. 27. Academic Press. ISBN 978-0-12-026127-7.
• Sammy De Grave; N. Dean Pentcheff; Shane T. Ahyong; et al. (2009). "A classification of living and fossil genera of decapod crustaceans"(PDF). Raffles Bulletin of Zoology. Suppl. 21: 1–109. Archived from the original(PDF) on 2011-06-06.
• Rodney Feldmann; Carrie Schweitzer (2010). "The oldest shrimp (Devonian: Famennian) and remarkable preservation of soft tissue". Journal of Crustacean Biology. 30 (4): 629–635. doi:10.1651/09-3268.1.
• Indian Aquaculture Authority (2001). "Shrimp Aquaculture and the Environment - An Environment Impact Assessment Report, chapter 2; IAA report"(PDF). Archived from the original(PDF) on 2011-07-16.
• Laxman Kanduri; Ronald A. Eckhardt (2002). "HACCP in shrimp processing". Food Safety in Shrimp Processing: a Handbook for Shrimp Processors, Importers, Exporters and Retailers. John Wiley and Sons. pp. 40–64. ISBN 978-0-85238-270-7.
• K. Y. Ma; T.-Y. Chan; K. H. Chu (2009). "Phylogeny of penaeoid shrimps (Decapoda: Penaeoidea) inferred from nuclear protein-coding genes". Molecular Phylogenetics and Evolution. 53 (1): 45–55. doi:10.1016/j.ympev.2009.05.019. PMID 19477284.
• J. W. Martin; G. E. Davis (2001). An Updated Classification of the Recent Crustacea(PDF). Natural History Museum of Los Angeles County. pp. 1–132.
• Frederick R. Schram; Shen Yanbin; Ronald Vonk; Rodney S. Taylor (2000). "The first fossil stenopodidean"(PDF). Crustaceana. 73 (2): 235–242. doi:10.1163/156854000504183. JSTOR 20106269.
• Carolina Tavares; Joel W. Martin (2010). "Suborder Dendrobranchiata Bate, 1888". In F. R. Schram; J. C. von Vaupel Klein; J. Forest; M. Charmantier-Daures. Eucarida: Euphausiacea, Amphionidacea, and Decapoda (partim)(PDF). Treatise on Zoology – Anatomy, Taxonomy, Biology – The Crustacea. 9A. Brill Publishers. pp. 99–164. ISBN 978-90-04-16441-3.

## Abstract

In the Crustacea, sex determination is generally considered to be cell non-autonomous, with a centralized sex differentiation signaling system. However, observed frequencies of axial (anterior-posterior) morphological gynandromorphs of Branchinecta lindahliPackard, 1883 suggest cell autonomous sex determination. Thoracic appendages of seven Branchinecta lindahli axial gynandromorphs with male second antennae and female genitalia were compared to normal male and female thoracic appendages to quantitatively determine if the putative gynandromorphs showed evidence of intersexuality. None of the gynandromorphs showed evidence of intersexuality, supporting the occurrence of cell autonomous sex determination in branchiopod crustaceans.

## Introduction

Gynandromorphs and intersexes have been observed at low frequencies in species spanning multiple phyla (Stern, 1968; Narita et al., 2010). These individuals exhibit a mixture of male and female characteristics in species that normally have discrete male and female morphologies. If the underlying genetics of the sexual heterogeneity is unknown, classification of an individual as a gynandromorph or intersex is based on the type of morphological heterogeneity. Gynandromorphs are individuals with clearly defined areas of male and female characteristics. Organisms with phenotypes intermediate to normal male and female characteristics are classified as intersexes. This morphological classification scheme may be occasionally unclear, especially in organisms where sexually dimorphic characteristics are limited and additional developmental abnormalities are present. However, in individuals with distinct areas of unambiguous male and female morphologies, it may provide information about the causal mechanisms of the sexual heterogeneity.

The distribution of male and female characteristics in morphological gynandromorphs may be bilateral (left/right), axial (anterior/posterior), or mosaic (patchily distributed). Bilateral and mosaic gynandromorphy are often observed in Lepidoptera, due to striking differences in male and female wing patterns. Bilateral gynandromorphs display one male wing and one female wing; mosaic gynandromorphs show patches of male and female coloration in their wings, usually resulting in asymmetric wing coloration (Scriber and Evans, 1988). Axial gynandromorphy (male anterior, female posterior) has been observed in the fairy shrimp Branchinecta lindahli (Sassaman and Fugate, 1997). The pattern of gynandromorphy observed depends on the sex determination mechanism of the species and the specific genetic or environmental cause of the sexual heterogeneity (Narita et al., 2010). Gynandromorphs can therefore be useful in research on sex determination, developmental pathways, and the mechanisms by which parasites affect sex determination in their hosts (Sassaman and Fugate, 1997; Olmstead and LeBlanc, 2007; Yang and Abouheif, 2011).

Bilateral and mosaic phenotypic gynandromorphs are generally attributed to random mutations (such as chromosomal non-disjunction) during development (Morgan and Bridges, 1919; Manley, 1971; Scriber and Evans, 1988). Axial (anterior-posterior) morphological gynandromorphs have been observed in a few species at much higher frequencies than expected if caused by random mutations. Sassaman and Fugate (1997) observed axial gynandromorphs at a frequency of 0.0020 in a laboratory reared population of Branchinecta lindahliPackard, 1883. Kamping et al. (2007) observed axial gynandromorphs at frequencies up to 0.0927 in field strains of the wasp Nasonia vitripennisWalker, 1836. If axial gynandromorphs were created by a single random mutation, they would be expected in frequencies similar to bilateral and mosaic gynandromorphs. If they require two separate mutations occurring in the cell lineages producing the left and right sides of the organism, they would be even rarer (Sassaman and Fugate, 1997). Another explanation is required to account for the occurrence of axial gynandromorphs at levels noticeably greater than expected by chance (Sassaman and Fugate, 1997).

In the parasitic wasps Nasonia vitripennis and Trichogramma spp., the presence of morphological axial gynandromorphs has been attributed, at least in part, to the actions of heritable cytoplasmic components (Stouthamer, 1997; Kamping et al., 2007; Tulgetske and Stouthamer, 2012). In insects, sex determination is generally considered to be cell autonomous, or individually determined by each somatic cell through a well known genetic cascade (Sanchez, 2008; Salz and Erickson, 2010). If a cytoplasmic factor affects each cell individually, then variability in the influence of a cytoplasmic factor across the anterior-posterior axis of an individual may produce discrete areas of male and female morphology, resulting in axial gynandromorphy.

In crustaceans, sex determination is generally considered to be cell non-autonomous, with sex differentiation signaling coming from a centralized source, the androgenic gland (Payen, 1991). The androgenic gland has been shown to mediate sex determination in the soma and gametes of isopods and amphipods cell non-autonomously, producing hormones that act on tissues throughout the body of the organism (Payen, 1991; Sagi et al., 1997). This has been confirmed by non-localized masculinization of tissues following androgenic gland implantation in females (Katakura, 1960; Negishi and Hasegawa, 1992; Taketomi and Nishikawa, 1996). In isopods and amphipods, when incomplete feminization by heritable cytoplasmic factors produces sexually heterogeneous individuals, it produces morphological intersexes instead of morphological gynandromorphs (Bulnheim, 1978; Rigaud and Juchault, 1998). In isopods, the intersexes vary greatly from fertile females bearing small male characters to sterile males with female genital orifices (Rigaud and Juchault, 1998).

In the fairy shrimp B. lindahli, a cytoplasmically inherited feminizing factor was proposed by Sassaman and Fugate (1997) as the cause of morphological axial gynandromorphism. They hypothesized that delayed feminization of a genetic male produced male characteristics in segments formed during embryogenesis and female characteristics in segments formed during larval development. This would result in male second antennae and female thoracic appendages and genitalia. Sassaman and Fugate (1997) examined thirty-one B. lindahli axial gynandromorphs, with normal male second antennae and normal female ovisacs. The thoracic appendages were examined on two individuals and found to have female morphologies.

If B. lindahli sex determination is controlled by a hormonal system similar to the androgenic gland in isopods and amphipods, a closer examination of the thoracic appendages of a greater number of gynandromorphs should show evidence of intersexuality in the thoracic appendages. If instead, the mechanism of sex determination is cell autonomous, the thoracic appendages should show no evidence of intersexuality. This would suggest a sex determination system more similar to that found in insects, providing evidence for the existence of both cell autonomous and cell non-autonomous mechanisms of sex determination within Crustacea.

This study will establish morphological measures of the thoracic appendages of B. lindahli to produce a clear and quantitative method of distinguishing between male and female morphologies. Branchinecta lindahli axial gynandromorph morphologies will be compared to normal male and female morphologies to determine if there is any evidence of intersexuality in the thoracic appendages of axial gynandromorphs.

## Materials and Methods

A sample of the cysts of B. lindahli was collected from a small temporary pool at the Robert J. Bernard Field Station operated by the Claremont Colleges in Claremont, CA, USA. Soil samples containing cysts were hydrated with deionized water at a ratio of $${\rm{5 \,c}}{{\rm{m}}^{\rm{3}}}$$ of soil to $${\rm{5}}\,{\rm{l}}$$ of water. Hydrated samples were placed in a dark incubator at approximately $${\rm{5}}^\circ {\rm{C}}$$ for 2-3 days, and transferred to constant light at $${\rm{18}} {\text{-}} {\rm{23}}^\circ {\rm{C}}$$. Nauplii hatched in 1-3 days, and were raised to adulthood in conditioned water. Conditioned water was produced by adding $${\rm{3}}0\,{\rm{c}}{{\rm{m}}^{\rm{3}}}$$ of soil from a roadside ditch off Ramona Expressway in Riverside, CA, USA (site no longer exists due to road construction) to approximately $$150\,{\rm{l}}$$ of deionized water at least 2 days before use. Conditioned water was screened through a sieve to remove any cysts or nauplii from the Ramona Expressway site.

Sexually mature males, females and axial gynandromorphs from the Bernard Field Station population were fixed in 10% formalin for $$24\,{\rm{hours}}$$ and transferred to 70% ethanol. Nine males, ten females and seven axial gynandromorphs were included in the study. One appendage from each thoracic segment was removed from the shrimp and transferred to a glycerol slide. Incomplete data was collected from one male and four gynandromorphs due to preexisting damage to limbs or damage during limb removal from the shrimp. One gynandromorph is missing data for thoracic limbs 4, 5, 10 and 11 (anterior to posterior), two gynandromorphs are missing data for thoracic limb 1, and one gynandromorph is missing data for thoracic limb 3. One male is missing data for thoracic limb 7. Digital images were taken of each intact appendage and analyzed for sexually dimorphic traits.

Two landmarks on each appendage were located based on setal type and location. The thoracic appendages of B. lindahli are used for filtering small particles from the water and for scraping fungal and algal growths from surfaces (Daborn, 1979). The setae on the medial edge of the endopod are short, stiff and brush or comb-like and are used for scraping. The setae on the ventral edge of the endopod are long and feather-like, and are used for filtering. The first landmark was the base of the last unmodified filter-type seta before the transition to scraping-type setae. Reference was made to the original slides to positively identify the first landmark because setal morphology was not always readily identifiable in the digital images. The second landmark was the base of the last scraping-type seta closest to the endites.

A line was drawn connecting the two setal landmarks. A second line was then drawn perpendicular to the first line, which extended to the edge of the endopod and maximized the total length of the second line. The second line, Line C, divided the first line into two lengths, Line A and Line B. Line A extended from the base of the last unmodified filter-type seta to the base of Line C. Line B extended from the base of the last scraping-type seta to the base of Line C (Fig. 1). The lengths of all three lines were measured and two morphological ratios were calculated for each appendage. Ratios of two measures were used to describe the shapes of the endopods to compensate for differences in total body size between individuals. The morphological ratios were: $${\rm{A/(A \,+\, B)}}$$ and $${\rm{C}}/\left( {{\rm{A }} + {\rm{ B}}} \right)$$.

Fig. 1

Morphological measures describing variation in limb morphologies in the endopods of the thoracic appendages of Branchinecta lindahli.

Fig. 1

Morphological measures describing variation in limb morphologies in the endopods of the thoracic appendages of Branchinecta lindahli.

Using SPSS 17, a repeated measures analysis of variance was used to analyze each of the morphological measures, using limb number as the repeated measures variable. Males, females, and gynandromorphs were treated as separate genders for the purposes of the analysis. Tamhane T2 post hoc comparisons were used to compare between male, female and gynandromorph morphologies. Due to missing data from several of the shrimp, three gynandromorphs, eight males, and ten females were included in the analysis of variance statistical test.

## Results

Twelve B. lindahli axial gynandromorphs and one mosaic gynandromorph were collected from a total of 4441 individuals reared under laboratory conditions. The frequency of gynandromorphs observed was 0.0029, which was similar to the frequency of 0.0020 observed by Sassaman and Weeks (1997) in laboratory reared individuals from a different population of B. lindahli. The mosaic gynandromorph displayed bilateral gynandromorphism in the second antennae, and normal female characteristics in all thoracic appendages and in the genitalia. All of the axial gynandromorphs had male second antennae and female genitalia.

Distinct morphological differences were observed between normal male and female thoracic appendages. The endopod extends further medially in males than in females and the tip curves back towards the body of the males in the medially located thoracic appendages (Fig. 2). Males and females had distinctly different endopod morphologies in all but the posterior-most thoracic appendage, based on the two morphological measures (Figs. 3 and 4).

Fig. 2

Endopod morphology of thoracic appendages of male and female Branchinecta lindahli (limbs numbered anterior to posterior). A, male limb 2; B, male limb 5; C, male limb 10; D, female limb 2; E, female limb 5; F, female limb 10. $${\rm{Scale\,bars}} = 0.2\,{\rm{mm}}$$.

Fig. 2

Endopod morphology of thoracic appendages of male and female Branchinecta lindahli (limbs numbered anterior to posterior). A, male limb 2; B, male limb 5; C, male limb 10; D, female limb 2; E, female limb 5; F, female limb 10. $${\rm{Scale\,bars}} = 0.2\,{\rm{mm}}$$.

Fig. 3

Means and 95% confidence intervals of endopod measure $${\rm{A/(A + B)}}$$ for females, males, and axial gynandromorphs of Branchinecta lindahli (females: all limbs $$n = 10$$; males: L7 $$n = 8$$, all other limbs $$n = 9$$; gynandromorphs: L1 $$n = 5$$, L3, 4, 5, 10, 11 $$n = 6$$, all other limbs $$n = 7$$).

Fig. 3

Means and 95% confidence intervals of endopod measure $${\rm{A/(A + B)}}$$ for females, males, and axial gynandromorphs of Branchinecta lindahli (females: all limbs $$n = 10$$; males: L7 $$n = 8$$, all other limbs $$n = 9$$; gynandromorphs: L1 $$n = 5$$, L3, 4, 5, 10, 11 $$n = 6$$, all other limbs $$n = 7$$).

Fig. 4

Means and 95% confidence intervals of endopod measure $${\rm{C/(A + B)}}$$ for females, males, and axial gynandromorphs of Branchinecta lindahli (females: all limbs $$n = 10$$; males: L7 $$n = 8$$, all other limbs $$n = 9$$; gynandromorphs: L1 $$n = 5$$, L3, 4, 5, 10, 11 $$n = 6$$, all other limbs $$n = 7$$).

Fig. 4

Means and 95% confidence intervals of endopod measure $${\rm{C/(A + B)}}$$ for females, males, and axial gynandromorphs of Branchinecta lindahli (females: all limbs $$n = 10$$; males: L7 $$n = 8$$, all other limbs $$n = 9$$; gynandromorphs: L1 $$n = 5$$, L3, 4, 5, 10, 11 $$n = 6$$, all other limbs $$n = 7$$).

There was a highly significant effect of gender from the repeated measures analysis of variance for both morphological measures (Table 1). Post hoc comparisons for morphological measure $${\rm{A/(A + B)}}$$ indicated a significant difference between males and females (Tamhane T2, $$P = 0.006$$) and between males and gynandromorphs (Tamhane T2, $$P = 0.036$$), but not between females and gynandromorphs (Tamhane T2, $$P = 0.624$$). Post hoc comparisons for measure $${\rm{C/(A + B)}}$$ produced the same pattern, with significant difference between males and females (Tamhane T2, $$P = \, \lt 0.001$$), between males and gynandromorphs (Tamhane T2, $$P = \, \lt 0.001$$), but not between females and gynandromorphs (Tamhane T2, $$P = 0.912$$). Overall, the gynandromorph endopods differed significantly in shape from male endopods, but did not differ significantly in shape from normal females.

The limbs changed significantly in shape along the anterior-posterior axis of the shrimp (Table 1). There was also a significant interaction between limb number and gender, indicating that the pattern of change along the thorax of the shrimp was not identical between males, females and gynandromorphs.

Table 1

Repeated measures ANOVA summaries for morphological measures $${\rm{A/(A + B)}}\,{\rm{and}}\,{\rm{C/(A + B)}}$$ in Branchinecta lindahli thoracic appendages (repeated measures variable was limb number; gender categories included males, females and gynandromorphs).

Source SS df  MS $$F$$-ratio $$P$$-value
$${\rm{A/(A + B)}}$$
Between Gender 0.929 0.464 14.586 $$\lt 0.00{\rm{1}}$$
Error 0.573 18 0.032
Within Limb 0.892 10 0.089 23.079 $$\lt 0.00{\rm{1}}$$1
Interaction $${\rm{(limb }} \times \,{\rm{gender)}}$$2.460 20 0.123 31.839 $$\lt 0.00{\rm{1}}$$1
Error 0.695 180 0.004
$${\rm{C/(A + B)}}$$
Between Gender 18.412 9.206 406.840 $$\lt 0.00{\rm{1}}$$
Error 0.407 18 0.023
Within Limb 3.458 10 0.346 123.709 $$\lt 0.00{\rm{1}}$$2
Interaction $${\rm{(limb }} \times \,{\rm{gender)}}$$2.560 20 0.128 45.780 $$\lt 0.00{\rm{1}}$$2
Error 0.503 180 0.003
Source SS df  MS $$F$$-ratio $$P$$-value
$${\rm{A/(A + B)}}$$
Between Gender 0.929 0.464 14.586 $$\lt 0.00{\rm{1}}$$
Error 0.573 18 0.032
Within Limb 0.892 10 0.089 23.079 $$\lt 0.00{\rm{1}}$$1
Interaction $${\rm{(limb }} \times \,{\rm{gender)}}$$2.460 20 0.123 31.839 $$\lt 0.00{\rm{1}}$$1
Error 0.695 180 0.004
$${\rm{C/(A + B)}}$$
Between Gender 18.412 9.206 406.840 $$\lt 0.00{\rm{1}}$$
Error 0.407 18 0.023
Within Limb 3.458 10 0.346 123.709 $$\lt 0.00{\rm{1}}$$2
Interaction $${\rm{(limb }} \times \,{\rm{gender)}}$$2.560 20 0.128 45.780 $$\lt 0.00{\rm{1}}$$2
Error 0.503 180 0.003

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## Discussion

The aim of this study was to determine if evidence of intersexuality was present in the thoracic appendages of B. lindahli axial gynandromorphs through quantitative morphological measures of the endopods. Intersexuality would be considered absent if the gynandromorph endopods were statistically indistinguishable from normal female endopods for all measures, or from normal male endopods for all measures. The results clearly indicated that there were no significant differences between gynandromorph endopods and female endopods, providing no evidence for intersexuality in B. lindahli. These results support the hypothesis of cell autonomous sex determination in B. lindahli.

The morphological pattern observed in axial gynandromorphs in B. lindahli from Bernard Field Station matched the pattern observed by Sassaman and Fugate (1997) in other populations of B. lindahli. It places the transition between anterior male morphology and posterior female morphology firmly between the segment bearing the second antennae and the first thoracic segment. Due to the high frequencies of axial gynandromorphs in B. lindahli populations relative to the frequencies expected if axial gynandromorphs were produced by random mutations, Sassaman and Fugate proposed the existence of an epigenetic feminizing factor. Axial gynandromorphs would result from incomplete feminization of genetic males, where transformation from male to female morphology occurred after the sex of the cephalic segments (including the segment bearing the second antennae) was established (Sassaman and Fugate, 1997).

This study proposed to take the analysis of axial gynandromorphs in B. lindahli a step further, looking specifically for evidence of intersexuality in the sexually dimorphic thoracic appendages. While the question of intersexuality in the thoracic appendages of gynandromorphs would not challenge Sassaman and Fugate’s (1997) hypothesis of an epigenetic feminizing factor, it may provide insight into the sex determining mechanisms that underlie feminization in B. lindahli.

In other species of Crustacea that carry epigenetic feminizing factors, partial feminization produces intersexuals (Bulnheim, 1978; Rigaud and Juchault, 1998). These individuals display combinations of male and female traits, but the traits are intermediate to normal sexually dimorphic traits. Additionally, partial feminization is not limited to specific areas of the soma. Feminizing factors act on the androgenic gland, modifying the production of the androgenic hormone which can produce changes in sexually dimorphic characteristics across the body of the organism (Rigaud, 1997). This indicates that a cell non-autonomous mechanism controls sex determination, because the sex of individual cells is not controlled by intrinsic characteristics of the cells, but is instead controlled by a hormonal system.

I propose the existence of a cell autonomous mechanism of sex determination in B. lindahli. My results indicate no evidence of intersexuality in B. lindahli as would be expected with a cell autonomous sex determining system interacting with a feminizing factor. If each cell of the B. lindahli soma is either male or female depending on the intrinsic characteristics of each individual cell, an epigenetic feminizing factor must feminize every cell individually. Branchinecta lindahli