Reproductive indicators and gonad development of the Panama brief squid Lolliguncula panamensis (Berry 1911) in the gulf of California, Mexico.
Article Type: Report
Subject: Squids (Physiological aspects)
Anestrus (Research)
Authors: Arizmendi-Rodriguez, D.I.
Rodriguez-Jaramillo, C.
Quinonez-Velazquez, C.
Salinas-Zavala, C.A.
Pub Date: 08/01/2012
Publication: Name: Journal of Shellfish Research Publisher: National Shellfisheries Association, Inc. Audience: Academic Format: Magazine/Journal Subject: Biological sciences; Zoology and wildlife conservation Copyright: COPYRIGHT 2012 National Shellfisheries Association, Inc. ISSN: 0730-8000
Issue: Date: August, 2012 Source Volume: 31 Source Issue: 3
Topic: Event Code: 310 Science & research
Product: Product Code: 0912198 Squid NAICS Code: 114112 Shellfish Fishing SIC Code: 0913 Shellfish
Geographic: Geographic Scope: Mexico Geographic Code: 1MEX Mexico
Accession Number: 303011407
Full Text: ABSTRACT The reproductive biology of the Panama brief squid was evaluated using reproductive indicators, and histological and histochemical analyses. A total of 2,460 squid were analyzed, which were captured during 15 exploratory fishing surveys in the Gulf of California during 2003 to 2006 and 2008. Of the total sample, 61% were female, 15% were male, and the rest were undifferentiated. Based on the frequency of the gonad developmental stages, the largest number of mature females was identified in February and October, whereas mature males were found in April and August. This result coincided with the gonadosomatic index. According to the histological analyses, we characterized 4 stages of oogenesis: previtellogenesis, vitellogenesis, postvitellogenesis with mature oocytes, and postspawning. We also characterized 6 substages of oocyte and 2 structural indicators of spawning: oogonia (Po0), previtellogenic oocyte primary (Pol), previtellogenic oocyte secondary (Po2), vitellogenic primary oocyte (Vol). secondary vitellogenic oocyte (Vo2), postvitellogenic oocyte (Pvo), postovulatory follicles, and atresia. Oocyte size among types showed significant differences (P < 0.05). The presence of postovulatory follicles, and oocytes of different sizes and various developmental stages throughout the study period indicates that the Panama brief squid has a synchronous ovarian development with multiple spawning. The size at first maturity ([L.sub.50]) indicates that males (mantle length, 51 ram) mature at lengths shorter than females (mantle length, 85 mm).

KEY WORDS: squid, multiple spawning, ovarian development, phospholipids, histochemistry, Loliginidae, Lolliguncula panamensis, mucopolysaccharides, oocyte, triglycerides, vitellogenic


The Panama brief squid Lolliguncula panamensis is distributed in the eastern Pacific from the Gulf of California, Mexico, to Peru, and is found in depths between 1 m and 70 m, at salinities between 15 ups and 23 ups, and at temperatures ranging from 21 27[degrees]C (Fischer et al. 1995, Sanchez 2002). In Mexico, this species is considered a potential resource and is recurrent in the bycatch of the shrimp trawl fishery (Hernandez-Vazquez 1987, Alejo-Plata et al. 2001). Biological information for this species is scarce, and there is none on the impact of the nonselective pressure by the shrimp fishery. Off the Colombian coast, Squires and Barragan (1979) analyzed the size structure, and Barragan (1977a, 1977b) described the feeding habits and reproductive aspects of L. panamensis.

In reproductive biology, it is important to identify the reproductive cycles of species, spawning periods, and the types and quality of the spawning products of the species. Changes in these factors influence fluctuations in recruitment. In several species of squid studies have been undertaken to understand reproductive behavior, such as the spawning periods in Loligo vulgaris reynaudii (D'Orbigny) and Loligo sanpaulensis (Brakonieci 1984) (Sauer et al. 1992, Costa & Fernandes 1993), and the male:female ratio in L. panamensis and L. plei (Blainville 1823) (Squires & Barragfin 1979, Perez et al. 2002) and L. vulgaris reynaudii (Sauer & Lipinski 1990, Sauer et al. 1999). In Loligo opalescens (Berry 1911), oocyte types have been described (Macewicz et al. 2004). The size of oocytes has been reported for Loligo pealei (Lesueur 1821), Loligo gahi (D'Orbigny 1835), and L. opalescens by Maxwell et al. (1998), Laptikhovsky and Arkhipkin (2001), and Macewicz et al. (2004). The relationships between energy reserves (lipids, carbohydrates, and proteins) and gonad development cycles have been documented poorly in cephalopods. Moltschaniwsky and Semmens (2000) found that the loliginid squid Photololigo sp. transfers energy for reproduction directly from the food consumed, without it being stored in somatic tissues such as the digestive gland or mantle.

For the Panama brief squid off the coast of Colombia, Barragfin (1977a) identified 2 annual peaks of reproduction and estimated the length at first maturity; females are mature at a mantle length (ML) between 73 mm and 76 mm, and males reach maturity at an ML between 38 mm and 40 mm.

In the northern part of the distribution range of the species, the biology of the Panama brief squid is virtually unknown. Recently, Arizmendi-Rodriguez et al. (2011), describing the trophic spectrum of L. panamensis in the Gulf of California, mentioned that the preferred prey is juvenile Sardinops sagax (Girard 1856). Our work addresses the reproductive strategy of the Panama brief squid in the Gulf of California using reproductive indicators, quantitative histology for the types of oocytes, and histochemistry to quantify the amount of lipid and glucidic reserves showing oocytes at different stages of development.


Biological material was collected during 15 exploratory fishing surveys in the Gulf of California from 2003 to 2006 and in 2008. The vessels BIP-XI and BIP-XII were used and were equipped with a trawl (90 ft in headline with a mesh size of 2-2.26 in. in the body and 1.5-1.75 in. in the cod end with otter boards (wooden doors) 10 ft x 60 in.). The sampling grid included 96 stations at depths of 7-165 m (Fig. 1). The tows were made horizontally at 2.5 knots.

The catch from each trawl was placed on deck, and a random sample of 40M5 kg was collected, which was then separated by taxonomic group (cephalopods, crustaceans, and fish), placing them in plastic bags, labeled properly and kept frozen.

In the laboratory, cephalopods were identified to the species level using diagnostic characteristics described in Fischer et al. (1995). For all Panama brief squid L. panamensis caught, we recorded the ML [+ or -] 0.1 cm, total weight (TW; [+ or -] 0.1 g), gender, and reproductive condition (immature, maturing, mature, spawning) from the morphochromatic properties of fresh gonads, according to the description of Barragan (1977a). The gonads were fixed in 10% formalin and, before histological processing, the ovarian and spermatophoric complexes were weighed.

To determine an annual cycle, the biological information collected during 2003 to 2006 and in 2008 was grouped by month to form a virtual year, because research cruises did not include all the months in each year of sampling. The average monthly sea surface temperature (SST) for each positive station was estimated for the period 2003 to 2008 from the remote sensor data of the Advanced Very High Resolution Radiometer Pathfinder 5, with a spatial resolution of 4 x 4 km ( The interannual differences in SST were evaluated to identify potential changes in the environment that limit the formation of an annual cycle of the biological data.

Gonadal Development

Gonadal development was analyzed by month and by males and females based on the percentage of organisms by maturity stage (immature, maturing, mature, and spawning) according to the morphochromatic scale of Barragfin (1977a) as

[FR.sub.i] = ([n.sub.i]/N) x 100,

where [FR.sub.i] is the percentage of gonads in the ith maturity stage, [n.sub.i] is the number of gonads in the ith maturity stage, and Nis the total number of gonads examined.


Gonadosomatic Index

The gonadosomatic index (GSI) by month and gender was obtained from the ratio of the TW of the ovary (TWG) and TW of the body (TW) in females, and the weight of the spermatophores complex (TWG) and the TW in males (Sauer et al. 1999, Markaida & Sosa-Nishizaki 2001), and was estimated based on the expression

GSI = (TWG/TW) x 100,

where TWG and TW are measured in grams.

Gender Ratio

Total gender ratio was determined. Statistical significance was tested with a chi-square test, testing a null hypothesis of 3 females per male ([H.sub.0] = 3:1), which was reported by Squires and Barragan (1979) in L. panamensis. The observed value was compared with the theoretical value of chi-square with a 95% confidence level (Zar 1999).

Quantitative Histology

For histological analysis, we used the ovaries of females collected during July and August 2008. Three transverse sections of the gonad (anterior, middle, and posterior), 5 mm thick, were placed in cassettes, then dehydrated in increasing concentrations of ethyl alcohol at 70%, 80%, 90%, 96%, and 100% (1 h at each concentration); clarified with xylene; and embedded in Paraplast X-Tra with a melting point of 54-56[degrees]C. Four tissue sections 4 [micro]m thick were made with a Leica RM 2155 rotating microtome and stained with Harris hematoxylin and counterstained with eosin-phloxine (HE) and with Masson's trichrome (Sheehan & Hrapchak 1980). After obtaining the histological preparations, ovarian stages and oocyte substages were determined through microscopic observations based on morphological characteristics of the ooplasm, oolemma, and follicular epithelium surrounding the oocyte. Histochemistry was done to determine the composition of the oocytes, and they were stained with periodic acid-Schiff (PAS) reagent for carbohydrates (Martoja & Martoja-Pierson 1970) and Sudan Black (SB) for lipids (phospholipids and triglycerides) (Rodriguez-Moscoso & Arnaiz 1998).

Size of Oocytes

The area of oocytes was obtained from digitized images of histological sections taken at 10x with Olympus microscopes BX41 and BX50, and images were captured with a CoolSnapPro digital camera connected to a computer using the program Image Pro Plus (v.7.0). A total of 50 oocytes were measured per female, differentiated by the substage of development. Gametes with a visible nucleus were measured, only in the case of a mature oocyte with a own nucleus that had migrated. The test was to measure the larger oocytes. Once the scale of the images was assigned and traced the perimeter of the oocyte manually, the program automatically calculated the area. The diameter is calculated from the formula

Dt = [square root of 4A/[phi]],

where Dt is the diameter, A is the area, and [pi] - 3.1416 (Briarty 1975, Saout et al. 1999). Then the mean was calculated with the SD for the substage during the main growth phase, previtellogenesis, vitellogenesis, or postvitellogenesis.

Quantification of Phospholipids and Triglycerides

To quantify the amount of phospholipids and triglycerides, the standardized methodology of Rodriguez-Jaramillo (2004) was followed, who proposed an index based on the lipid-specific staining of the lipid dye Sudan Black B (SB), and quantified by the digital image analysis of the ooplasm lipids present in the oocyte through the selection of tones of each pixel-specific color staining, expressed as a coverage area (Rodriguez-Jaramillo et al. 2008). The SB staining technique stains triglycerides in blue to black and phospholipids in shades of gray (Bayliss 1984, Rodriguez-Moscoso & Arnaiz 1998). We selected 10 female gonads and stained the histological sections with SB. For each gonad, 3 images were digitized at 10 x. In each female, the oocytes were measured, and 30 areas of the gray pixel tones that correspond to phospholipids (Pls) and the dark blue to black corresponding to triglycerides (Tgs) were quantified by adding the occupied pixels expressed in area. These areas are recorded in a spreadsheet, and the lipid levels (PlI and Tgl) were then determined using the equations of Rodriguez-Jaramillo (2004):

PlI = (Pls coverage area/Oocyte area) x 100


TgI = (Tgs coverage area/Oocyte area) x 100

To identify other components, such as carbohydrate reserves in the ovary of the female Panama brief squid, we used the ovaries of 18 females stained with PAS, which stains carbohydrates and glycoproteins pink-magenta (Martoja & Martoja-Pierson 1970).

CrI = (Carbohydrates coverage area/Oocyte area) x 100

Size at First Maturity ([L.sub.50])

TO determine the population size at first maturity in females, we used 2 approaches. The first was by grouping 44 females who were in the Vol, Vo2, and Pro ovarian stages, as determined by histological analysis, and a second using 1,170 mature females according to the macroscopic scale. This was done to evaluate the differences in assessing the size at maturity using both methods. In males, only the morphochromatic scale method was used based on levels of maturity; histological analysis was not done. For both genders, the organisms were grouped into class intervals and the cumulative relative frequency was calculated for estimating [L.sub.50], defined as the length interval where 50% of the population showed reproductive activity. The data were input into the logistic equation (Sparre & Venema 1995).


where P% is the proportion of mature organisms in the ith size interval, [S.sub.1] and [S.sub.2] are model parameters, and L is the ML in millimeters. [L.sub.50] is equal to the ratio [S.sub.1]/[S.sub.2].


We sampled a total of 1,887 organisms with a mean ML of 67 [+ or -] 17 mm and a mean TW of 16.9 [+ or -] 10.3 g. During the study period 2003 to 2008, the SST showed a similar annual pattern (ANOVA: F = 4.96, P > 0.05). This result strengthens the decision to group the information by simulating an annual cycle with the aim of assessing changes in the reproductive status of Panama brief squid.

Gonadal Development

Of the total of organisms collected, the number of females was 1,507 and males numbered 380, with more than 50% of the organisms having mature gonads (Fig. 2). Females had the highest reproductive activity in February, September, and October. The males had the highest reproductive activity in March, May, and September when most organisms are sexually active. Throughout the year there were individuals in maturing and mature stages; however, there was no spawning stage. No male squid were collected in November. In June and December, no research cruises occurred.

Gonadosomatic Index

The average GSI of females ranged from 0.01-1.76 and in males was 0.001-.07, with the differences in the values because the reproductive system of females was larger and heavier than the males (Fig. 3). In male squid, there were 2 GSI peaks in April and August. In females, the GSI peaks were in July and, to a lesser extent, February.

Gender Ratio

The gender ratio during the study period was 4 females: 1 male, which is different from the reference value according to Barragan (1977b). Significant monthly differences (chi-square, P < 0.05) were identified in January, February, March, May, August, and October, whereas in April and July there were no differences when comparing the ratio with 3 females: 1 male (chi-square, P > 0.05; Table 1).

When assessing the differences in the gender ratio by interval length for the 3 females:1 male ratio (Table 2), significant differences were found in the ML in intervals ranging from 30-39 mm and 100-109 mm. Males dominated in ML sizes smaller than 50 ram, and females dominated in ML sizes larger than 50 mm. In ML interval ranges of 20 29 mm and 110-119 mm, the sample size of organisms was small and the results are, to some extent, suspect.

Quantitative Histology

Histological analysis was accomplished using HE to stain the ovaries of 100 females collected in July and August 2008. There was a synchronous ovarian development by groups, characterized by the simultaneous presence of 6 types of oocytes in different substages of growth.

Four stages of oogenesis were characterized: previtellogenesis (oogonia, Po0; early previtellogenic oocyte, Pol; late previtellogenic oocyte, Po2), vitellogenesis (early vitellogenic oocyte, Vol; late vitellogenic oocyte, Vo2), postvitellogenesis (postvitellogenic oocyte, Pvo), and 2 structural indicators of spawning (postovulatory follicles (FP) and atretic follicles (FA)) (Fig. 4, Table 3). To identify the incorporation of lipids in all types of oocytes, SB was used. The result of this histochemical staining showed histological sections of the previtellogenic ovaries that were gray, indicating that at this stage of growth oocytes are composed mainly ofphospholipids, both oolemma and ooplasm. For vitellogenic oocytes, they accumulate small granules that are stained dark blue to black, indicating that their composition is made up of triglycerides as an energy reserve. In postvitellogenic oocytes we observed that the composition of the membranes of the egg, both the yolk sac and the follicular cells, are composed mainly of phospholipids (polar lipids), whereas the ooplasm contains a large amount of neutral lipids (triglycerides). Similarly, PAS staining showed a magenta-pink color, indicating the presence of carbohydrates as a reserve in the previtellogenic oocytes, and that form part of the yolk in the postvitellogenic oocytes at mature stage of the female squid.


Morphological criteria used to assign the developmental stages of oocytes according to the progress of gametogenesis (previtellogenesis, vitellogenesis, postvitellogenesis, postspawning) were the abundance of ovarian stroma, the thickness of the follicular layer of oocytes, the presence of lipid and carbohydrate inclusions in the ooplasm, invaginations of the follicle, the presence and thickness of the corium, and the presence of FP and oocytes in reabsorption. Based on these characteristics, 4 stages of ovarian development were defined along with 6 oocyte substages (Table 3).


Size of Oocytes

A total of 3,122 oocytes were measured, including the 6 defined stages, and to assess the differences in diameter were detected significant differences between the stages of the oocyte (ANOVA: [F.sub.(5, 3,116)] = 5934,7, P < 0.05; Fig. 5).

Size at Maturity

The size range in which mature females were found was an ML of 30 115; male ML ranged from 27-100 ram. In females, the size at first maturity ([L.sub.50]) based on the morphochromatic scale was an ML of 75 mm and according to oocyte stages, Ovl, Ov2, and Opv, was at 85 mm in ML (Fig. 6). In males, [L.sub.50] was estimated at an ML of 51 mm. Females mature at lengths greater than males.

Quantification of Phospholipids and Triglycerides

We found significant differences in the lipid index of oocytes at different stages of gonadal development. The maximum percentage of structural phospholipids was estimated at stage I (20%) and the minimum (7.1%) was found at stage 1V (partial spawning and postspawn). For the reserve lipids or triglycerides, the percentage increased from stage I at 2.4% to stage III at 6.4%, with a maximum at maturity of 25%. Carbohydrates were present in stage I (3.1%), increasing to 42% in stage II, and reaching a maximum in stage III (67%). In stage IV, partially spawned organisms still contained previtellogenic oocytes and early vitellogenic oocytes (mature oocytes were spawned), so the levels of lipids (phospholipids, 4.5%: triglycerides, 25%) and carbohydrates (3.1%) were reduced (Fig. 7).


Gonadal development

The reproductive conditions of the Panama brief squid L. panarnensis, based on the morphochromatic scale proposed by Barragan (1977b), had more than 50% of the organisms (both genders) with mature gonads during the study period. This could be considered a reproductive tactic, allowing the species to reproduce and maximize favorable environmental conditions. This same behavior was reported by Barragan (1977a) for the species along the Colombian coast, where he found males and females with reproductive activity throughout the year.

The frequency of the mature stage showed peaks that are associated with increased reproductive activity, with a greater number of females at this stage being found in February and October, whereas males reached their most mature stage in April and August. The peak of the reproductive cycle of females and males were found to be out of phase, and it is believed that it is not necessarily required that gonadal development of males and females be synchronized for copulation or spawning because, in squid, the males place spermatophore packages into juvenile females, which in reality is when the moment of fertilization is defined (Hanlon & Messenger 1996). Using this scheme, the use of the spennatophores occurs between 3-4 mo after mating.

The high proportion of mature organisms (both genders) throughout the year indicates that temperature is not limiting for maturation of L. panamensis. It suggests that small changes in temperature serve as a stimulus for the greatest number of organisms to reach maturity and spawn. Giese & Pearse (1974), assuming that temperature is not a determining factor for the maturity in cephalopods, hypothesized that the accumulation of nutrients and hormonal activity are the mechanisms that determine the frequency of maturation (synchronous and intermittent periods). In addition, these same authors comment that reproductive activity is regulated according to daylight hours (photoperiod).

Gonadosomatic Index

The GSI is considered to be an indicator of gonadal development (Saborido 2004). In our work, this ratio was used as an indicator of spawning in the Panama brief squid. The average values of intermonthly GSI variations in females ranged from 0.01-1.76, and in males from 0.001 0.07. The highest values of the GSI were found in females because the ovary is heavier than the male spermatophoric sack. The maximum values of GSI coincide with the most mature organisms.

The minimum values of the GSI indicate spawning--in females, in January, March, May, August, October, and November; in males, in January, May, July, and September. This suggests that the Panama brief squid spawn throughout the year. There was no temporal synchrony in sexual maturation between genders, indicating that the males mate with females who have not reached maturity. This was already reported in L. gahi females (Pineda et al. 1998).

In other species of squid, as in L. panamensis, reproductive activity has been reported throughout the year with 2 well-defined spawning peaks. Sauer et al. (1992) found that L. vulgaris reynaudii spawns throughout the year, peaking in the spring and summer with greater intensity. Costa and Fernandes (1993) also reported 2 peaks of spawning in L. sanpaulensis, one in winter-spring and another summer autumn.

Gender Ratio

For the Panama brief squid in the study area, we found a ratio of 4 females:1 male. According to Emery et al. (2001a), a greater number of females compared with males increases genetic diversity positively, influencing survival. One possible explanation for this gender ratio was raised by Sauer et al. (1992), who noted that L. vulgar& reynaudii is grouped by gender in the vicinity of spawning areas. In our study, the highest proportion of females compared with males is similar to that reported for L. vulgaris reynaudii (Sauer et al. 1999), suggesting a spatial segregation by gender and area of male distribution has not been sampled efficiently. In contrast, Perez et al. (2002) reported 1 H: 1 male for Loligo plei at the time of spawning, and after this event the number of males increased, which was also found by Rodrigues and Gasalla (2008). Through genetic testing, Emery et al. (2001a, 2001b) confirmed that females of Loligo forbesi (Steenstrup 1856) mated with up to 4 males. The egg-level identification and paralarvae suggested that differences in the ML in the paralarvae were the result of genetic variability.


Timing differences in the gender ratio indicated that the Panama brief squid made migrations into the study area. This behavior has been documented in other species such as L. vulgaris reynaudii (Sauer et al. 1992) and L. plei (Perez et al. 2002). The migration is more intense when spawning is imminent.

The gender ratio by length interval showed significant differences. We found that in smaller sizes (ML, 30-40 mm) males predominated, a situation that was reversed at sizes greater than an ML of 50 mm. These differences may be because the males mature at shorter lengths, and have intercourse and increased mortality after mating. Females reach lengths greater than males, possibly increasing their reproductive potential.

Quantitative Histology

There are few reports for loliginid squid in which histological analyses have been used to describe oocyte types. Sauer and Lipinski (1990) identified in L. vulgaris reynaudii 4 types of oocytes: (1) oogonia, (2) oocytes with follicle cells that change from squamous to cuboidal, (3) oocytes surrounded by follicular cells, (4) follicular oocyte folds displaced to the periphery by the formation of the yolk, and (5) mature oocytes without follicle cells.

We describe 6 types of oocyte, 1 more than Sauer and Lipinski (1990) and 2 less than Sauer et al. (1999) for L. vulgaris reynaudii. In L. vulgaris reynaudii, FP, atresia, and oocyte development were identified in the same ovary. The presence of these structures suggested that the species is a multiple spawner. Macewicz et al. (2004) identified 3 stages of ovarian development in L. opalescens: immature (oogonia and oocytes with follicular invaginations), mature preovulatory (oocytes with yolk), and mature (postovulatory ovarian follicles). This description is too general because histological analysis allows one to describe the different types of oocytes in greater detail.

Although there is much discrepancy between the number of stages and types of oocytes in the assigned nomenclature in the scientific literature, most agree in establishing various stages with different types of oocytes. By using morphological and histochemical stages and types of oocytes, a more rigorous analysis is possible that allows for a more uniform nomenclature to describe oogenesis. This can be done by focusing on the stages of oogenesis--previtellogenesis, vitellogenesis, and postvitellogenesis--thereby avoiding confusion and allowing for standardization of the studies of gonadal development and allowing for a better understanding of the reproductive strategies of cephalopods.

The presence of different types of oocytes in the ovary of the Panama brief squid suggests that the species has a synchronous oocyte development by groups with intermittent spawning such as L. vulgaris (Lamarck 1798), L.Jorbesi, Loligo pealeii, and L. gahi (Rocha et al. 2001). Rocha et al. (2001) describe the interspecies reproductive strategies, considering the type of ovulation, spawning, and growth pattern of individuals during the reproductive period. The squid L. opalescens is classified as a terminal spawner because it has synchronous ovulation, and egg laying occurs during a short period at the end of its life cycle. The squid Loligo bleekeri (Keferstein 1866) has a highly asynchronous development of eggs, spawning several batches during the breeding season. Sauer et al. (2000) referred to L. vulgaris reynaudii as an iteroparous, unitemporal species because it deposits several capsules in various locations during an extended spawning season.

The presence of FP indicates that the Panama brief squid matures and spawns at least once in its life. Macewicz et al. (2004) argue that the presence of FP in the ovary indicates that ovulation is not a continuous process that develop and mature oocyte cohorts. Melo and Sauer (2007) found FP in the ovary of L. reynaudii (Orbigny 1845) 14 h after spawning, whereas Laptikhovsky et al. (2002) noted that these structures are absorbed quickly. The presence of FP in ovaries of female Panama brief squid suggests that they had spawned a few hours before their capture.

Atresia in oocytes is structurally degenerating cells but with energy reserves (Melo & Sauer 1998). These structures were found along with previtellogenic oocytes in the ovary of L. vulgaris reynaudii and L. opalescens (Melo & Sauer 1998, Macewicz et al. 2004). Melo and Sauer (1998) mentioned that the presence of atresia is caused by an excess of oocytes in the ovary or insufficient energy to ripen the eggs. In our work, atresia was found in maturing and mature females, which suggests that an energy deficit and reabsorption of oocytes will ensure that other oocytes mature and are released.

The Panama brief squid has a synchronous oocyte growth by groups, and the presence of FP and atresia in the ovary. This is highly suggestive evidence that female Panama brief squid mature and spawn more than once during their reproductive period.

Quantification of Phospholipids and Triglycerides

In our study, semiquantitative techniques based on digital image analysis were used to determine the proportion of energy reserves in previtellogenic, vitellogenic, and postvitellogenic oocytes. Vitellogenesis is an essential event for the development of female gametes. The accumulation of yolk in the oocytes of oviparous animals is essential for embryonic development after fertilization, and is therefore a key process in reproductive success. The general rule in all oviparous organisms is that there are 2 phases of vitellogenesis depending on the nature of the molecules incorporated. During the first phase, called early vitellogenesis, carbohydrates and lipids are incorporated. During the second phase, proteins are incorporated; the major molecule incorporated is vitellogenin (Raven 1966).


The characterization of the processes of previtellogenesis, vitellogenesis, and postvitellogenesis is described through histological examination of gametogenesis. During maturation of gametes, the main energy components are glycoproteins and triglycerides, which are the main source of energy for the future embryos. Our results suggest that glycoproteins and phospholipids are the main components of the oocyte at the beginning of gametogenesis. As this progresses, the ooplasm changes its composition to incorporate significant amounts of neutral lipids (triglycerides) and phospholipids, forming membranes and follicle cells. The abundance of glycoproteins and triglycerides stored in mature oocytes of the Panama brief squid provide important information about the relationship between energy reserves and gonadal development cycles, which have so far been documented poorly in cephalopods.



Size of Oocytes

The presence of oocytes of different sizes is an indicator of continuing development and supports the hypothesis that Panama brief squid females spawn more than once during their reproductive period. Squires and Barragan (1979), found different-size oocytes in the ovary of the Panama brief squid female and concluded that it is possible for the female to release more than 1 capsule (which envelops the oocytes) and spawn more than once in its reproductive period. This has also been reported in the ovaries of females of L. vulgaris reynaudii (Melo & Sauer 1998).

Based on the size of the oocytes in L. pealeii, Maxwell et al. (1998) concluded that this species is characterized as a multiple spawner. Similar results were found in our study. We identified 3 different sizes of oocytes in the same ovary. By analyzing the frequency of the size of oocytes, we identified 2 significant increases in size. The first change came when vitellogenesis began (average size, 381 [micro]m), attributed to the accumulation of triglycerides such as small lipid inclusions observed in the cytoplasm of the oocyte by staining with SB. The second increase was observed at the beginning of postvitellogenesis (average size, 697 [micro]m). We found no reports in the literature on significant increases in the size of the oocytes. Melo and Sauer (1999) reported that the vitellogenic-stage oocytes of L. vulgaris reynaudii have a size of 980 [micro]m, Macewicz et al. (2004) for L. opalescens and Laptikhovsky and Arkhipkin (2001) for L. gahi document that maturing oocytes reached 1,100 [micro]m. In other species of loliginid squid, the oocyte size is larger than the oocytes of the Panama brief squid, which is the result of differences in size of mature females.

Size at Maturity

By comparing the [L.sub.50] size of the female Panama brief squid using a morphochromatic scale (ML, 75 mm) and histology (ML, 85 mm), a difference in ML of 10 mm was found, which indicates the usefulness of histological analysis and which identifies microscopically the oocyte developmental stage and determines the minimum length for capture for the Panama brief squid as a means to manage the resource and ensure fishery sustainability. In males, the [L.sub.50] size was determined at 51 mm from a morphochromatic scale, which is less than that determined in females. This difference may be a tactic that impacts the reproductive potential of the spawning stock positively.

This difference in size at maturity between genders in the Panama brief squid has been reported at other latitudes. Barragan (1977b) noted that females of L. panamensis mature at lengths greater than an ML of 75 mm, whereas males reach maturity at smaller MLs (40 mm), attributes these differences to the existence of a sexual dimorphism that also has been reported for L. plei (Perez et al. 2002). In both L. gahi and L. sanpaulensis, males and females mature at similar sizes (Pineda et al. 1998).

The largest size at maturity in females of the Panama brief squid can be interpreted as a tactic of the species to maximize its reproductive potential. The length is a factor that determines the ability to develop and accumulate oocytes in the ovary and oviduct. In contrast, mature males at smaller lengths are less invested developmentally in energy; hence, they reach maturity sizes more quickly to direct energy to mating.


Biological material for this study was obtained from the exploratory surveys of the project "Evaluation of shrimp stocks in marine waters of the Mexican Pacific during the period of closure and the effect of the bycatch of shrimp bycatch in the ecosystem." For technical help in the laboratory we thank Maria Eulalia Meza Chavez, and Jesus Padilla Serrato for the preservation of the samples. D. I. A. R. was a scholarship recipient from the Consejo Nacional de Ciencia y Tecnologia (CONACyT) and the Programa Institucional de Formacion de Investigadores (PIFI-IPN). C. Q. V. received fellowships from the EDI and COFAA programs. Thanks to Ellis Glazier for editing our English.


Alejo-Plata, M. C., G. Cerdenares-Ladron de Guevara & J. E. Herrera-Galindo. 2001. Cefalopodos loliginidos en la fauna de acompanamiento del camaron. Cienc. Mar. 5:41-46.

Barragan, V. J. 1977a. Estudio de la nutricion del calamar del Pacifico Colombiano, Lolliguncula panamensis, Berry (Cephalopoda: Myopsida). Div. Pesq. 10:1-7.

Barragan, V. J. 1977b. Estudio de la maduracion sexual del calamar del Pacifico Colombiano, Lolliguncula panamensis, Berry (Cephalopoda: Myopsida). Div. Pesq 10:8-14.

Bayliss, H. O. 1984. Lipid histochemistry. Department of Pathology, Guy's Hospital Medical School London. London: Oxford University Press. 68 pp.

Briarty, L. G. 1975. Stereology methods for quantitative light and electron microscopy. Sci. Prog. 62:1-32.

Costa, P. A. S. & F. C. Fernandes. 1993. Reproductive cycle of Loligo sanpaulensis (Cephalopoda: Loliginidae) in the Cabo Frio region, Brazil. Mar. Ecol. Prog. Ser. 101:91-97.

Emery, A. M., I. J. Wilson, S. Craig, P. R. Boyle & L. R. Noble. 2001a. Assignment of paternity groups without access to parental genotypes, multiple mating and developmental plasticity in squid. Mol. Ecol. 10:1265-1278.

Emery, A. M., L. R. Noble & P. R. Boyle. 2001b. Squid family values: multiple paternity of Loligo forbesi egg strings examined. B. Mar. Sci. 71. 1119 (poster abstract).

Fischer, W. & F. Krupp, W. Schneider, C. Sommer, K. E. Carpenter & V. H. Niem. 1995. Guia FAO para la identificacion de especies para los fines de pesca. Rome: FAO Pacifico Centro-oriental. 1813 pp.

Giese, A. C. & J. S. Pearse. 1974. Introduction: general principles. In: A. C. Giese & J. S. Pearse, editors. Reproduction of marine invertebrates, vol. 1. New York: Academic Press. pp. 1-49.

Hanlon, R. & J. Messenger. 1996. Cephalopod behavior. Cambridge: Cambridge University Press. 232 pp.

Hernandez-Vazquez, S. 1987. Pesquerias pelagicas y neriticias de la costa occidental de Baja California, Mexico. CalCOFI 28: 53-56.

Laptikhovsky, V. V. & A. I. Arkhipkin. 2001. Oogenesis in the squid Loligo gahi in the southeast shelf of the Falkland Islands. J. Mollusc. Stud. 67:475-482.

Laptikhovsky, V., A. Salman, B. Onsoy & T. Katagan. 2002. Systematic position and reproduction of squid of the genus Alloteuthis (Cephalopda: Loliginidae) in the eastern Mediterranean. J. Mar. Biol. Assoc UK 82:983-985.

Macewicz, B. J., J. R. Hunter., N. C. H. Lo & E. L. LaCasella. 2004. Fecundity, egg deposition, and mortality of market squid (Loligo opaleseens). Fish. Bull. 102:306-327.

Markaida, U. & O. Sosa-Nishizaki. 2001. Reproductive biology of jumbo squid Dosidicus gigas in the Gulf of California, 1995-1997. Fish. Res. 54:63-82.

Martoja, R. & M. Martoja-Pierson. 1970. Tecnicas de histologia animal. Barcelona: Toray-Masson. 350 pp.

Maxwell, M. R., W. K. Macy, S. Odate & R. T. Hanlon. 1998. Evidence for multiple spawning by squids (Loligo pealei) in captivity. Biol. Bull. 195:22-226.

Melo, Y. C. & W. H. H. Sauer. 1998. Ovarian atresia in cephalopods. S. Afr. J. Mar. Sci. 20:143-151.

Melo, Y. C. & W. H. H. Sauer. 1999. Confirmation of serial spawning in the chokka squid Loligo vulgaris reynaudii off the coast of South Africa. Mar. Biol. 135:307-313.

Melo, Y. & W. H. H. Sauer. 2007. Determining the daily spawning cycle of the chokka squid, Loligo reynaudii off the South African Coast. Rev. Fish Biol Fisheries. 17:247-257.

Moltschaniwskyj, N. A. & J. M. Semmens. 2000. Limited use of stored energy reserves for reproduction by the tropical loliginid squid Photololigo sp. J. Zool. (Lond.) 251:307-313.

Perez, J. A. A., D. C. Aguiar & U. C. Oliveira. 2002. Biology and population dynamics of the long-finned squid Loligo plei (Cephalopoda: Loliginidae) in southern Brazilian waters. Fish. Res. 58:267-279.

Pineda, S. E., N. E. Brunetti & N. A. Scarlato. 1998. Calamares loliginidos (Cephalopoda, Loliginidae). In: E. E. Boschi, editor. Los moluscos de interns pesquero: cultivos y estrategias reproductivas de bivalvos y equinodermos, pp. 13-36.

Raven, C. P. 1966. Morphogenesis: the analysis of molluscan development. Oxford: Pergamon Press. 365 pp.

Rocha, F., A. Guerra & A. F. Gonzalez. 2001. A review of the reproductive strategies in cephalopods. Biol. Rev. Camb. Philos. Soc. 76:291-304.

Rodrigues, A. R. & M. A. Gasalla. 2008. Spatial and temporal patterns in size and maturation of Loligo plei and Loligo sanpaulensis (Cephalopoda:Loliginidae) in southeastern Brazilian waters, between 23[degrees]S and 27[degrees]S. Sci. Mar. 72:631-643.

Rodriguez-Jaramillo, C. 2004. Efecto de la temperatura sobre la gametogenesis en el callo de hacha Atrina maura (Sowerby, 1835) (Bivalvia: Pinnidae). MS thesis, Centro Interdisciplinario de Ciencias Marinas del IPN, La Paz, BCS, Mexico. 74 pp.

Rodriguez-Jaramillo, C., M. Hurtado, E. Romero-Vivas, J. L. Ramirez, M. Manzano & E. Palacios. 2008. Gonadal development and histochemistry of the tropical oyster, Crassostrea corteziensis (Hertlein, 1951) during an annual reproductive cycle. J. Shellfish Res. 27:1129-1141.

Rodriguez-Moscoso, E. & R. Arnaiz. 1998. Gametogenesis and energy storage in a population of grooved carpet-shell clam, Tupes decussurus (Linne, 1787), in northwest Spain. Aquaculture 162:125-139.

Saborido F. 2004. Ecologia de la reproduccion y potencial reproductivo en las poblaciones de peces marinos. Curso doutoramento do bienio 2002-2004. Universidad de Vigo. Inst. de Invest. Mar. Vigo, Espana. 71 pp.

Sanchez, P. 2002. Cephalopods from off the Pacific coast of Mexico: biological aspects of the most abundant species. Sci. Mar. 67: 81-90.

Saout, C., Y. M. Paulet & A. Duinker. 1999. Histological study on the early stages of oogenesis in Pecten maximits: a new approach with quantitative semithin histology. Bergen, Norway, 12th Inter. Pectinid Workshop. pp. 129-130.

Sauer, W. H. H. & M. R. Lipinski. 1990. Histological validation of morphological stages of sexual maturity in chokker squid Loligo vulgaris reynaudii D'Orb (Cephalopoda: Loliginidae). S. Afr. J. Mar. Sci. 9:189-200.

Sauer, W. H. H., M. R. Lipinski & C. J. 2000. Tag recapture studies of the chokka squid Loligo vulgaris revnaudii d'Orbigny, 1845 on inshore spawning grounds on the south-east coast of South Africa. Fish. Res. 45:283-289.

Sauer, W. H. H., Y. C. Melo & W. de Wet. 1999. Fecundity of the chokka squid Loligo vulgaris reynaudii on the southeastern coast of South Africa. Mar. Biol. 135:315 319.

Sauer, W. H. H., M. J. Smale & M. R. Lipinski. 1992. The location of spawning grounds, spawning and schooling behaviour of the squid Loligo vulgaris reynaudii (Cephalopoda: Myopsida) off the eastern Cape coast, South Africa. Mar. Biol. 114:97-107.

Sheehan, D. & B. B. Hrapchak. 1980. Theory and practice of histotechnology, 2nd edition. OH: Battelle Press. 481 pp.

Sparre, P. & S. C. Venema. 1995. Introduccion a la evaluacion de recursos pesqueros tropicales. Parte I. Manual FAO Doc. Tec. de Pesca, no. 306.1, rev. 1. Rome. 440 pp.

Squires, H. J. & J. H. Barragan. 1979. Lolliguncula panamensis from the Pacific coast of Colombia. Veliger 22:67-74.

Zar, J. H. 1999. Biostatistical analysis, 4th edition. Englewood Cliffs, N J: Prentice-Hall. 123 pp.


(1) CICIMAR-IPN, A venida Instituto Politecnico Nacional sin Col. Playa Palo de Santa Rita, Apartado Postal 592, La Paz, Baja California Sur, Mexico, CP 23090; (2) CIBNOR, Mar Bermejo 195 Col. Playa Palo de Santa Rita, CP 23090

* Corresponding author. E-mail:

DOI: 10.2983/035.031.0328
Number of males and females per month and chi-square
statistic values to evaluate differences.

             Males   Females
Month         (n)      (n)     Statistical Analysis

January          8        45   Chi-square = 8.20, df = 1, P < 0.05 *
February       150       638   Chi-square = 72.71, df = 1, P < 0.05 *
March           11        67   Chi-square = 12.98, df = 1, P < 0.05 *
April          101       237   Chi-square = 1.87, df = 1, P > 0.05
May              1        17   Chi-square = 6.25, df = 1, P < 0.05 *
June            --        --                      --
July            57       147   Chi-square = 2.66, df = 1, P > 0.05
August          35       244   Chi-square = 54.25, df = 1, P < 0.05 *
September        1         0                      --
October         16       105   Chi-square = 21.74, df = 1, P < 0.05 *
November         0         5                      --
December        --        --                      --

* Significant difference.

Evaluation of the differences between the number of male and
female organisms by size range and chi-square statistic values.

Length Size
Interval     Males  Females
(mm)          (n)     (n)              Statistical Analysis

 20              1        2  Chi-square = 0.00, df = 1, P > 0.05
 30             31       23  Chi-square = 12.79, df = l, P < 0.05 *
 40            144       60  Chi-square = 136.37, df = 1, P < 0.05 *
 50             98      134  Chi-square = 7.68, df = 1, P < 0.05 *
 60             55      223  Chi-square = 32.61, df = 1, P < 0.05 *
 70             42      469  Chi-square = 425.21, df = 1, P < 0.05 *
 80              6      368  Chi-square = 2398.43, df = 1, P < 0.05 *
 90             --      182                     --
100              2       42  Chi-square = 88.32, df = 1, P < 0.05 *
110              1        4  Chi-square = 1.11, df = 1, > P 0.05

* Significant difference.

Description of ovarian oocyte stages and substages of the female
Panama brief squid Lolliguncula panamensis.

Ovarian State        Type of Oocyte   Description and Diameter

I (immature)         Po0              The oogonia are spherical cells,
                                      with scant ooplasm lacking
                                      follicular cells, and are
                                      embedded in the ovarian stroma,
                                      consisting of connective tissue
                                      (diameter, 36.82 [+ or -] 6.16
                                      [micro]m (SD)).

Previtellogenesis    Pol              The oocytes are irregular in
                                      shape with large, round nuclei
                                      and scant basophilic cytoplasm.
                                      Nucleoli are not observed in the
                                      nucleoplasm. The oocyte is still
                                      embedded in the connective
                                      tissue (diameter, 84 [+ or -] 28
                                      [micro]m). The ooplasm is
                                      composed of phospholipids (Sudan
                                      Black (SB)) and neutral
                                      carbohydrates (periodic
                                      acid-Schiff reagent (PAS)).

Primary growth       Po2              Polygonal oocytes, there is an
                                      increased volume of ooplasm that
                                      remains basophilic. The
                                      histochemical composition of
                                      ovoplasma is equal to Pol
                                      (diameter = 142, SD = 35
                                      diameter, Crl = 9.7%, PII = 20%,
                                      TgI = 2.4 IV.).

II                   Vol              In these oocytes the process of
                                      vitellogenesis has begun, there
                                      is the presence of follicular
                                      cells (early folliculogenesis),
                                      the nucleoplasm has reached its
                                      maximum growth, the ooplasm
                                      increases and becomes
                                      acidophilic (diameter = 185, SD
                                      = 68, CrI = 42%, PII = 17%, TgI
                                      = 6.4%).

Vitellogenesis       Vo2              The ooplasm is acidophilic and
Secondary growth                      granular. There is a change in
                                      the composition of ooplasm
                                      caused by the accumulation of
                                      phospholipids and triglycerides
                                      stained with SB and small lipid
                                      inclusions are observed. The egg
                                      takes an irregular shape
                                      (lobed), has an elongation at
                                      the ends, and is attached to the
                                      ovarian stroma. There are deep
                                      invaginations of the follicular
                                      cell layer (final
                                      folliculogenesis) and the
                                      nucleus moved to animal pole of
                                      the oocyte (diameter = 381, SD =
                                      124, CrI = 67%, P11 = 7.5%, Tg1
                                      = 25%).

III                  Pvo              Exogenous vitellogenesis, the
Postvitellogenesis                    oocyte is completely filled with
Tertiary growth                       yolk which is rich in
                                      triglycerides (SB) and
                                      carbohydrates (PAS). The
                                      invaginations of the follicular
                                      cells become less pronounced and
                                      are composed of phospholipids.
                                      At the end of maturity,
                                      invaginations of follicular
                                      cells disappear and oocyte are
                                      shaped hexagonal. Although at
                                      this stage is dominated by
                                      mature oocytes, some
                                      previtellogenic oocytes can be
                                      found (diameter = 697, SD = 114,
                                      CrI = 3.1%, P11 = 7.1 %, TgI =

IV                   FP and FA        Spawning can be partial,
Spawning and                          postovulatory follicles
postspawning                          characterized this stage with
                                      pyknotic follicular cells. Is
                                      also observed the presence of
                                      atretic oocytes in resorption.
                                      Atresia occurs in vitellogenic
                                      oocytes, are irregular shape,
                                      the nucleus is amorphous,
                                      follicular cell forms clusters.

Cr, carbohydrate; FA, atretic follicles; FP, postovulatory
follicles; PI, phospholipid; Tg, triglyceride.
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